Structural organization of HBV pgRNA genome driven by phase separation in capsid confinement

Domain: chemistry
Published: 2026-02-19
Editorial outcome: major_revision

Abstract
Viruses rely on the precise packaging of their genomes within a capsid to execute essential life-cycle events, yet the principles governing genome structural organization in this confined environment remain elusive. Here, we reveal that the hepatitis B virus (HBV) pregenomic RNA (pgRNA) exploits liquid-liquid phase separation (LLPS) inside the capsid to sculpt its architecture. Multiscale molecular dynamics (MD) simulations, supplemented by biochemical assays, show that pgRNA coalesces into a hollow, shell-like condensate along the inner capsid surface, with coexisting low- and high-density regions. Electrostatic interactions between pgRNA and the disordered C-terminal domain of capsid protein primarily govern condensate formation. LLPS drives the establishment of microphases composed of nematically aligned RNA hairpin arrays interspersed by domains rich in flexible single-stranded RNA linkers, achieving an optimal balance between structural order and dynamic flexibility. Intriguingly, although the ensemble-averaged pgRNA density exhibits icosahedral symmetry, individual simulation snapshots display pronounced heterogeneity, indicating symmetry breaking at the single-particle level. In addition, LLPS-induced hollow-shell architecture of pgRNA genome promotes long-range RNA base-pairing and enhances polymerase mobility, which may facilitate the functional dynamics of polymerase during reverse transcription. Our findings uncover a capsid-confined LLPS mechanism that orchestrates viral genome structure and dynamics, offering new targets for antiviral intervention.

Introduction
Viruses are nanoscale pathogens with their genetic material encapsulated in a protein capsid. Due to the polyanionic nature of nucleic acids and the restricted volume of the capsid, the viral genome is often tightly compacted with significant electrostatic repulsion1–3. Some viruses even execute key biochemical events, e.g., genome replication, within this densely crowded environment4,5. Consequently, a fundamental question arises: how is the viral genome structurally organized to fit the confined capsid space while still enabling essential biochemical functions? Addressing this question holds profound significance, because it not only advances our understanding of the key molecular mechanisms driving viral life cycle but also provides critical insights for designing targeted vaccines and antiviral therapies6–9.

The structural organization of viral genomes has long been a focus in structural biology research10–15. Breakthroughs in cryo-electron microscopy (cryo-EM) have enabled the determination of viral genome architectures at subnanometer resolution for several viruses, although the atomic resolved structural determination of viral genome remains unfeasible11,16. Complementing these experimental advances, molecular dynamics (MD) simulations have emerged as a powerful tool for probing the dynamic processes underlying viral genome packaging, offering insights that are otherwise difficult to capture experimentally17–22. For instance, Coshic et al. performed multiscale MD simulations for the bacteriophage HK97, which is a pressurized double-stranded DNA (dsDNA) virus with its genome compactly packaged within a preformed capsid under the assistance of packaging motor19. They uncovered a loop-extrusion mechanism of the motor-powered dsDNA encapsulation and produced all-atom structural models for the entire virion. The genomes of the densely packed dsDNA viruses often show clear order in structures within their capsids23,24. In contrast, for some viruses containing single-stranded RNA (ssRNA), e.g., the pregenomic RNA (pgRNA) intermediate of Hepatitis B virus (HBV) and the majority of icosahedral ssRNA viruses, their genomes can exhibit high conformational flexibility10. This inherent dynamics complicates traditional structural characterization methods, leaving the organizational principles of these viral genomes poorly understood. In this study, we investigate the structural organization and dynamics of HBV pgRNA genome within the viral capsid through multiscale MD simulations and experimental validation.

HBV is an icosahedral virus with its capsid composed of 240 capsid core protein (Cp) monomers arranged in T=4 symmetry (Fig. 1a). The Cp comprises two domains: a well-structured N-terminal domain (NTD; residues 1–149) and an intrinsically disordered C-terminal domain (CTD; residues 150–185) (Fig. 1b). It is important to note that HBV serotypes may exhibit distinct lengths (e.g., 183 aa). The NTDs assemble into the outer capsid shell, while the unstructured CTDs, which are enriched in arginine residues (termed as R-arm or ARM), shape up a positively charged inner capsid surface essential for genome packaging25,26. Although HBV is a dsDNA virus, its replication cycle involves a pregenomic RNA intermediate. During early stage of replication, viral pgRNA and polymerase co-assemble with capsid proteins to form immature virions27. DNA synthesis takes place in capsid via reverse transcription with pgRNA functioning as the template, during which the dynamic remodeling of pgRNA conformations plays a crucial role. Along with the DNA synthesis, pgRNA is hydrolyzed by the ribonuclease domain of the polymerase, which is then followed by synthesis of the complementary DNA strand to produce mature HBV. The structural organization and dynamics of pgRNA in capsid is central to its role in reverse transcription, making it a prime target for antiviral therapies28. Cryo-EM revealed an icosahedrally ordered density distribution of the encapsulated pgRNA with the CTDs acting as a nucleic acid chaperone29. In addition, MD simulations have elucidated asymmetric fluctuations of capsid subunits and ion transport dynamics across the capsid18,30. However, how pgRNA is structurally organized to enable its functional dynamics within capsid remains elusive.

To address this question, we investigate the structural organization and dynamics of HBV pgRNA within the capsid combining all-atom MD simulations, coarse-grained (CG) MD simulations, and biochemical experiments. Strikingly, the results demonstrate that the organization of the pgRNA genome is governed by liquid-liquid phase separation (LLPS) mediated protein-RNA co-condensation. The electrostatic interactions are the dominant driving force of the co-condensation between the unstructured CTDs and the pgRNA chain along the inner surface of the capsid. These CTD-pgRNA condensates exhibit liquid-like dynamics, with the pgRNA retaining high mobility. The viral polymerase tends to partition out of the condensate and into the dilute regions of the capsid interior, enabling rapid diffusion, which is a prerequisite for efficient reverse transcription. Intriguingly, while the density distributions of the CTD-pgRNA condensates in the individual snapshots sampled by MD simulations are highly heterogeneous, the ensemble-averaged density distribution recapitulates the icosahedral symmetry patterns as observed in cryo-EM maps, which suggests symmetry breaking of pgRNA organization in the individual virions. Notably, CTD-pgRNA condensates formed by LLPS exhibit microphase structure featured by nematically aligned RNA hairpin arrays interspersed by flexible single-stranded RNA linkers, achieving an optimal balance between structural order and dynamic flexibility. This study provides the first instance of a virus harnessing phase separation to orchestrate genome organization within a confined capsid space, though phase separation is well-documented in wide range of host-cell processes like ssRNA-mediated capsid assembly31–34. Targeting the phase behavior of CTD-pgRNA condensates could thus represent a novel antiviral strategy.

Results
Structural organization of pgRNA genome in HBV capsid
We first performed all-atom MD simulations with explicit solvent to elucidate the global structural characteristics of pgRNA within the capsid. The missing CTDs in the PDB structure of HBV capsid (PDB code: 3J2V) were reconstructed to generate a complete capsid. In the initial configuration of the MD simulations, the pgRNA with the wild-type sequence was placed inside the capsid in a compact conformation without pre-assigned secondary structures (Fig. 1c and Methods). During the simulations, pgRNA quickly condensed along the inner surface of the capsid, facilitating the emergence of a hollow conformation (Fig. 1d), which is in close agreement with the cryo-EM data observed in previous experimental studies29. The radial distribution of atomic density offers a direct demonstration of this structural feature (Fig. 1e). Notably, the density distributions of the CTDs and pgRNA are largely overlapping, indicating their co-condensation driven by CTD-pgRNA interactions. Furthermore, the density distribution within this spherical shell is non-uniform, with the coexistence of low and high density regions (Fig. 1f and Supplementary Movie 1).

To further assess the dependence of the results on the initial structure and force fields, we conducted two additional sets of all-atom MD simulations initiated from pgRNA structures without pre-assigned secondary structures and with predicted secondary structures, respectively. Both simulations employed an alternative force field (amber14sb_OL15 force field with NBFIX correction)35,36. Consistent with the above result (Fig. 1d), these simulations also revealed rapid condensation of pgRNA along the inner surface of the capsid, forming a shell-like arrangement (Supplementary Fig. 1a–d and Supplementary Movies 2, 3). These results indicate that the observed pgRNA condensation is a robust process, largely insensitive to the choice of initial pgRNA structures and force fields.

By analyzing ion concentrations from the all-atom MD simulations, we found that the number of mobile ions flowing in and out of the capsid becomes comparable within 10 ns (Supplementary Fig. 2a). Moreover, time-dependent radial distributions of ion concentrations converge within the simulation timescale (Supplementary Fig. 2b, c). These results suggest that the Donnan equilibrium can be established within the timescale of the all-atom MD simulations. The converged radial distributions further demonstrate that the distribution of mobile ions inside the HBV capsid exhibits notable heterogeneity (Supplementary Fig. 2d, e). In addition, the average concentration of Na+ inside and outside the capsid are 0.20 M and 0.086 M, respectively. The resulted relative ratio of the Na+ concentrations between the interior and exterior of the capsid (~2.3) is considerably smaller than the values reported for other dsDNA virus (>10)19. A likely explanation for this weaker Donnan equilibrium effect is that only a single RNA strand is present at the pgRNA intermediate stage of the HBV lifecycle. In this case, the positive charges of the CTD segments (+3600 in total) and the negative charges of pgRNA (−3200) are approximately balanced. With the progression of reverse transcription of pgRNA into dsDNA, the negative charges of the genome will significantly exceed the positive charges of CTDs, which would lead to more pronounced Donnan equilibrium effect.

It is intriguing to further investigate the biophysical mechanism driving the formation of the above observed structural organization of pgRNA genome. Physically, the observed coexistence of low- and high-density regions is often a hallmark of phase separation. Notably, LLPS of biomolecules has been widely implicated in many biological processes, such as chromatin organization, stress responses, and DNA damage repair37–46. Aberrant condensation is linked to the pathogenesis of cancer and numerous neurodegenerative diseases. To elucidate that the above observed structural organization of pgRNA within the capsid is an outcome of phase separation, we further investigated its structure and dynamics in response to typical environmental modulators of LLPS, including salt concentration (Csalt) and temperature (T) in the subsequent sections.

PhaseseparationofpgRNAgenomewithincapsidconfinement
Because all-atom MD simulations of the phase behaviors of biomolecules is computationally challenging, CG MD simulations were utilized to explore the phase behavior of pgRNA by manipulating the salt concentration Csalt and temperature T. In the CG simulations, each residue is represented by one spherical bead located at the Cα position (Fig. 2a), and each nucleotide is represented by three beads corresponding to the phosphate group (P), sugar ring (S), and base (B), respectively (Fig. 2b). The force fields are described by the AICG2+ model and 3SPN.2 model47–49, which have been successfully used in modeling the structural dynamics of protein-nucleic acid complexes50,51, such as the nucleosome assembly and remodeling52,53, the protein binding induced DNA bending54, the dynamics of parental H3/H4 recycling at the replication fork55, and the LLPS of protein-ssDNA mixture56. More details of the CG model are given in Methods.

Initiating from a uniformly distributed random conformation of pgRNA in the capsid (Fig. 2c), we performed three independent MD simulations across varying Csalt and T. In all simulations, pgRNA rapidly condensed into a spherical shell colocalized with CTDs. We also conducted CG simulations initiated from pgRNA structures with predicted secondary structures, and comparable results were observed (Supplementary Fig. 1e, f and Supplementary Movie 4). Under physiological conditions, concurrent segregation of pgRNA and CTDs into coexisting low- and high-density regions along this spherical shell was clearly observed (Fig. 2d), which is consistent with the results of all-atom MD simulations. CG simulations under different conditions further revealed that phase separation extent is sensitive to Csalt or T (Fig. 2d–f and Supplementary Figs. 3, 4a). At lower Csalt or T, pgRNA exhibited more pronounced phase separation, forming grid-like morphology with heterogeneous density along the spherical shell, whereas an increment in Csalt or T leads to a more uniform morphology.

To characterize phase separation behavior, we categorized nucleotides into “free” and “condensed”, depending on whether they form large clusters. As shown in a representative MD simulation trajectory under physiological conditions (Fig. 2g), the nucleotides in the free state were observed to progressively coalesce into condensed state. By the end of the simulation trajectory, more than half of the nucleotides coalesced into condensates, while a substantial fraction remained free. Concurrently, low-density pores emerged throughout the spherical shell. These observations clearly illustrate a phase separation that results in the formation of substructures with different densities. Furthermore, under conditions of low salt concentration and temperature (Fig. 2h and Supplementary Fig. 4b), the nucleotides exhibited a more pronounced phase separation with increased content of condensates, consistent with the above conformational analysis. We then quantified phase behavior by dividing the pgRNA spherical shell into discrete bins and computing pgRNA densities within each bin (Methods). Phase diagram displays the coexistence of low- and high-densities (ρ l and ρ h) across wide range of Csalt and T (Fig. 2i and Supplementary Fig. 4c). This bimodal density distribution and its environmental dependence support again the phase separation of pgRNA genome.

Given that biomolecular condensates formed via LLPS typically exhibit liquid-like properties, we further investigated the fluidity of the encapsidated pgRNA genome. By calculating the density of RNA nucleotides within a bin located at a fivefold symmetry site over time (Fig. 2j), we observed significant fluctuations, indicating that the pgRNA genome is highly dynamic and the nucleotides are exchangeable between low- and high-density regions. By dividing the pgRNA sequence into 100-nucleotide segments, we also calculated the mean squared displacement (MSD) averaged among these segments from the aforementioned CG MD simulations. The MSD results reveal substantial diffusivity (Fig. 2k and Supplementary Fig. 4d), particularly under physiological conditions (Supplementary Movie 5). At longer lag-timescale, the slope of the MSD plot decreases due to confinement within the capsid (Supplementary Fig. 5). These observations suggest that the pgRNA maintains a high degree of fluidity despite its association with the inner surface of the capsid, supporting a liquid-like condensate organization, rather than a fully ordered self-assemble structure under physiological conditions.

PhaseseparationofpgRNAgenomeinbulksystem
To directly assess the LLPS propensity of CTD-RNA mixtures, we carried out slab coexistence simulations, which is a benchmark method for characterizing biomolecular phase behavior57–59. The simulation system contains 216 protein chains corresponding to the disordered CTD segments and 216 15-mer polyadenosine oligonucleotides (A15), recapitulating the protein-RNA stoichiometry found in pgRNA-encapsulated HBV capsids. By this setup, the geometric restraints arising from capsid anchors and contiguous RNA chain are eliminated, and the simulation results will be dominated by the intrinsic physicochemical properties of the two components. At 300K and 150mM salt, the z-axis density profiles reveal two coexisting phases: a dense condensate enriched in inter-chain CTD-RNA contacts, and a dilute phase of freely diffusing molecules (Fig. 3a, b and Supplementary Fig. 6; Supplementary Movie 6). This bimodal demixing persists across a wide range of salt concentrations, as shown by the distinct bimodal boundary in the phase diagram (Fig. 3c). These findings clearly establish that the CTD-RNA system is capable of undergoing phase separation to yield coexisting dense and dilute phases.

We then experimentally validated phase separation in CTD-RNA solutions composed of CTD peptides and A15 RNA chains. At 10 μM concentration for each of the two components, we observed characteristic LLPS features including droplet growth and spontaneous droplet fusion events by brightfield light microscopy (Fig. 3d, e and Supplementary Movie 7), confirming liquid-like condensate formation. Further variation of salt concentration revealed a strong dependence of phase separation on electrostatic interactions (Fig. 3f). Condensates formed readily at low and moderate salt concentrations but were completely suppressed at 500 mM salt, demonstrating that the electrostatic interactions between CTDs and RNA molecules play a crucial role in driving condensation. To probe hydrophobic contributions, we added 1,6-hexanediol, a compound known to disrupt hydrophobic interactions, into the solution at the Csalt of 150 mM. Phase separation persisted even at a high concentration of 1,6-hexanediol (20% v/v) (Fig. 3g), indicating that hydrophobic interactions are not the primary driving force behind phase separation.

We next assessed how CTD-RNA stoichiometry modulates LLPS by varying CTD concentration against a fixed 10 μM RNA background at 150mM NaCl (Fig. 3h and Supplementary Fig. 7). Condensates formed over a broad CTD:RNA ratio. However, when the ratio dropped below 1:15, phase separation diminished, which may be attributed to the electrostatic repulsion from excess anionic RNA molecules. These observations further underscore that attractive electrostatic interactions between cationic CTDs and anionic RNA are the principal forces driving condensate formation.

Surface-inducedcondensationofpgRNAgenome
While LLPS-driven protein-RNA co-condensation has been extensively characterized in systems with freely diffusion components, HBV CTDs are tethered to the icosahedrally arranged capsid core. This suggests that the phase separation observed in our study represents a surface-induced CTD-pgRNA condensation. To investigate whether the observed coexistence of low- and high-density regions of pgRNA genome in capsid is related to the spatial heterogeneity of CTDs due to the icosahedral arrangement, we performed control simulations with CTDs uniformly arranged in a two-dimensional planar array. In this two-dimensional system, the NTD of the capsid protein was omitted, and the N-terminus of the peptides corresponding to CTDs were anchored to a two-dimensional plane by applying a harmonic potential. The reference positions were uniformly distributed at a density approximately equivalent to that of the CTDs in the native capsid. Molecular simulations across varying salt concentrations at 300K revealed phase behavior analogous to capsid-confined systems upon the addition of pgRNA (Fig. 4b–f). Under physiological conditions, clear phase separation emerged, manifesting as coexisting high- and low-density regions (Fig. 4e and Supplementary Movie 8). The phase separation became more pronounced at low Csalt (e.g., 100 mM), while it was nearly indiscernible at high Csalt (e.g., 350 mM). In addition, no phase separation was observed for the CTD-only system (Supplementary Fig. 8). Similar phase-separation behaviors were observed when much shorter RNA segments (A15 RNA segments) were added (Supplementary Fig. 9). Interestingly, we observed clear coarsening events, in which two small clusters coalesced into a larger cluster (Supplementary Fig. 10 and Supplementary Movie 9), a hallmark feature of macroscopic phase separation. However, because one terminus of each CTD is anchored, the sizes of the high-density clusters remain limited and continuous coalescence into larger droplets becomes difficult. These findings, corroborated with the corresponding phase diagram (Fig. 4b), imply that the structural organization of pgRNA genome driven by surface-induced phase separation is the intrinsic propensity of the CTD-pgRNA mixture system, which is independent of the icosahedral arrangement of capsid proteins.

To further examine the surface-induced condensation of pgRNA genome, we analyzed its density distribution along the two-dimensional surface. As expected, ensemble-averaged profiles from MD simulations at Csalt = 150 mM yielded a uniform pattern (Fig. 4g), reflecting global symmetry. In contrast, individual simulation snapshots revealed pronounced heterogeneity and asymmetry in pgRNA organization, consistent with phase separation-driven patterning (Fig. 4h, i). This variability across conformations underscores the dynamic and liquid-like nature of pgRNA-CTD condensates under physiological conditions. Interestingly, the highly fluid feature of HBV-RNA interactions within the capsid has been observed by Taji et al through nuclear magnetic resonance (NMR) studies,60 consistent with the liquid-like dynamics of the CTD-RNA condensate described in this work.

Structural analysis of the high- and low-density regions in the two-dimensional condensate layer reveals that, although one terminus of each CTD is uniformly grafted to a planar surface, the overall CTD density distribution is highly heterogeneous. Typically, the free ends of CTD chains tend to orientate from low density regions toward high density regions (Supplementary Fig. 11a, b), leading to the formation of nanometer-sized clusters. Consequently, high-density regions are enriched in both CTD and RNA, whereas low-density regions contain relatively fewer CTD moieties due to covalent anchoring. In the high-density regions, close contacts between positively charged CTD segments and negatively charged RNA segments promote favorable electrostatic attractions. Meanwhile, the larger inter-CTD distances (due to outward orientation) in low-density regions reduce unfavorable electrostatic repulsion. Thus, RNA dewetting is facilitated by the enhanced electrostatic attraction in high-density regions and the reduced electrostatic repulsion in low-density regions. Further investigations established that the two-dimensional phase separation of the CTD-RNA mixture system depend critically on the surface density of CTDs. Altering CTD density by changing inter-chain distances under physiological conditions diminished phase separation (Supplementary Figs. 11, 12), likely due to alteration of the electrostatic balance.

The two-dimensional planar system described above is analogous to a polymer brush. Previous studies have showed that polyelectrolytes grafted onto a two-dimensional substrate can segregate into coexisting domains in response to electrostatic regulation61–63, a phenomenon often referred to as “lateral phase separation”64 or “planar phase separation”65. Compared to typical polyelectrolyte brush, RNA in the HBV capsid is not only a participant in the phase behavior as an electrostatic regulator, but also a central component of the condensates. To further support the two-dimensional phase separation of the CTD-RNA system, we employed atomic force microscope (AFM) to characterize the morphological transition. Because of the short length of CTD peptides, the resulting condensate are typically nanometer in size, which is too small to be detected by fluorescence microscope or AFM. To overcome this limitation, we grafted polyethyleneglycol (PEG) onto a planar substrate and decorated the free PEG terminus with CTD segments. This extended the effective polymer length, enabling the formation of larger clusters upon RNA addition. AFM measurements revealed that RNA addition promotes morphological heterogeneity in the two-dimensional system (Supplementary Fig. 13), resembling the lateral phase separation observed in early studies61. See Supplementary Material for more details. Because AFM has limited resolution and it is difficult to absolutely rule out alternative origins for the observed morphological transition beyond phase separation, we also carried out additional all-atom MD simulations of the two-dimensional CTD layer mixed with RNA segments. These all-atom MD simulations clearly demonstrated RNA dewetting (Supplementary Fig. 14a, b). Correspondingly, the distributions of Na+ and Cl− around the condensate layer are highly heterogeneous (Supplementary Fig. 14c, d) and closely correlated with the spatial organization of the CTD-RNA condensate (Supplementary Fig. 14e).

Asymmetry in pgRNA genome of individual virion
Previous cryo-EM studies using multi-particle averaging revealed that the pgRNA genome adopts an icosahedrally symmetric conformation adapted to the HBV capsid geometry29. To examine whether our simulations recapitulate this characteristic symmetry observed in experiment, we computed three-dimensional density distributions of pgRNA and CTDs from the MD sampled conformational ensemble. Following the experimental protocol, we applied rotational averaging based on icosahedral symmetry19. The resulting density distributions for both CTDs and pgRNA components exhibit clear icosahedral symmetry (Fig. 5a), closely resembling prior cryo-EM data29. The icosahedral arrangement can be demonstrated more clearly by the projection of the pgRNA density distribution onto the two-dimensional spherical coordinates (Fig. 5b). Specifically, the populations are much enriched around the fivefold axis and quasi-sixfold vertices, while densities around the threefold axis are relatively lower. This high symmetry is also evident in the volume slice plot (Fig. 5c), consistent with experimental findings29. The enrichment of pgRNA around the fivefold and quasi-sixfold symmetry sites is attributed to the co-condensation of CTDs and pgRNA. As CTDs are relatively more concentrated at the fivefold and quasi-sixfold symmetry sites on the inner surface (Supplementary Fig. 15), the average density of pgRNA at these sites is consequently higher.

To assess whether the icosahedral density distributions are solely the results of rotational averaging, we also calculated the ensemble-averaged density distribution without applying rotational symmetry operations, using multiple independent MD simulations. One can see that the icosahedral symmetry is still evident, although it becomes much weaker (Fig. 5d). This indicates that the pgRNA genome organization is inherently modulated by the symmetric arrangement of capsid proteins. Notably, this pattern of reduced symmetry aligns with experimental reconstructions of HBV virions using asymmetric reconstruction methods66. Additionally, we analyzed the structural characteristics of pgRNA of the individual snapshots without ensemble-averaging. The pgRNA configurations from the individual snapshots of the MD simulations exhibit no discernible symmetry in their three-dimensional density profiles (Fig. 5e, f), suggesting that symmetry breaking occurs dynamically at the single-particle level despite global structural constraints.

Base-pairing and microphase separation of pgRNA genome
The above combined MD simulations and experimental observations reveal that HBV exploits LLPS to regulate the spatial organization of its pgRNA genome within the capsid. To probe how LLPS influences pgRNA structural dynamics, we further analyzed the conformational features of pgRNA genome under LLPS regulation. A principal outcome of LLPS is the localized concentration of nucleotides, generating a microenvironment that favors secondary structure folding in pgRNA. Both all-atom and CG simulations demonstrate that base-pairing preferentially occurs in densely packed regions, facilitating the formation of dsRNA structures (Fig. 6a and Supplementary Fig. 16). Typical processes involving the formation of base-pairing interactions are presented in Supplementary Fig. 17 and Supplementary Movies 10–13. Representative CG trajectory (Fig. 6b) shows a marked increase in the number of base pairs (Nbp) coinciding with pgRNA condensation along the inner surface of HBV capsid, indicating that LLPS enhances pgRNA secondary structure. We also conducted additional CG simulations with the initial structure of the capsid-confined pgRNA possessing pre-assigned secondary structures, and similar role of LLPS in promoting base-pair formation was observed (Supplementary Fig. 18). Furthermore, Nbp correlates with the extent of phase separation. Conditions promoting LLPS (i.e., low salt and low temperature) result in significantly elevated Nbp values, whereas conditions that suppress LLPS reduce Nbp accordingly (Fig. 6c and Supplementary Fig. 19). These results demonstrate again a mechanistic link between LLPS and pgRNA base-pairing within the HBV capsid.

The formed base-pairs exhibit pronounced dynamics in the CG simulations. Unwinding events of dsRNA can be clearly observed in the simulation trajectories (Supplementary Fig. 17b and Supplementary Movie 11). Moreover, substantial variation in the number of formed dsRNA segments during the CG MD simulations support the highly dynamic nature of the dsRNA conformation (Supplementary Fig. 17e). The stability of dsRNA is length-dependent. Segments containing more base pairs exhibit longer lifetimes (Supplementary Fig. 17f), which were estimated using a maximum-likelihood method. While the mobility of the CTDs is constrained by their anchoring to the inner capsid surface, the encapsulated RNA displays considerable fluidity, as evidenced by dynamic changes in local density at specified capsid site (Fig. 2j) and by its significant mean square displacement (Fig. 2k). The dsRNA segments also exhibit pronounced mobility, although markedly reduced compared to the ssRNA region (Supplementary Fig. 20e). This structural plasticity is functionally important for HBV pgRNA, as overly stable or rigid secondary structures could interfere with subsequent reverse transcription. Furthermore, the formation of secondary structures through local base-paring in pgRNA, particularly sites involving packaging signals, has been proposed to play a critical role in the assembly of the HBV capsid.67–70 Notably, Patel et al. demonstrated that folding of packaging sites into stem-loop structures promotes capsid assembly67. In line with this, our CG simulations showed that the segment corresponding to a potential packaging site (PS3) can spontaneously form stem-loop-like structure (Supplementary Fig. 17d). In addition, another important protein recognition motif, the cis-acting element ε, can also fold to stem-loop-like structure, although it involves frequent folding and unfolding transitions (Supplementary Fig. 17c).

To further substantiate the link between phase separation and pgRNA base-pairing, we conducted three control CG simulations designed to impair LLPS: (i) ΔCTD, (ii) CTD-Neu, and (iii) CTD-Free (Fig. 6d). In the ΔCTD simulation, CTD of capsid protein was truncated; in the CTD-Free simulation, the covalent linkage between CTD and NTD was severed, allowing CTD to diffuse freely; and in CTD-Neu, all charged residues of CTD were neutralized. As anticipated, all three conditions resulted in marked reductions in base-pair formation. Notably, the CTD-Free simulation retained co-condensation between CTD and pgRNA, although the condensates were diffusely distributed throughout the capsid interior and failed to adopt the characteristic spherical shell morphology (Supplementary Fig. 21). These findings indicate that the CTD-induced alignment of pgRNA along the inner capsid surface is critical for stabilizing base-pairing interactions. In addition, both ΔCTD and CTD-Neu abolished phase separation, reinforcing the importance of electrostatic interactions in driving CTD-pgRNA co-condensation. Interestingly, the arginine-rich CTD has been suggested to act as a specific nucleic acid chaperone to facilitate the structural organization of the viral genome.29,71 The CTD-induced pgRNA condensation and base-pairing observed in this study is in line with the chaperone role of CTDs as proposed in previous studies.

The detection of dsRNA within the HBV pgRNA genome indicates that its conformation is not fully disordered. Detailed structural analysis revealed a spatial segregation between dsRNA and ssRNA domains (Fig. 6a, e), with regions enriched in dsRNA distinctly separated from ssRNA-dominant areas (Fig. 6f), suggesting the emergence of microphase separation during pgRNA organization. This microphase separation was strongly modulated by ionic strength, with the segregation increasingly pronounced under lower salt conditions (Fig. 6f–h). Meanwhile, the dsRNA becomes shorter as the salt concentration increases (Supplementary Fig. 22a–c). Notably, spatially adjacent dsRNA segments tend to display nematic alignment, suggesting orientational ordering within these structured domains. To confirm this finding, we calculated the scalar order parameter S, defined as the largest eigenvalue of the local Q-tensor that describes the orientational order of anisotropic molecules in a nematic liquid crystal. Specifically, S was calculated from the orientation of each spatially adjacent pair of dsRNA segments and then averaged across all pairs. Across different salt concentrations, the average value of S ranged between 0.3 and 0.5 (Fig. 6i), which is characteristic of systems in the nematic phase.72 As expected, the average S value decreased with increasing salt concentration, reflecting the enhanced flexibility of dsRNA under high-salt conditions. Contact probability map further revealed that dsRNA predominantly arises from short-range base-pairing events, although long-range interactions also contributed substantially (Fig. 6j). For instance, ~80% of dsRNA elements adopt hairpin structures with the sequence length smaller than 100 bp (Fig. 6k). Under physiological conditions, these clustered RNA hairpins with nematic alignment, which are interspersed by flexible ssRNA linkers, assemble into a tree-like architecture (Fig. 6l). This higher-order organization likely minimizes chain entanglement and enhances molecular recognition, while the ssRNA segments provide the flexibility needed for polymerase mobility and effective reverse transcription. Together, these results indicate that pgRNA adopts a partially ordered conformation comprising distinct dsRNA and ssRNA substructures, modulated by its interactions with the capsid inner surface.

To further characterize the structural organization of the pgRNA genome within the HBV capsid, we estimated the averaged maximum ladder distance (MLD) among the sampled structural ensemble of pgRNA in CG simulations, following previous works.73,74 The average MLD value ranges between 110 and 120 bp under conditions where phase separation is prominent (Supplementary Fig. 22d). This value is significantly lower than the MLD of a randomly permuted single-stranded RNA of similar sequence length (~300 for a 3000-nt RNA), consistent with previous findings that viral RNAs are more compact than their randomized counterparts.74,75 We also analyzed the interaction characteristics between RNA and CTD. Structural visualization showed that CTDs tend to be embedded between adjacent dsRNA segments, acting as bridges (Supplementary Fig. 20a, b). Although CTDs can also fit into the major or minor grooves of dsRNA, such specific interaction modes are not dominant. Notably, the CTD-RNA interactions are also highly dynamic, as evidenced by substantial variations in the number of CTD-RNA contacts within the clusters (Supplementary Fig. 20d). More detailed analysis revealed that the arginine-RNA ratio in the dense phase stabilizes at a characteristic value of ~1.05–1.11 across varying salt concentrations (Supplementary Fig. 20c). This value is lower than the overall system average of 1.13, indicating a fractionation effect typical of multicomponent phase separation system56.

Role of phase separation on long-range base-pairing and polymerase mobility
The reverse transcription of the pgRNA genome requires multiple template switching events within the HBV capsid, during which polymerase translocates between distant sites along pgRNA template. Prior experimental evidence has demonstrated that long-range base-pairing is essential for enabling the template switches76–79. In addition, efficient template switching requires sufficient mobility of the polymerase to navigate the pgRNA landscape and locate specific interaction sites. Thus, the LLPS-mediated structural organization of pgRNA genome within the capsid likely contributes to the molecular events that govern reverse transcription, particularly template switching. As an illustration, we performed additional simulations wherein the polymerase, an essential component for the reverse transcription process, was introduced to the pgRNA-encapsulated capsid (Fig. 7a, c). The resulting CTD-pgRNA condensates in the presence of polymerase mirrored those observed without it, forming a distinct hollow morphology characterized by coexisting low- and high-density regions (Fig. 7a and Supplementary Fig. 24a). Base-pair formation accompanied the CTD-pgRNA co-condensation (Supplementary Fig. 24b). In contrast, simulations in which CTD charges were neutralized abolished phase separation and eliminated the shell-like organization (Fig. 7b).

Under phase-separation conditions, contact probability maps revealed frequent intramolecular base pairing interactions (Fig. 7d). Notably, Abraham et al. demonstrated that interactions between the cis-acting element ϵ and the distal ϕ site facilitate template switching from ϵ to DR1,69 highlighting the importance of long-range base pairing in reverse transcription. Here, we can observe an increase of long-range base pairing probability (Fig. 7d and Supplementary Fig. 23), which is essential for enabling the template switches as discussed above. In this study, the base pairs with sequence separation larger than 100 are classified as long-range base pairs. These long-range contacts were largely absent when phase separation was suppressed. Moreover, disruption of phase separation led to dense encapsulation of the polymerase by pgRNA, which significantly reduced polymerase mobility (Fig. 7e) and may impair its ability to efficiently locate template-switching sites. Collectively, these results demonstrate that phase separation not only facilitates structural organization of pgRNA genome but also enhances essential dynamics required for efficient template switching during HBV reverse transcription (Fig. 7f).

Methods
All atom MD simulations
The HBV capsid was built from an atomic model resolved by cryo-EM (PDB code: 3J2V), where the missing residues in CTD was reconstructed by Modeller100. The initial structure of the 3200-nt pgRNA was generated using the Nucleic Acids Builder (NAB) script in Ambertools101 with the wild-type pgRNA sequence. The pgRNA sequence without the polyA tail was retrieved from GenBank (accession number V00866.1). The 5’- and 3’-termini of the pgRNA sequence were identified by referring to the information documented in a previous study102. The pgRNA conformation generated by NAB without pre-assigned secondary structures was subsequently relaxed though MD simulations at elevated temperature to produce a more randomized initial structure, thereby minimizing potential bias from the starting configuration. As a control, we also used an initial structure of pgRNA with rich secondary structures modeled by AlphaFold3103. Finally, the pgRNA was further relaxed by applying a spherical wall potential in MD simulation, resulting in a relatively compact structure with dimensions suitable for integration into the capsid.

The pgRNA-containing capsid was solvated within a periodic box (390 Å × 390 Å × 390 Å) of 1,812,893 TIP3P water molecules. Na+ and Cl− were added in the box to neutralize the charge and achieve a salt concentration of 150 mM, leading to a system containing 6,278,802 atoms. Here the salt concentration corresponds to the average salt concentration of the whole system. The amber14sb_OL21 force field was used, which combines the amber14sb force field for protein and χ OL3 force field for RNA104,105. To assess the force field dependence of the results, we performed additional all-atom MD simulations employing the amber14sb_OL15 force field with NBFIX correction.35,36 The electrostatic interaction was treated by the particle mesh Ewald (PME) method with a distance cutoff of 10 Å. The same cutoff value was also used for the van der Waals (VDW) interactions. The MD timestep was 2 fs and all bonds involving hydrogen atoms were constrained using the LINCS algorithm. The initial structure was first minimized for 100,000 steps using the steepest decent algorithm, which was followed by a 5-ns equilibrium simulation with an NPT ensemble at 1 atm and 300 K. The production simulations with the length of 100 ns were then performed at 1 atm and 300 K, with atomic coordinates recorded every 10 ps. V-rescale thermostat and Parrinello-Rahman barostat were used for temperature and pressure coupling,106,107 with the time constants of 0.1 ps and 2 ps, respectively. The simulations were conducted with Gromacs (version 2020.4).108,109.

Two additional all-atom molecular dynamics simulations were performed to characterize phase separation on a two-dimensional planar membrane composed of CTD segments. One system contained a 15×16 array of CTD chains (240 chains total), while the other consisted of a 10×10 array (100 chains). The initial all-atom configuration was generated by structure reconstruction from CG structure in which A15 RNA molecules were uniformly distributed across the anchored CTD layer.110,111 The corresponding systems contained 219 and 71 RNA chains, respectively. Each setup was solvated in a periodic box with TIP3P water molecules, and Na+ and Cl− were added to neutralize the system and achieve corresponding salt concentrations. The amber14sb_OL15 force field with NBFIX correction was used, with all other settings consistent with the above pgRNA-containing capsid simulations. During production simulations, the temperature was first raised to 500 K and then gradually reduced to 300 K over 20 ns. Subsequently, production simulations were conducted for the two systems for 100 ns and 200 ns, respectively, in the NPT ensemble at 1 atm and 300 K, with atomic coordinates saved every 10 ps.

To identify Watson-Crick base pairs, we applied geometric criteria to complementary nucleotides with a sequence separation greater than one. A pair of nucleotides was considered to form a base pair when the bases of two complementary nucleotides established hydrogen bonds, defined by a donor-acceptor heavy atom distance of less than 3.5 Å and the N-H-O or N-H-N angle exceeded 120°.

Residue-resolved coarse-grained MD simulations
The residue-resolved coarse-grained models were employed to characterize the protein and nucleotide acids involved in the HBV virion. For the capsid protein, each residue was represented by one particle, which is centered at the Cα position. The atomic interaction-based coarse-grained model with flexible local interactions (AICG2+)47,48 was used to describe the dynamics of the folded NTD domain of capsid protein, while the hydrophobicity scale (HPS) model57 was employed to characterize the interactions involving the intrinsic disordered CTD domain. The AICG2+ model has the following function form:

V(R|R0) = V bond + Vl o lp c + λV SB + V exv (1)

The first term, the bond potential (V bond= Σ i kb (ri,i+1 - r0 i,i+1 )2 ) describes covalent connectivity between consecutive residues. The second term, the flexible local potential (Vf lo lp c =Vs a tat+Vs d tat), characterizes the sequence dependent flexibility and secondary structure propensity of the protein chain. Here, Vs a tat and Vs d tat denotes statistical potentials for bond angles and dihedral angles, respectively, derived from a loop-segment dataset112. The third term V SB represents the structure-based potential,113 defined as:

V SB = Σ i,j=i+2 ϵi 1 j,3 exp(- (r-r0)2 / 2W2 ij) + Σ i,j=i+3 ϵi 1 j,4 exp(- (ϕ-ϕ0)2 / 2W2 ϕ) (2)
+ Σ j>i+3 ϵi n j, loc [5(r0 ij/rij)12 - 6(r0 ij/rij)10]

This potential combines local interactions with non-local interactions. rij denotes the distance between residues i and j in a given conformation, while r0 ij represents the corresponding distance in the reference structure. Similarly, ϕ i corresponds to the dihedral angle defined by four consecutive residues (i, i+1, i+2, and i+3), with ϕ0 i being its reference value. The force field parameters kb, ϵi 1 j,3, ϵi 1 j,4, ϵi n j, loc, W 1,3, and W ϕ were assigned their default values from the GENESIS 1.7.1 software package.114–116 In this study, the cryo-EM resolved structure (PDB code: 3J2V)117 was used as the reference of the capsid structure. The final term, V exv, accounts for excluded volume interactions between all non-native residue pairs. To better capture the flexibility of the HBV capsid, we introduced a scaling factor λ for the structure-based potential. At λ = 0.7, the coarse-grained model accurately reproduced the average root mean square fluctuation (RMSF) of HBV capsid protein dimers obtained from previous atomistic molecular dynamics simulations.18.

For the nucleotide acids, the three-site-per-nucleotide model (3SPN.2)49 was adopted. Each nucleotide was simplified as three particles representing the phosphate group, the sugar group and the base group, respectively. To accurately depict the flexibility of the RNA molecule, the magnitude of the dihedral energy term was adjusted according to the experimentally measured persistence length value for a polyU sequence (U40) (Supplementary Fig. 25)118. The Debye–Hükel type electrostatic potential was used to characterize the salt-concentration-dependent interactions of charged particles. It is worth mentioning that with such an implicit solvent model, the Donnan equilibrium cannot be explicitly captured. While neglecting this effect in the coarse-grained model may limit the quantitative interpretation, the major results are expected to be less sensitive to this limitation as the positive charges of CTDs and negative charges of pgRNA are roughly balanced.

In the simulations of polymerase-containing system, the full-length structure of polymerase was modeled from AlphaFold2119. Langevin dynamics was used to control the temperature. The timestep was set to 0.01 ps. The friction parameter of the Langevin barostat was selected as 0.1 ps−1. The length of the simulation was set to 1×108 MD steps and three independent simulations were performed for each case with different random seeds. All simulations were conducted with GENESIS (version 1.7.1)114–116.

In the coexistence simulation of CTD peptides and A15 RNA molecules, a slab simulation methodology was implemented. The system was placed in a periodic box with a long z-axis (194 Å × 194 Å × 3600 Å), which contained 216 CTD chains and 216 A15 chains. The initial compact conformation was derived from a shrinking simulation wherein the box was compressed to dimensions of 194 Å × 194 Å × 194 Å over 1×106 MD steps, at the temperature of 300 K and salt concentration of 150 mM. The simulation lasted for 1.0×108 MD steps. All other simulation parameters matched those described above.

Here, a contact was defined between two CG particles separated by ≤ 6.5 Å. Using this definition, contacting nucleotides were grouped into clusters. Nucleotides were then classified as “free" or “condensed" based on cluster size. In addition, a Watson-Crick base-pair was assigned when the distance between the base beads of two complementary nucleotides (with a sequence separation greater than one) was less than a cutoff value of 6.5 Å.

To estimate phase diagrams, the simulation space was divided into discrete bins, and the density within each bin was calculated. Dense (high-density) and dilute (low-density) phases were defined based on the average density, using threshold scaling factors of 1.2 and 0.8. Specifically, bins with densities exceeding 1.2 times the average were assigned to the dense phase, while those below 0.8 times the average were assigned to the dilute phase.

Experimental materials
CTD-peptide and RNA polyA (A15) were synthesized by GenScript, Nanjing, China. 1,6-hexanediol (1,6-HD) was purchased from Aladin, Shanghai, China. The peptide was dissolved in TE buffer (10 mM Tris-HCl, pH 7.5 and 2 mM EDTA). The concentration of CTD-peptide was double checked from the absorbance at 280 nm using a Nano-500 Micro-spectrophotometer (Allsheng, Shanghai, China). The A15 was also dissolved in TE buffer and the concentration was measured with the same spectrophotometer. All solutions were filtered using 0.22 μm syringe filters before use.

The microscope coverslips for the observation of droplets were obtained from VWR. Before observation, the coverslips were ultrasonic cleaned with 5% detergent for 30 minutes, followed by 2 cycles of mPEG-SVA (Laysan Bio., USA) passivation treatments to reduced nonspecific bindings. The coverslips were dried in a drying oven afterwards. The observation wells (radius ~3 mm, height ~5 mm) were home-made using plastic rings glued to the passivated surface.

Brightfield microscopy imaging of droplets
A stock solution of CTD-peptide (~1 mM) and A15 (~200 μM) was used to prepare the sample. Total volume of a 15 μl solution containing 10 μM A15 and variable concentrations of CTD-peptide in TE buffer (pH = 7.5) with different salt concentrations was mixed in a centrifuge tube, and transferred to the observation well immediately after a brief vortex-mix. The samples were observed after incubation for 30 min. Images were obtained using an inverted microscope equipped with a Basler camera (Nexcope NIB610-FL) and official Basler software. All experiments were repeated 3 times.

Atomic force microscope experiments
No. 1.5 glass coverslips (VWR) were used as substrates. The surfaces were first functionalized with (3-aminopropyl)triethoxysilane (APTES, Fisher Scientific) to generate an amine-terminated layer. Subsequently, the heterobifunctional crosslinker Maleimide-PEG2000-SVA (20 mg dissolved in 60 μL of buffer) was conjugated to the surface amines via its NHS ester (SVA) group. A cysteine-terminated peptide (CTD, 5 mM) was then coupled to the maleimide end of the tethered PEG chains. This conjugation step was performed in the presence of 2 mM TCEP as a reducing agent. Finally, 10 μL of a 5 μM RNA sample (A15) was deposited onto the peptide-modified surface, incubated for 5 min, rinsed thoroughly with 2 mL of deionized water, and dried under a gentle stream of nitrogen gas. Atomic force microscopy (AFM) was performed using a Bruker Dimension Icon system. Imaging was conducted in tapping mode in air using NSG30 probes (Golden Silicon Probes) with a nominal spring constant of 20–100 N/m. The images were captured at a scan size of 1×1 μm with a scan rate of 1 Hz. All experiments were performed at room temperature.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present a comprehensive and well-structured study on the structural organization of the hepatitis B virus (HBV) pregenomic RNA (pgRNA) genome, driven by phase separation within the confined capsid environment. Employing multi-scale molecular dynamics simulations alongside biochemical assays, they demonstrate that the pgRNA–C-terminal domain (CTD) condensate exhibits coexisting low- and high-density regions, leading to the formation of hollow, shell-like condensates along the arginine-rich inner surface of the capsid. These interactions are predominantly electrostatic, consistent with expectations for RNA viruses. While the study is thorough, many aspects of the manuscript remain speculative, necessitating further justification of the underlying hypotheses and in particular the modeling approaches used. Provided that the authors adequately address these concerns, this work will be suitable for publication in Nature Communications, as it offers valuable insights of broad relevance.\nMajor concerns:\n• The description of the molecular dynamics methods lacks critical details necessary for clarity and reproducibility. For instance, the modeling of the pgRNA genome is insufficiently described; the only mention is at line 298, stating that “the initial structure of a 3200-nt pgRNA was created by AmberTools.” The authors should specify which AmberTools feature or protocol was used to generate the random placement of RNA beads. Moreover, before relying on a randomly placed pgRNA as a foundational assumption, the authors must justify this choice by referencing published studies that support and validate this approach.\n• The authors have used the Berendsen algorithm for both temperature and pressure coupling. This is dangerous because it is well known that the Berendsen coupling does not provide adequate Boltzmann sampling. For this reason, GROMACS even warns against using Berendsen coupling for anything beyond initial equilibration. Redoing simulations would be beyond the scope, and hence, the authors must provide a reasonable reason why their findings would be unaffected.\n• Looking at the all-atom structure of the RNA (input files provided at the link) I found the RNA to be completely unstructured. This is an important assumption that needs to be discussed: that the authors (i) model the RNA as a random polymer and (ii) the RNA is unstructured, i.e. devoid of any secondary structure to begin with. Both assumptions require a reference. Looking at the trajectory, it was also very difficult to convince myself that Watson-Crick base-pairing was spontaneously observed in the pgRNA genome, something I was curious having seen the unstructured RNA at the beginning of the simulation. Could the authors pin-point to specific residues that validate the observation of RNA base-pairing? This is an important result that has a section on its own (Figure 6). How exactly are base-pairs defined would be an important information to mention in the methods section. This needs to be done for both all-atom and coarse-grained (CG) trajectory analysis (see line 192). The definition of base pairs in the CG model may overestimate true Watson-Crick base pairing.\n• Line 70: The “pgRNA quickly condensed along the inner surface of the capsid”: Could this be because of bad initial configuration of the RNA whereby any internal stabilization via base-pairing is completely dominated by strong nucleic acid-Arginine interactions? A movie of the initial spherical confinement to this shell-like arrangement would be a useful addition.\n• Traditionally, MD force fields can overestimate interactions between highly charged moieties, that includes Arginine residues binding strongly to the phosphate groups on nucleic acids. Conventional practice prevails the use of specific NBFIXs or charge rescaling to tackle this, none that were used in this study. Could the authors comment on the choice of force field, whether it can adequately represent these interactions. This issue is particularly critical because the pgRNA-CTD condensate formation is driven by electrostatics, and overestimating these interactions could result in an artificial state that mimics condensate properties. Nonetheless, the slab simulations provide a robust representation.\n• Line 114: Using only a single 50nt segment out of the 3200nts is questionable as “randomly selected”. Also Fig. S2b’s caption mentions this segment as nucleotides 1-50. If so, the “randomly selected” in line 115 should be removed. The RNA is confined and one may argue that the true MSDs can be very sensitive to the choice of the fragment, in particular due to the observation of both voids and high density regions. The same analysis should therefore be based on an average over several such fragments. Why can’t the analysis be done for all such possible fragments?\n• Line 174-177: The authors mention that the RNA density is much lower at the threefold symmetry sites, compared to fivefold and sixfold. This is an interesting result. Could the authors comment on why this may be so? Also, it is not clear whether the analysis done in Figure 5 used only the all-atom trajectories or does it also include coarse-grained trajectories. Accordingly the caption needs to be updated, that only says “MD simulations.”\n• Line 193-195: The authors argue that a marked increase in the number of base pairs indicates that LLPS enhances the pgRNA secondary structure. Could the increase of base pairs be just due to a bad initial structure of the pgRNA that is devoid of secondary structure at the beginning? If so, that LLPS enhances this formation would be purely speculative.\n• How is long-range base pairing defined? This is hard to convince from nucleotide contacts plots (Fig. 6j) because nucleotides distant w.r.t. their index/residue may still be spatially located in relative proximity and vice versa. Accordingly, the text may need to be updated (foe e.g. line 215). The use of the word “significant” on line 235 is speculative.\nMinor concerns:\n• Line 17: I would recommend citing Bloomfield, V. A. DNA condensation by multivalent cations. Biopolymers 44, 269–282 (1997), as the first to suggest “significant electrostatic repulsions”.\n• Figure 2c-f: The colors are not defined in the caption.\n• Figure 4a: It is not clear how the anchored CTD is modeled. Is it all the 37 residues of the CTD? From the image, the anchored NTD is not fully modeled and it is unclear where the anchor point is. This should be updated in line 152 (“N-termini fixed”).\n• Figure 5a: The isosurface density scale is missing. From the image it may falsely appear that there are no RNA residues in the threefold symmetry sites, whereas this is likely only due to the isosurface density used to create the image.\n• Figure 5c: Color scale is missing\n• Line 199-203: Are these simulations all-atom or coarse grained?\n• The MSD plots (Figure 2k and 7e) suggest the diffusion is very much Brownian like (straight lines at longer lag times). This is a bit counter-intuitive at longer lag times because of the capsid confinement.\n• Line 92: The “an” should be “a”", "reviewer_2": "The manuscript describes results of a detailed study, mostly by means of multiscale molecular dynamics computer simulations and partly by experimentation, of the structural organization and condensation of the pregenomic RNA in the capsid of the virus HBV. The authors focus on the role of the positively charged, structurally disordered C-terminal RNA binding domain of the coat protein, often called Arginine Rich Motif or ARM, in relation with their spatial confinement in the capsid. Parenthetically, in the manuscript the ARMs are called CTD, short for C terminal domain.\nThe main point the authors try to convince the readers of is that the structural arrangement of the RNA in HBV is driven by phase separation, arguing that viruses exploit what is known as liquid-liquid phase separation or LLPS. LLPS has attracted a lot of attention in biophysical literature recently. Often (but not always) driven by interaction of, e.g., RNAs with positively charged, structurally disordered proteins, and I understand why seeing a correspondence between virus assembly and LLPS in biology is such an attractive one.\nI find the paper hugely interesting, and I am particularly impressed by the comprehensiveness of the simulation study. The findings are highly significant, and, if accurate, would further our understanding of the structure of viruses and appeal to a broad readership. Also, the paper is very well written, well-structured and easy to read. I do find pushing the LLPS narrative so forcefully as is done in the manuscript at times a little too much. As a matter of fact, I am not yet convinced that the case made by the authors actually hold water, irrespective of the interesting findings they present.\nSome might view this a semantic issue, but in my view absorption of what in the end is a polymer (the ssRNA) into a structured polymer brush (the ARMs emanating into the cavity of a capsid) is not the same as macroscopic phase separation, in the same way that condensation of a gas in bulk is not the same as condensation of a gas onto an adsorbing surface. Strictly speaking, the assembly of coat proteins around an RNA cannot represent a true phase transition on account of its finite size, noting that the droplet phases typically found in LLPS must be transient. The physics of adsorption of RNA onto immobilized ARMs part of a capsid is very different from the co-condensation of ARMs with RNA in free solution – the authors are aware of this but seem to downplay this.\nThere are a few other issues that I have with the manuscript that need to be addressed before I would advise in favour of publication.\n1. There is a lot of theoretical and simulation literature on the structure of (model) RNAs in virus capsids, and the readers should be aware of that. I am referring to, e.g., the work of Hagan, Zandi and Podgornik to name but a few. Of particular importance for the topic of the manuscript is the work of Hagan in eLife 2013; 2: e00632, and Zandi and Podgornik in Phys. Rev. E 2014; 89: 032707 and Zandi in Phys. Rev. E 2020; 102: 062423. These, and other works, highlight the importance of the localization of the positive charges, but also the configurational (base-pairing) response of the RNA to the binding to the ARMs. See also the review paper of Zandi and collaborators, Physics Reports 2020; 847: 1–102.\n2. I do not believe that the observed coexistence of high- and low-density regions in the absorption layer near the ARMs indicates phase separation, as is implied on page 3, lines 77 and 78. The difference in RNA density in the cavity and that in the absorption layer would be in closer to correspondence to complex coacervation of ARMs and RNA, discussed also in the main text. I am very skeptical about the findings from the simulations that phase separation takes place in the absorption layer in the virus (fig. 2), and on the flat surface with end-grafted ARMs (fig. 4) unless the salt concentration is sufficiently high. The reason is that this leaves the regions with low RNA density with nearly naked ARMs, so without the benefit of attractive interaction with the RNA whilst the higher-density regions must suffer from higher self-repulsion of the RNA. No experimental verification has been done, as was with the bulk phase separation of RNAs and ARMs (fig. 3). I would strongly recommend experimental verification, knowing full well that this is a major endeavor. The discovery of dewetting of a polyanion such as RNA adsorbed on a polycationic surface would represent a major surprise in the field.\n3. The in-layer phase separation shown in fig. 4 was studied for different ionic strengths, but only a single RNA length. It would be useful to investigate how the length of the RNA influences this phase separation. Also, the phase separation, if real, should lead to inhomogeneous distribution of mobile ions. Not much is said about this in the manuscript. Note also, that if phase separation indeed takes place, the high- and low-density regions should increase in size in time and coarsen, e.g., by coalescence. Does coarsening take place?\n4. The structure of the RNA in particular in relation to base pairing is discussed on page 9 and 10 (figure 6). There is quite some work on the structure of viral ssRNAs, and why they are different from random RNAs or cellular RNAs. See, e.g., the work of Gelbart and others on this topic in PLoS ONE 2014; 9: e105875 and Biophys. J. 2017; 113: 339-347, to cite a few of many papers these authors wrote on this topic. Also, theoretical work (e.g. of Zandi, see earlier) indicates that interaction with the ARMs changes the base pairing. It would be interesting to get more information on the structure of the RNA in the virus, such as maximum ladder distances, length distributions of hair pin structures, and so on. The authors also mention nematic alignment of clustered hairpins but do not elaborate on how this was established: by eye or by calculating the largest eigenvalue and associated eigenvector of the local Q tensor? This would also produce a director field configuration – in that case one would expect four half-integer disclination defects to appear. There is quite a bit of literature on this.\n5. Viruses tend to be overcharged, that is, there is more negative charge on the RNA than needed to compensate for the positive charges on the ARMs in the virus. This causes a Donnan equilibrium to establish itself for the mobile ions inside and outside the virus, and between the empty core of the lumen of the capsid and the region where ARMs and RNA interact. This equilibrium takes time to set up, and I wonder if the all-atom simulations are sufficiently long to let this equilibrium establish itself. Also, the Debye-Hückel potential used by the authors for the coarse-grained simulations most certainly cannot deal with this kind of phenomenology. It would be good to see if for physiological conditions Donnan effects can indeed be neglected by calculating time dependent ion density distributions in the atomistic simulations.", "reviewer_3": "Bian et al computationally show that interaction of RNA with the basic C terminal domain of the HBV core protein results in a condensed phase. The condensate resembles the hollow shell of density seen in image reconstructions of HBV with the pregenome and with random RNA from the expression system. In the condensed phase the RNA is able to diffuse. As one would expect, neutralisation of phosphates allows base pairing. Unsurprisingly, the dsRNA segments shows nematic organisation. Unsurprisingly, the heterogeneity of the layer follows the symmetry of the capsid. To support their in silico studies, the authors demonstrated that isolated CTDs were able to form a phase when mixed with 15 nucleotide poly A oligomers.\nThere are several issues the authors need to address. The approach to adding pgRNA to a capsid is novel; do all trajectories lead to the same result? How much do they differ? While the peptide – RNA oligomer complex is experimentally shown to form a liquid-liquid phase separation, the two dimensional nature of the inner layer of the capsid stretches the definition of a phase separation. This needs to be addressed. The phase needs to be better described. Do the high RNA density clusters show a characteristic arginine-RNA ratio? How fluid are the CTD-RNA clusters ? How fluid are the dsRNA segments? If sufficiently stable, they will halt reverse transcription. A quantitative view of their stability will be appreciated.\nOne of the advantages of molecular dynamics, even with coarse graining, is the ability to visualize molecular contacts. How do the CTDs interact with the RNA? Do they interdigitate between strands? Do the fit in major grooves of dsRNA? The authors appear to use the HBV pgRNA in their study but do not identify the sequence used. It is not clear if they have a polyA tail. The means of adjusting ionic strength are not clearly defined. Are the authors holding the capsid interior to the stated bulk ionic strength ? is the exterior held at the stated ionic strength? If the latter, what is the interior ionic strength or total number of ions?\nThere are several papers that are germane to this study that ought to be discussed to provide deeper context. Most importantly, Harati Taji et al (DOI: 10.1021/jacs.1c12439) showed that HBV-RNA interactions were highly fluid in NMR studies. How does your study conform to Harati Taraji predictions? Chu et al (DOI: 10.1128/JVI.03235-13) showed that HBV CTDs could act as RNA chaperones. Is this consistent with your results? Do dsRNA duplexes readily break in your simulations? Patel et al (DOI: 10.1038/nmicrobiol.2017.98) identify putative packing sites; are they folded in your study and how would a stable fold affect your predictions. Abraham and Lob (DOI: 10.1128/JVI.80.9.4380-4387.2006) predict long-range base pairing ; how would such base pairing bias the packaging of RNA."}, "author_response": "Responses to Reviewer's Questions and list of changes\nReviewer #1 (Remarks to the Author):\nThe authors present a comprehensive and well-structured study on the structural\norganization of the hepatitis B virus (HBV) pregenomic RNA (pgRNA) genome, driven\nby phase separation within the confined capsid environment. Employing multi-scale\nmolecular dynamics simulations alongside biochemical assays, they demonstrate that\nthe pgRNA–C-terminal domain (CTD) condensate exhibits coexisting low- and high-\ndensity regions, leading to the formation of hollow, shell-like condensates along the\narginine-rich inner surface of the capsid. These interactions are predominantly\nelectrostatic, consistent with expectations for RNA viruses. While the study is thorough,\nmany aspects of the manuscript remain speculative, necessitating further justification\nof the underlying hypotheses and in particular the modeling approaches used.\nProvided that the authors adequately address these concerns, this work will be suitable\nfor publication in Nature Communications, as it offers valuable insights of broad\nrelevance.\nReply:\nWe thank the reviewer for the insightful comments.\nMajor concerns:\n1）The description of the molecular dynamics methods lacks critical details necessary\nfor clarity and reproducibility. For instance, the modeling of the pgRNA genome is\ninsufficiently described; the only mention is at line 298, stating that “the initial structure\nof a 3200-nt pgRNA was created by AmberTools.” The authors should specify which\nAmberTools feature or protocol was used to generate the random placement of RNA\nbeads. Moreover, before relying on a randomly placed pgRNA as a foundational\nassumption, the authors must justify this choice by referencing published studies that\nsupport and validate this approach.\nReply:\nWe thank the reviewer for the thoughtful comments and suggestions. In this revised\nmanuscript, we have expanded the Methods section to provide additional details on\nthe MD simulation steps. In this work, the initial structure of pgRNA was generated\nusing the Nucleic Acids Builder (NAB) script in AmberTools. The pgRNA sequence\ncorresponds to the wild-type sequence obtained from GenBank (accession number\nV00866.1), with the 5′- and 3′-terminal regions set according to the work by Haines et\nal. (J. Mol. Biol. 2007, 370:471–480). The NAB-generated pgRNA conformation was\nsubsequently relaxed through molecular dynamics (MD) simulations at elevated\ntemperatures to yield a more randomized initial structure, thereby reducing potential\nbias from the starting configuration. We have added the above description of the\nmolecular simulation methods to further enhance clarity and reproducibility in this\nrevised manuscript.\nCorresponding changes:\n1. (page 3, line 71) The following sentence was refined to clarify the initial structure of\nMD simulations in this revised manuscript:\n“In the initial configuration of the MD simulations, the pgRNA with the wide-type\nsequence was placed inside the capsid in a compact conformation without pre-\nassigned secondary structures (Fig. 1c and Methods).”\n2. (page 16, line 418) The following sentences were added in the Methods section to\nfurther clarify the used tool and pgRNA sequence in the construction of the initial\nstructure of pgRNA model:\n“The initial structure of the 3200-nt pgRNA was generated using the Nucleic Acids\nBuilder (NAB) script in Ambertools101 with the wild-type pgRNA sequence. The pgRNA\nsequence without the polyA tail was retrieved from GenBank (accession number\nV00866.1). The 5’- and 3’- termini of the pgRNA sequence were identified by referring\nto the information documented in a previous study102. The pgRNA conformation\ngenerated by NAB without pre-assigned secondary structures was subsequently\nrelaxed though MD simulations at elevated temperature to produce a more randomized\ninitial structure, thereby minimizing potential bias from the starting configuration. As a\ncontrol, we also used an initial structure of pgRNA with rich secondary structures\nmodelled by AlphaFold3103. Finally, the pgRNA was further relaxed by applying a\nspherical wall potential in MD simulation, resulting in a relatively compact structure with\ndimensions suitable for integration into the capsid.”\n3. (page 16, line 427) To enhance clarity and reproducibility, more details of MD\nsimulations were added in the Methods section:\n“The pgRNA-containing capsid was solvated within a periodic box (390 Å× 390 Å× 390\nÅ) of 1,812,893 TIP3P water molecules. …… Here the salt concentration corresponds\nto the average salt concentration of the whole system. …… To assess the force field\ndependence of the results, we performed additional all-atom MD simulations\nemploying the amber14sb_OL15 force field with NBFIX correction.35, 36 …… The initial\nstructure was first minimized for 100000 steps using the steepest decent algorithm,\nwhich was followed by a 5-ns equilibrium simulation with an NPT ensemble at 1 atm\nand 300K. The production simulations with the length of 100 ns were then performed\nat 1 atm and 300K, with atomic coordinates recorded every 10 ps. V-rescale thermostat\nand Parrinello-Rahman barostat were used for temperature and pressure coupling,106,\n107 with the time constants of 0.1 ps and 2 ps, respectively.”\n2）The authors have used the Berendsen algorithm for both temperature and pressure\ncoupling. This is dangerous because it is well known that the Berendsen coupling does\nnot provide adequate Boltzmann sampling. For this reason, GROMACS even warns\nagainst using Berendsen coupling for anything beyond initial equilibration. Redoing\nsimulations would be beyond the scope, and hence, the authors must provide a\nreasonable reason why their findings would be unaffected.\nReply and corresponding changes:\nWe thank the reviewer for the kind comment. Upon re-examining the input file of the\nall-atom MD simulations, we realized that the description of the temperature and\npressure coupling in the original version of the manuscript was not entirely accurate.\nFor temperature coupling, we employed the v-rescale thermostat, which is an improved\nversion of the Berendsen thermostat by introducing a stochastic term to the rescaling\nfactor. It can better generate correct sampling of the canonical ensemble (J. Chem.\nPhys. 126, 014101, 2007). For the pressure coupling, we used the Parrinello-Rahman\nalgorithm, which is a widely adopted option of pressure coupling in GROMACS. We\napologize for any confusion caused by the earlier description. In the coarse-grained\nsimulations, the Langevin thermostat was applied. In this revised manuscript, we have\ncorrected and clarified the description of both temperature and pressure coupling.\n3）Looking at the all-atom structure of the RNA (input files provided at the link) I found\nthe RNA to be completely unstructured. This is an important assumption that needs to\nbe discussed: that the authors (i) model the RNA as a random polymer and (ii) the RNA\nis unstructured, i.e. devoid of any secondary structure to begin with. Both assumptions\nrequire a reference. Looking at the trajectory, it was also very difficult to convince\nmyself that Watson-Crick base-pairing was spontaneously observed in the pgRNA\ngenome, something I was curious having seen the unstructured RNA at the beginning\nof the simulation. Could the authors pin-point to specific residues that validate the\nobservation of RNA base-pairing? This is an important result that has a section on its\nown (Figure 6). How exactly are base-pairs defined would be an important information\nto mention in the methods section. This needs to be done for both all-atom and coarse-\ngrained (CG) trajectory analysis (see line 192). The definition of base pairs in the CG\nmodel may overestimate true Watson-Crick base pairing.\nReply:\nWe sincerely appreciate the reviewer’s valuable comments and suggestions. In this\nstudy, the wild-type pgRNA sequence was used in the all-atom MD simulations,\nenabling more realistic modeling of the inter-nucleotide interactions, particularly the\nWatson-Crick base-pairing. The initial pgRNA conformation was chosen to be\nunstructured, allowing us to more clearly identify base pairs that formed spontaneously\nduring the simulations within the HBV capsid.\nFollowing the reviewer’s suggestion, we conducted additional all-atom MD\nsimulations with different initial structures and performed more in-depth analyses (Fig.\nR1). First, we conducted two sets of new all-atom MD simulations, in which the\nunstructured initial conformation and the structured initial conformation with rich\nsecondary structure are used, respectively. The structured initial conformation was\nmodelled by using AlphaFold3. In both sets of all-atom MD simulations, we observed\nthe molecular events of canonical Watson-Crick base-pairing, although it is still unlikely\nto obtain converged sampling of pgRNA secondary structures due to timescale limit.\nTo illustrate these processes, we have added new figures and video showing the time\ncourse of RNA base-pair formation for a representative nucleotide pair (Fig. R1a).\nSecond, we extended our analyses of the coarse-grained simulation trajectories and\npresented an additional figure and video that more specifically highlight the base-\npairing processes observed in the simulations (Fig. R1b,c). We also highlighted the\nspecific sequence segments forming the dsRNA in Fig.6a, Fig. S16 and Fig. S17 in\nthis revised manuscript. Together, the results of these new simulations and analyses\nfurther corroborate our observation that Watson-Crick base-pairing can occur within\nthe HBV capsid.\nIn all-atom MD simulations, a pair of nucleotides was considered to form a base pair\nwhen the bases of two complementary nucleotides established hydrogen bonds,\ndefined by a donor–acceptor heavy atom distance of less than 3.5 Å and an N–H–O\nor N-H-N angle greater than 120°. In the coarse-grained simulations, base-pair\nformation was defined when the distance of the base beads of two complementary\nnucleotides (with a sequence separation greater than 1) was less than a cutoff value\nof 6.5 Å. In this revised manuscript, we have incorporated these new results and\ndiscussions. The definition of base-pair formation has been explicitly added to the\nMethods section to improve clarity and reproducibility.\nFigure R1. Dynamics of base pairs in MD simulations. (a, b) Snapshots of\nrepresentative base-pairing events from all-atom (a) and coarse-grained (CG)\nsimulations (b). (c) Representative folding and unfolding transitions of the cis-acting\nelement ε observed in CG simulations. (d) Representative folding event of packaging\nsite, PS3. (e) Time evolution of the number of dsRNA segments from CG simulations.\n(f) Lifetimes of dsRNA segments with lengths (LdsRNA) ≤ 10 and > 10. The lifetimes\nwere estimated from a maximum-likelihood method.\nCorresponding changes:\n1. (Supplementary Material and Figure 6) We have added Fig. S17 and Movies 10–13\nto illustrate the base-pairing events observed in both all-atom and coarse-grained\nsimulations. We also highlighted the specific sequence segments forming the dsRNA\nin Fig. 6a, Fig. S16 and Fig. S17.\n2. (page 12, line 254) The following sentence was added in this revised manuscript to\ndescribe the base pair dynamics:\n“Typical processes involving the formation of base-pairing interactions are presented\nin Fig. S17 and Supplementary Movies 10–13.”\n3. (page 12, line 263) The following sentences were added in this revised manuscript\nto describe the base pair dynamics:\n“The formed base-pairs exhibit pronounced dynamics in the CG simulations.\nUnwinding events of dsRNA can be clearly observed in the simulation trajectories (Fig.\nS17b and Supplementary Movie 11). Moreover, substantial variation in the number of\nformed dsRNA segments during the CG MD simulations supports the highly dynamic\nnature of the dsRNA conformation (Fig. S17e).”\n4. (page 16, line 445) The following sentences were added in the Methods section to\nclarify the definition of base pairs for all-atom MD simulations:\n“To identify Watson-Crick base pairs, we applied geometric criteria to complementary\nnucleotides with a sequence separation greater than one. A pair of nucleotides was\nconsidered to form a base pair when the bases of two complementary nucleotides\nestablished hydrogen bonds, defined by a donor–acceptor heavy atom distance of less\nthan 3.5 Å and the N–H–O or N-H-N angle exceeded 120o.“\n5. (page 17, line 475) The following sentence was added in the Methods section to\nclarify the definition of base pairs for coarse-grained MD simulations:\n“Watson-Crick base-pair was assigned when the distance between the base beads of\ntwo complementary nucleotides (with a sequence separation greater than one) was\nless than a cutoff value of 6.5 Å.”\n4）Line 70: The “pgRNA quickly condensed along the inner surface of the capsid”:\nCould this be because of bad initial configuration of the RNA whereby any internal\nstabilization via base-pairing is completely dominated by strong nucleic acid-Arginine\ninteractions? A movie of the initial spherical confinement to this shell-like arrangement\nwould be a useful addition.\nReply:\nWe thank the reviewer for the helpful comment. In this revised manuscript, we have\nincluded new MD simulations initiated from pgRNA structures with predicted secondary\nstructures, using both all-atom and coarse-grained models. Consistent with our earlier\nsimulations starting from unstructured pgRNA conformation, these new simulation\nresults show that pgRNA rapidly condenses along the inner surface of the capsid,\nforming a shell-like arrangement (Fig. R2). These results indicate that pgRNA\ncondensation along the capsid interior is a robust process, largely insensitive to the\nchoice of initial pgRNA structure. Following the reviewer’s suggestion, we prepared\nnew movies and figures to more clearly illustrate the formation process of the shell-like\nconformation of pgRNA. In this revised manuscript, we have added the above results\nand discussion.\nFigure R2. Conformations of pgRNA from the MD simulations with different initial\nstructures. (a,b) The starting and final configurations (a), and the corresponding radial\ndistribution functions of NTD, CTD, and pgRNA in the final configuration (b), from the\nall-atom MD simulation initialed from an unstructured pgRNA. (c,d) Same as (a,b),\nbut for the all-atom MD simulation initiated from a pgRNA structure predicted by\nAlphaFold3. (e,f) Same as (a,b) but for the CG MD simulation initiated from a pgRNA\nstructure predicted by AlphaFold3.\nCorresponding changes:\n1. (Supplementary Material) We have added Fig. S1 and Movies 2-4 from the MD\nsimulations with different initial structures to illustrate that the results are insensitive to\nthe initial structures.\n2. (page 3, line 79) The following paragraph was added in the main text of this revised\nmanuscript to describe the results of all-atom MD simulations with different initial\nstructures:\n“To further assess the dependence of the results on the initial structure and force fields,\nwe conducted two additional sets of all-atom MD simulations initiated from pgRNA\nstructures without pre-assigned secondary structures and with predicted secondary\nstructures, respectively. Both simulations employed an alternative force field\n(amber14sb_OL15 force field with NBFIX correction)35, 36. Consistent with the above\nresult (Fig. 1d), these simulations also revealed rapid condensation of pgRNA along\nthe inner surface of the capsid, forming a shell-like arrangement (Fig. S1a,b and\nSupplementary Movies 2-3). These results indicate that the observed pgRNA\ncondensation is a robust process, largely insensitive to the choice of initial pgRNA\nstructures and force fields.”\n3. (page 5, line) The following sentences were added in the main text of this revised\nmanuscript to describe the results of CG MD simulations with different initial structures:\n“We also conducted CG simulations initiated from pgRNA structures with predicted\nsecondary structures, and comparable results were observed (Fig. S1c and\nSupplementary Movie 4).”\n5）Traditionally, MD force fields can overestimate interactions between highly charged\nmoieties, that includes Arginine residues binding strongly to the phosphate groups on\nnucleic acids. Conventional practice prevails the use of specific NBFIXs or charge\nrescaling to tackle this, none that were used in this study. Could the authors comment\non the choice of force field, whether it can adequately represent these interactions.\nThis issue is particularly critical because the pgRNA-CTD condensate formation is\ndriven by electrostatics, and overestimating these interactions could result in an\nartificial state that mimics condensate properties. Nonetheless, the slab simulations\nprovide a robust representation.\nReply:\nIn this work, the ff14sb and χ force fields were used for the protein and RNA,\nOL3\nrespectively. Following the reviewer’s comment, we added additional all-atom MD\nsimulations employing an Amber force field (amber14sb_OL15) with NBFIX correction\nto assess the force field dependence of the results. The key observations, such as the\nformation of shell-like RNA arrangement and RNA base-pairing, were consistently\nreproduced in these new simulations, indicating that the observed condensate\nproperties are robust (Figs. R1a and R2). In this revised manuscript, we have added\nthe above results and discussions.\nCorresponding changes:\n1. (page 16, line 431) The following sentence was added in the Methods section of this\nrevised manuscript:\n“To assess the force field dependence of the results, we performed additional all-atom\nMD simulations employing the amber14sb_OL15 force field with NBFIX correction.35,\n36”\n2. (page 3, line 79) The following paragraph was added in the main text of this revised\nmanuscript to describe the results of all-atom MD simulations with different initial\nstructures (The same change as in the reply of comments 4):\n“To further assess the dependence of the results on the initial structure and force fields,\nwe conducted two additional sets of all-atom MD simulations initiated from pgRNA\nstructures without pre-assigned secondary structures and with predicted secondary\nstructures, respectively. Both simulations employed an alternative force field\n(amber14sb_OL15 force field with NBFIX correction)35, 36. Consistent with the above\nresult (Fig. 1d), these simulations also revealed rapid condensation of pgRNA along\nthe inner surface of the capsid, forming a shell-like arrangement (Fig. S1a,b and\nSupplementary Movies 2-3). These results indicate that the observed pgRNA\ncondensation is a robust process, largely insensitive to the choice of initial pgRNA\nstructures and force fields.”\n6）Line 114: Using only a single 50nt segment out of the 3200nts is questionable as\n“randomly selected”. Also Fig. S2b’s caption mentions this segment as nucleotides 1-\n50. If so, the “randomly selected” in line 115 should be removed. The RNA is confined\nand one may argue that the true MSDs can be very sensitive to the choice of the\nfragment, in particular due to the observation of both voids and high density regions.\nThe same analysis should therefore be based on an average over several such\nfragments. Why can’t the analysis be done for all such possible fragments?\nReply:\nWe thank the reviewer for the helpful comments and suggestion. We agree that\ncalculating the average MSD across multiple RNA segments provides a more\nappropriate characterization of diffusion ability. Accordingly, we divided the pgRNA\nchain into segments of 100 nucleotides and computed the average MSD among these\nsegments (Figs. R3, R4). Consistent with our original results, the average MSD\nindicates significant diffusion of pgRNA along the shell-like condensate. In this revised\nmanuscript, we updated the corresponding figures and refined the related description\nof the analysis method to reflect this improvement.\nFigure R3. Mean-squared displacement (MSD) of pgRNA, averaged over different\n100-nt segments of the full-length pgRNA, under varying salt conditions.\nFigure R4. Mean-squared displacement (MSD) of pgRNA, averaged over different\n100-nt segments of the full-length pgRNA, under varying temperatures.\nCorresponding changes:\n1. (Main text figure and Supplementary Material) The Fig.2k and Fig. S2d were\nupdated in this revised manuscript.\n2. (page 5, line 137) The following sentences in the main text of this revised manuscript\nwere refined to reflect the MSD results with updated computing method:\n“By dividing the pgRNA sequence into 100-nucleotide segments, we also calculated\nthe mean square displacement (MSD) averaged among these segments from the\naforementioned CG MD simulations. The MSD results reveal substantial diffusivity\n(Figs. 2k and S4d), particularly under physiological conditions (Supplementary Movie\n5).”\n7）Line 174-177: The authors mention that the RNA density is much lower at the\nthreefold symmetry sites, compared to fivefold and sixfold. This is an interesting result.\nCould the authors comment on why this may be so? Also, it is not clear whether the\nanalysis done in Figure 5 used only the all-atom trajectories or does it also include\ncoarse-grained trajectories. Accordingly the caption needs to be updated, that only\nsays “MD simulations.”\nReply:\nWe thank the reviewer for the helpful comment and suggestion. Due to the co-\ncondensation of CTDs and pgRNA, the density distribution of pgRNA is influenced by\nthe spatial locations of CTDs along the inner surface of the capsid. Based on the\nicosahedron arrangement of the core proteins, CTDs are more densely localized near\nthe fivefold and sixfold symmetry axes (Fig. R5). Consequently, pgRNA density tends\nto be enriched around these symmetry sites, reflecting the underlying organization of\nthe capsid proteins.\nIn this study, the RNA density map and its distribution were estimated from the\ncoarse-grained trajectories. In this revised manuscript, we have added the related\ndiscussion on the pgRNA distribution. The caption of Figure 5 has also been modified\naccordingly to improve clarity.\nFigure R5. Density of CG beads at different symmetry sites estimated from CG\nsimulations.\nCorresponding changes:\n1. (Supplementary Material) We have added Fig. S15 to demonstrate the distribution\nof CTDs across different symmetry sites.\n2. (page 9, line 235) The following sentences were added in the main text of this\nrevised manuscript:\n“The enrichment of pgRNA around the fivefold and sixfold symmetry sites is attributed\nto the co-condensation of CTDs and pgRNA. As CTDs are relatively more\nconcentrated at the fivefold and sixfold symmetry sites on the inner surface (Fig. S15),\nthe average density of pgRNA at these sites is consequently higher.”\n3. (Figure 5 caption) The Figure 5 caption was updated to clarify that the results were\nobtained from CG MD simulations.\n8）Line 193-195: The authors argue that a marked increase in the number of base\npairs indicates that LLPS enhances the pgRNA secondary structure. Could the\nincrease of base pairs be just due to a bad initial structure of the pgRNA that is\ndevoid of secondary structure at the beginning? If so, that LLPS enhances this formation\nwould be purely speculative.\nReply:\nWe thank the reviewer for the constructive comments. To further validate the\nobservation that LLPS enhances base-pairing, we conducted two additional sets of\ncoarse-grained simulations with the initial structure of the capsid-confined pgRNA\npossessing secondary structures. In the first set of simulations, electrostatic\ninteractions between RNA and the RNA and the CTDs of capsid proteins were turned off, thereby\npreventing phase separation. Under this condition, the number of base pairs (N ) have\nbp\nno significant changes during the simulations (Fig. R6). In the second set of simulations,\nelectrostatic interactions between RNA and CTDs were retained. In this case, N\nbp\nincreased continuously over the time course of ~1×107 MD steps (Fig. R6). These\nresults validate that LLPS promotes base-pair formation, and that this effect is robust\nand insensitive to the choice of initial pgRNA structure. For each condition, two\nindependent simulations were conducted. In this revised manuscript, we have added\nthe above results and discussions to more clearly illustrate the effect of LLPS on the\nformation of pgRNA base pairs.\nFigure R6. Temporal evolution of the number of base pairs (N ) from CG simulations\nbp\nof the pgRNA-containing HBV capsid under conditions with and without the occurrence\nof phase separation, starting from a pgRNA conformation with predicted secondary\nstructures. Two independent simulations were conducted for each case.\nCorresponding changes:\n1. (Supplementary Material) We have added Fig. S18 to demonstrate that the observed\nrole of LLPS in prompting base pairing is independent of the initial pgRNA\nconformations.\n2. (page 12, line 257) The following sentence was added in the main text of this\nrevised manuscript.\n“We also conducted additional CG simulations with the initial structure of the capsid-\nconfined pgRNA possessing pre-assigned secondary structures, and similar role of\nLLPS in promoting base-pair formation was observed (Fig. S18).”\n9）How is long-range base pairing defined? This is hard to convince from nucleotide\ncontacts plots (Fig. 6j) because nucleotides distant w.r.t. their index/residue may still\nbe spatially located in relative proximity and vice versa. Accordingly, the text may need\nto be updated (foe e.g. line 215). The use of the word “significant” on line 235 is\nspeculative.\nReply:\nWe thank the reviewer for the comment and suggestion. In this work, the long-range\nbase pairing is defined on the basis of the sequence separation, sequence\ncomplementarity, and distance. When the distance between the base beads of two\ncomplementary nucleotides in the structures of the coarse-grained simulations is less\nthan a cutoff value (6.5 Å), they form a base pair. The long-range base pairs refer to\nthose formed by nucleotides with large “sequence separation”. In this study, base pairs\nwith sequence separation larger than 100 were defined as long-range base pairs. We\ncalculated average number of long-range base pairs for the simulations with and\nwithout phase separation. The results show that the number of long-range base pairs\nincreases from 31 to 787 with the introduction of electrostatic interactions between\npgRNA and CTDs, suggesting that LLPS favors long-range base-pairing (Fig. R7). The\nnumber of long-range base pairs is also higher than that of the predicted pgRNA\nstructure (410). In this revised manuscript, we updated the related text to more\naccurately describe the results, and the word “significant” was rephrased.\nFigure R7. Number of base pairs (N ) formed by long-range nucleotides (with\nbp\nsequence separation greater than 100) from CG simulations of the pgRNA-containing\nHBV capsid under conditions with and without the occurrence of LLPS.\nCorresponding changes:\n1. (Supplementary Material) We have added Fig. S23 to quantify the role of phase\nseparation in prompting long-range base pairing.\n2. (page 13, line 335) The following sentences were added in the main text of this\nrevised manuscript:\n“Here, we can observe an increase of long-range base pairing (Fig. 7d and Fig. S23),\nwhich is essential for enabling the template switches as discussed above. In this study,\nthe base pairs with sequence separation larger than 100 are classified as long-range\nbase pairs.”\nMinor concerns:\n1) Line 17: I would recommend citing Bloomfield, V. A. DNA condensation by\nmultivalent cations. Biopolymers 44, 269–282 (1997), as the first to suggest “significant\nelectrostatic repulsions”.\nReply and corresponding changes:\nWe thank the reviewer for bringing this important paper into our attention. In this\nrevised manuscript, we added the citation of this paper.\n2) Figure 2c-f: The colors are not defined in the caption.\nReply and corresponding changes:\nIn Figure 2c-f, the NTDs, CTDs, and pgRNA are depicted in gray, blue and wheat,\nrespectively. In this revised manuscript, we added the color definition for Figure 2c-f in\nthe caption.\n3) Figure 4a: It is not clear how the anchored CTD is modeled. Is it all the 37 residues\nof the CTD? From the image, the anchored NTD is not fully modeled and it is unclear\nwhere the anchor point is. This should be updated in line 152 (“N-termini fixed”).\nReply and corresponding changes:\nIn the simulations of the two-dimensional system, the N-terminal domain of capsid\nprotein was omitted, and the peptides corresponding to CTDs (residues 150-185) were\nanchored to a two-dimensional plane by applying a harmonic potential between the N-\nterminal beads of CTDs and the reference positions in the two-dimensional plane. The\nreference positions are uniformly distributed with the density roughly equivalent to that\nof the CTDs in the capsid. In this revised manuscript, we updated the related\ndescription to improve clarity.\n1. (page 7, line 175) The following sentences were refined in this revised manuscript\nto clarify the protocol for the construction of the two-dimensional system:\n“In this two-dimensional system, the NTD of the capsid protein was omitted, and the\nN-terminus of the peptides corresponding to CTDs were anchored to a two-\ndimensional plane by applying a harmonic potential. The reference positions were\nuniformly distributed at a density approximately equivalent to that of the CTDs in the\nnative capsid.“\n4) Figure 5a: The isosurface density scale is missing. From the image it may falsely\nappear that there are no RNA residues in the threefold symmetry sites, whereas this\nis likely only due to the isosurface density used to create the image.\nReply and corresponding changes:\nWe thank the reviewer for the comments and suggestion. In this revised manuscript,\nwe added the isosurface density scale in Figure 5a. To improve clarity, we also\nemphasized that the density less than the lower-limit of the density scale is not plotted.\n5) Figure 5c: Color scale is missing.\nReply and corresponding changes:\nIn this revised manuscript, we added the color scale in Figure 5c.\n6) Line 199-203: Are these simulations all-atom or coarse grained?\nReply and corresponding changes:\nThese simulations were conducted with the coarse-grained models. In this revised\nmanuscript, we have refined the related text to explicitly describe the simulation\nmethod and improve clarity.\n7) The MSD plots (Figure 2k and 7e) suggest the diffusion is very much Brownian like\n(straight lines at longer lag times). This is a bit counter-intuitive at longer lag times\nbecause of the capsid confinement.\nReply and corresponding changes:\nIn Figure 2k and 7e, the lag-time scale of the diffusion is limited to 1.5x107 MD step,\nduring which the capsid confinement is less important. When the lag-time scale of the\ndiffusion becomes longer, the slope of the MSD plot gets smaller because of the capsid\nconfinement (Fig. R8). In this revised manuscript, we have added Fig. S5 to illustrate\nthe diffusion behavior at longer lag time (3x107 MD step) and the effect of capsid\nconfinement.\nFigure R8. Mean squared displacement (MSD) of pgRNA, averaged over different\n100-nt segments of the full-length pgRNA from CG simulations at temperature of 300K\nand salt concentration of 150 mM. The slope of the MSD plot gets smaller at longer\nlag-time because of the capsid confinement.\n8) Line 92: The “an” should be “a”.\nReply and corresponding changes:\nWe thank the reviewer for pointing out the grammatical error. In this revised manuscript,\nwe corrected this grammatical error.\nReviewer #2 (Remarks to the Author):\nThe manuscript describes results of a detailed study, mostly by means of multiscale\nmolecular dynamics computer simulations and partly by experimentation, of the\nstructural organization and condensation of the pregenomic RNA in the capsid of the\nvirus HBV. The authors focus on the role of the positively charged, structurally\ndisordered C-terminal RNA binding domain of the coat protein, often called Arginine\nRich Motif or ARM, in relation with their spatial confinement in the capsid.\nParenthetically, in the manuscript the ARMs are called CTD, short for C terminal\ndomain.\nThe main point the authors try to convince the readers of is that the structural\narrangement of the RNA in HBV is driven by phase separation, arguing that viruses\nexploit what is known as liquid-liquid phase separation or LLPS. LLPS has attracted a\nlot of attention in biophysical literature recently. Often (but not always) driven by\ninteraction of, e.g., RNAs with positively charged, structurally disordered proteins, and\nI understand why seeing a correspondence between virus assembly and LLPS in\nbiology is such an attractive one.\nI find the paper hugely interesting, and I am particularly impressed by the\ncomprehensiveness of the simulation study. The findings are highly significant, and,\nif accurate, would further our understanding of the structure of viruses and appeal to a\nbroad readership. Also, the paper is very well written, well-structured and easy to read.\nI do find pushing the LLPS narrative so forcefully as is done in the manuscript at times\na little too much. As a matter of fact, I am not yet convinced that the case made by the\nauthors actually hold water, irrespective of the interesting findings they present.\nReply:\nWe thank the reviewer for the kind comments.\nSome might view this a semantic issue, but in my view absorption of what in the end\nis a polymer (the ssRNA) into a structured polymer brush (the ARMs emanating into\nthe cavity of a capsid) is not the same as macroscopic phase separation, in the same\nway that condensation of a gas in bulk is not the same as condensation of a gas onto\nan adsorbing surface. Strictly speaking, the assembly of coat proteins around an RNA\ncannot represent a true phase transition on account of its finite size, noting that the\ndroplet phases typically found in LLPS must be transient. The physics of adsorption of\nRNA onto immobilized ARMs part of a capsid is very different from the co-condensation\nof ARMs with RNA in free solution – the authors are aware of this but seem to downplay\nthis. （polymer brush phase separation）\nReply:\nWe fully agree with the reviewer’s view that the condensation of pgRNA with CTDs\ninside the HBV capsid is very different from the macroscopic LLPS in free solution.\nFirst, the CTD is anchored to the inner surface of capsid, and therefore it cannot diffuse\nfreely to form larger droplet by co-condensation with pgRNA. Second, as the reviewer\nnoted, the finite size of the system makes the condensate structure distinct from bulk-\nphase LLPS, where low- and high-density phase are well separated to form round-\nshaped droplets.\nAt the same time, this CTD-RNA co-condensation shares some important features with\ntypical macroscopic LLPS in free solution: 1) coexistence of high- and low-density\nregions; 2) liquid-like dynamics as shown by the high molecular mobility of condensate\nand cluster coalescence events; and 3) high sensitivity to perturbation in temperature\nand salt concentration. Inspired by the reviewer’s comments, we emphasized these\ndifferences and similarities in this revised manuscript, highlighting how CTD-pgRNA\nco-condensation within the HBV capsid contrasts with LLPS in free solution.\nInspired by the reviewer’s comments, we carefully surveyed the physical studies on\npolymer brush. Interestingly, numerous interesting computational and experimental\nworks have examined the electrostatic regulation of the phase behavior of\npolyelectrolyte brush (Macromolecules 2009, 42, 7194-7202; PNAS, 2010, 107:5300-\n5; Sci. Adv. 2017, 3: eaao1497). Previous studies have shown that polyelectrolyte\nbrush can undergo morphological transition by modulating electrostatic interactions,\nfor example, through the absorption of multivalent ions (Sci. Adv. 2017, 3: eaao1497)\nor by pH-dependent modulation of the charge states of charged groups (PNAS, 2010,\n107:5300-5). Such electrostatically regulated morphology transition of uniformed\ndistributed polyelectrolyte brushes is often referred to as “lateral phase separation” or\n“planar phase separation” (Biointerphases, 2022, 17:060802; J. Polym. Sci. B. 2017,\n55: 370–377). Compared to typical polymer brush system, RNA in the HBV capsid is\nnot only a participant in the phase behavior as an electrostatic regulator, but also a\ncentral component of the condensates. Notably, a previous NMR study revealed that\nthe HBV-RNA interactions within the capsid are highly fluidity (JACS, 2022, 144:8536-\n8550.), in line with the liquid-like dynamics of the CTD-RNA condensate formed by\nLLPS as discussed in this work. In this revised manuscript, we have extended the\ndiscussion to relate the organization of the HBV pgRNA genome to the phase\nseparation of polymer brush described in the polymer physics literature. Furthermore,\nwe have added experiment data and supplementary all-atom simulations to support\nthe morphology transition observed in the two-dimensional CTD-RNA system.\nCorresponding changes:\n1. (page 15, line 371) The following paragraph was added in this revised manuscript\nto emphasize the difference between the phase separation of the CTD-RNA system\nfrom the macroscopic phase separation in free solution:\n“It is also worth emphasizing that the above discussed phase separation of the pgRNA\ngenome within the HBV capsid differs from the macroscopic LLPS in free solution in\nthe following two important aspects: i) the CTD is anchored to the inner surface of\ncapsid and therefore cannot diffuse freely to form larger droplets through co-\ncondensation with pgRNA; and ii) the finite size of the system makes the condensate\nstructure distinct from bulk-phase LLPS, where low- and high-density phases are\ntypically well separated to form round-shaped droplets. At the same time, this CTD-\nRNA co-condensation shares key features with typical macroscopic LLPS, including\nthe coexistence of high- and low-density regions, liquid-like condensate dynamics, and\nsensitivity to temperature and salt concentrations.”\n2. (page 9, line 210) The following sentences were added in this revised manuscript to\ndiscuss the similarity between the phase separation of the two-dimensional CTD-RNA\nsystem and the lateral phase separation observed in polymer brush:\n“The two-dimensional planar system described above is analogous to a polymer brush.\nPrevious studies have showed that polyelectrolytes grafted onto a two-dimensional\nsubstrate can segregate into coexisting domains in response to electrostatic\nregulation61–63, a phenomenon often referred to as “lateral phase separation”64 or\n“planar phase separation”65. Compared to typical polyelectrolyte brush system, RNA in\nthe HBV capsid is not only a participant in the phase behavior as an electrostatic\nregulator, but also a central component of the condensates.\nThere are a few other issues that I have with the manuscript that need to be addressed\nbefore I would advise in favour of publication.\n1. There is a lot of theoretical and simulation literature on the structure of (model) RNAs\nin virus capsids, and the readers should be aware of that. I am referring to, e.g., the\nwork of Hagan, Zandi and Podgornik to name but a few. Of particular importance for\nthe topic of the manuscript is the work of Hagan in eLife 2013; 2: e00632, and Zandi\nand Podgornik in Phys. Rev. E 2014; 89: 032707 and Zandi in Phys. Rev. E 2020; 102:\n062423. These, and other works, highlight the importance of the localization of the\npositive charges, but also the configurational (base-pairing) response of the RNA to\nthe binding to the ARMs. See also the review paper of Zandi and collaborators, Physics\nReports 2020; 847: 1–102.\nReply:\nWe thank the reviewer for bringing these interesting papers to our attention. These\nworks clearly demonstrate the close interplay between the secondary structural\norganization of RNA, the localization of positive charges within the arginine-rich motifs\nof capsid proteins, and the optimal length of encapsulated RNA genome, thereby\nhighlighting the importance of physics-based approaches in elucidating virion\norganization. In this revised manuscript, we have incorporated a discussion of these\nprevious studies to better contextualize our findings.\nCorresponding changes:\n1. (page 14, line 393) The following sentences were added to discuss the existing\ntheoretical studies of virial assembly:\n“The key role of phase separation in the structural organization of HBV pgRNA\ngenome demonstrated in this work underscores the importance of physical principles\nin elucidating the viral life cycle. Indeed, substantial advances in understanding viral\nstructure and assembly have been achieved through physics-based methodologies,\nwhich have successfully explained a wide range of experimental observations.96 For\ninstance, by using a mean-field theory, Zandi and coworkers have illustrated the effects\nof charge distribution of capsid protein and RNA topology on the amount of packaged\nRNA based on the genome configurational entropy and electrostatics.97, 98 Base-pairing\nin RNA can trigger a conformational transition of RNA genome from an extended coil\nto a compact globule.99 In addition, kinetic and thermodynamic analysis of viral capsid\nassembly by Hagan et al revealed the importance of genome tertiary structure in\ncapsid assembly and elucidated the key factors contributing to overcharging effect.73\nCollectively, these studies highlight the close interplay between RNA secondary\nstructure, the electrostatic regulation, and the optimal length of the encapsulated\ngenome, emphasizing the value of physics-based approaches in elucidating virion\norganization.”\n2. I do not believe that the observed coexistence of high- and low-density regions in\nthe absorption layer near the ARMs indicates phase separation, as is implied on page\n3, lines 77 and 78. The difference in RNA density in the cavity and that in the absorption\nlayer would be in closer to correspondence to complex coacervation of ARMs and RNA,\ndiscussed also in the main text. I am very skeptical about the findings from the\nsimulations that phase separation takes place in the absorption layer in the virus (fig.\n2), and on the flat surface with end-grafted ARMs (fig. 4) unless the salt concentration\nis sufficiently high. The reason is that this leaves the regions with low RNA density with\nnearly naked ARMs, so without the benefit of attractive interaction with the RNA whilst\nthe higher-density regions must suffer from higher self-repulsion of the RNA. No\nexperimental verification has been done, as was with the bulk phase separation of\nRNAs and ARMs (fig. 3). I would strongly recommend experimental verification,\nknowing full well that this is a major endeavor. The discovery of dewetting of a\npolyanion such as RNA adsorbed on a polycationic surface would represent a major\nsurprise in the field.\nReply:\nWe thank the reviewer for the insightful comments and suggestion. By carefully\nexamining the structural features of the high- and low-density regions in the absorption\nlayer, we found that although one terminus of the CTD is uniformly grafted to the planar\nsurface, the overall CTD density distribution is highly heterogeneous. Typically, the\nfree-ends of the CTD chains tend to orientate from low density regions toward high\ndensity regions (Fig. R9a,b), leading to the formation of nanometer-sized clusters.\nAs a result, high-density regions are enriched in both CTD and RNA, whereas low-\ndensity regions contain relatively few CTD moieties due to covalent anchoring (Fig.\nR9a,b). In the high-density regions, close contacts between positively charged CTD\nsegments and negatively charged RNA segments promote favorable electrostatic\nattraction. Meanwhile, in the low-density regions, the larger inter-CTD distances (due to\nthe outward orientation) reduce unfavorable electrostatic repulsion between CTD\nsegments. Thus, RNA dewetting is facilitated by the enhanced electrostatic attraction\nin high-density regions and the reduced electrostatic repulsion in low-density regions.\nWe agree with the reviewer’s comment that when low-density regions are instead\nenriched in naked CTD segments, electrostatic interactions would become unfavorable\nfor phase separation. This scenario arises when the CTD density is much higher than\nthe conditions studied in this work. Indeed, in newly added simulations with elevated\nCTD densities, phase separation gradually becomes invisible by increasing CTD\ndensity (Fig. R9c-f), likely due to unfavorable electrostatic repulsion among CTD\nsegments in the low-density region when RNA is segregated into clusters, as\ncommented by the reviewer.\nEncouraged by the reviewer’s suggestions, we tried to perform new experiments to\nfurther support the CTD-RNA microphase separation in the two-dimensional system.\nBecause CTD segments are short (36 amino acid) and the resulting clusters are\ntypically nanometer in size, which is too small to be detected by fluorescence\nmicroscope or atomic force microscope (AFM), we adopted an alternative strategy.\nSpecifically, we grafted PEG onto a substrate and decorated the free PEG terminus\nwith CTD segments. This extended the effective polymer length of the two-dimensional\nbrush, enabling the formation of larger clusters upon RNA addition. We then employed\nAFM to examine the morphological changes induced by adding RNA to the constructed\nPEG-CTD layer. As expected, the AFM measurements revealed that RNA addition\npromotes morphological heterogeneity in the hybrid PEG–CTD layer (Fig. R10),\nresembling the lateral phase separation observed in early studies (Sci. Adv. 2017, 3:\neaao1497). Because AFM morphology has limited resolution and it is difficult to\nabsolutely rule out alternative origins for the observed morphological transition beyond\nphase separation, we also carried out additional all-atom MD simulations of the\ntwo-dimensional CTD layer with RNA segments (Figs. R11a,b). Consistent with the coarse-\ngrained MD simulations, these all-atom MD simulations demonstrated RNA dewetting\nin the two-dimensional system, although the simulation length is limited due to\ncomputational demands.\nIn this revised manuscript, we have incorporated these new results and discussions\nto more clearly elucidate the molecular mechanism underlying two-dimensional phase\nseparation.\nFigure R9. Effect of CTD density on the CTD-RNA condensation in the two-\ndimensional system. (a,b) Representative snapshots for the CG simulations with the\naddition of pgRNA (a) and A RNA (b) to the two-dimensional system with the native\nconcentration of 150mM. (e,f) Same as (a,b) but with increased CTD density at the salt\nconcentration of 150mM. d refers to the distance between the N-terminal anchoring\nN\nbeads of neighboring CTDs.\nFigure R10. AFM characterization of RNA induced mophological transition of PEG-\nCTD layer. (a,b) AFM tapping mode height images of PEG-CTD layer before (a) and\nafter (b) the addition of A RNA. (c,d) AFM phase images of PEG-CTD layer before (c)\n15\nand after (d) the addition of A RNA.\nRNA system. (a) Representative snapshot of all-atom MD simulation for the two-\ndimensional system containing 15×16 array of CTD chains with the simulation length\nof 100 ns. (b) Representative snapshot of all-atom MD simulation for the two-\ndimensional system containing 10×10 array of CTD chains with the simulation length\nof 200 ns. (c,d) Distribution of Na+ (c) and Cl− (d) across the two-dimensional layer for\nthe two-dimensional system containing 15×16 array of CTD chains. (e) Fraction of ions\nwithin the high- and low-density regions of CTD-RNA clusters.\nCorresponding changes:\n1. (Supplementary Material) We have added Fig. S11-14 to illustrate the results of\ncoarse-grained MD simulations, AFM characterization, and all-atom MD simulations of\nthe phase separation for a two-dimensional system.\n2. (page 9, line 198) The following paragraph was added to discuss the physical basis\nof the phase separation for the two-dimensional CTD-RNA system.:\n“Structural analysis of the high- and low-density regions in the two-dimensional\ncondensate layer reveals that, although one terminus of each CTD is uniformly grafted\nto a planar surface, the overall CTD density distribution is highly heterogeneous.\nTypically, the free ends of CTD chains tend to orientate from low density regions toward\nhigh density regions (Fig. S11a,b), leading to the formation of nanometer-sized clusters.\nConsequently, high-density regions are enriched in both CTD and RNA, whereas low-\ndensity regions contain relatively fewer CTD moieties due to covalent anchoring. In the\nhigh-density regions, close contacts between positively charged CTD segments and\nnegatively charged RNA segments promote favorable electrostatic attractions.\nMeanwhile, the larger inter-CTD distances (due to outward orientation) in low-density\nregions reduce unfavorable electrostatic repulsion. Thus, RNA dewetting is facilitated\nby the enhanced electrostatic attraction in high-density regions and the reduced\nelectrostatic repulsion in low-density regions. Further investigations established that\nthe two-dimensional phase separation of the CTD-RNA mixture system depends\ncritically on the surface density of CTDs. Altering CTD density by changing inter-chain\ndistances under physiological conditions diminished phase separation (Figs. S11,\nS12), likely due to alteration of the electrostatic balance.”\n3. (page 9, line 214) The following sentences were added to describe the AFM\nexperiment and all-atom MD simulation results of the phase separation of the two-\ndimensional system:\n“To further support the two-dimensional phase separation of the CTD–RNA system,\nwe employed atomic force microscope (AFM) to characterize the morphological\ntransition. Because of the short length of CTD peptides, the resulting condensate are\ntypically nanometer in size, which is too small to be detected by fluorescence\nmicroscope or AFM. To overcome this limitation, we grafted polyethylene glycol (PEG)\nonto a planar substrate and decorated the free PEG terminus with CTD segments.\nThis extended the effective polymer length, enabling the formation of larger clusters\nupon RNA addition. AFM measurements revealed that RNA addition promotes\nmorphological heterogeneity in the two-dimensional system (Fig. S13), resembling the\nlateral phase separation observed in early studies61. Because AFM morphology has\nlimited resolution and it is difficult to absolutely rule out alternative origins for the\nobserved morphological transition beyond phase separation, we also carried out\nadditional all-atom MD simulations of the two-dimensional CTD layer mixed with RNA\nsegments. These all-atom MD simulations clearly demonstrated RNA dewetting (Fig\nS14a,b).”\n3. The in-layer phase separation shown in fig. 4 was studied for different ionic strengths,\nbut only a single RNA length. It would be useful to investigate how the length of the\nRNA influences this phase separation. Also, the phase separation, if real, should lead\nto inhomogeneous distribution of mobile ions. Not much is said about this in the\nmanuscript. Note also, that if phase separation indeed takes place, the high- and low-\ndensity regions should increase in size in time and coarsen, e.g., by coalescence.\nDoes coarsening take place?\nReply:\nIn this revised manuscript, we have added a series of new coarse-grained simulations\nwith much shorter RNA segments, specifically A RNA segments (Fig. R12). Similar\n15\nphase-separation behaviors were observed under varying salt concentrations, phase\nseparation becoming more pronounced at lower salt concentrations due to stronger\nelectrostatic interactions. Following the reviewer’s comments, we also analyzed the\ncoarsening dynamics in these simulations and identified clear coalescence events (Fig.\nR13). Because one terminus of each CTD is anchored, the sizes of the high-density\nclusters remain limited and continuous coalescence of smaller clusters into larger ones\nbecomes difficult.\nFollowing the reviewer’s comments, we also characterized the distribution of mobile\nions. Since the influence of salts is implicitly accounted for through the Debye-Hükel\nmodel in the coarse-grained simulations, we performed additional analysis using all-\natom MD simulations to examine whether ions exhibit inhomogeneous distribution\n(Figs. R11c-e and R14d,e). The results revealed that the distributions of Na+ and Cl-\naround the condensate layer are highly heterogeneous and closely correlated with the\nspatial organization of the CTD-RNA condensate (Fig. R11e), owing to the greater\naccumulation of ions in high-density CTD–RNA regions.\nIn this revised manuscript, we have added the above new results and discussion to\nillustrate the in-layer phase separation for shorter RNA segments, mobile ion\ndistributions, and the coarsening of CTD-RNA condensate.\nFigure R12. Representative simulation snapshots from CG simulations of the two-\ndimensional system at varying salt concentrations for A RNA molecules along the\nFigure R13. Fusion events captured in CG simulations of A RNA molecules along\n15\nthe two-dimensional CTD layer at the salt concentration of 50 mM.\nFigure R14. Distribution of mobile ions estimated from the all-atom MD simulations.\n(a) Ratio of the cumulative number of Na+ (red) and Cl− (blue) ions flowing into and out\nof the capsid over successive 5 ns intervals. (b) Radial distribution functions of mobile\nions relative to the capsid center. For comparison, the distribution of NTD, CTD, and\npgRNA were also shown. (c) Radial distribution functions of mobile ions relative to the\ncapsid center calculated at three different simulation times (80ns, 90ns, and 100ns).\n(d,e) Representative two-dimensional density distributions of Na+ (a) and Cl− (b)\nplotted along spherical coordinates (θ, ϕ). The density distributions in (d,e) were\ncalculated for the region indicated by two vertical lines in (b,c).\nCorresponding changes:\n1. (Supplementary Material) Fig. S2,S9-S10, and Supplementary Movie 9 were\nadded.\n2. (page 7, line 183) The following sentences were added in this revised manuscript:\n“Similar phase-separation behaviors were observed when much shorter RNA\nsegments (A15 RNA segments) were added (Fig. S9). Interestingly, we observed clear\ncoarsening events, in which two small clusters coalesced into a larger cluster (Fig. S10\nand Supplementary Movie 9), a hallmark feature of macroscopic phase separation.\nHowever, because one terminus of each CTD is anchored, the sizes of the high-density\nclusters remain limited and continuous coalescence into larger droplets becomes\ndifficult.”\n3. (page 9, line 224) The following sentence was added to describe the heterogeneous\ndistributions of Na+ and Cl- in all-atom simulations:\n“Correspondingly, the distributions of Na+ and Cl− around the condensate layer are\nhighly heterogeneous (Fig. S14c,d) and closely correlated with the spatial organization\nof the CTD-RNA condensate (Fig. S14e).”\n4. The structure of the RNA in particular in relation to base pairing is discussed on\npage 9 and 10 (figure 6). There is quite some work on the structure of viral ssRNAs,\nand why they are different from random RNAs or cellular RNAs. See, e.g., the work of\nGelbart and others on this topic in PLoS ONE 2014; 9: e105875 and Biophys. J. 2017;\n113: 339-347, to cite a few of many papers these authors wrote on this topic. Also,\ntheoretical work (e.g. of Zandi, see earlier) indicates that interaction with the ARMs\nchanges the base pairing. It would be interesting to get more information on the\nstructure of the RNA in the virus, such as maximum ladder distances, length\ndistributions of hairpin structures, and so on. The authors also mention nematic\nalignment of clustered hairpins but do not elaborate on how this was established: by\neye or by calculating the largest eigenvalue and associated eigenvector of the local Q\ntensor? This would also produce a director field configuration – in that case one would\nexpect four half-integer disclination defects to appear. There is quite a bit of literature\non this.\nReply:\nWe thank the reviewer for bring these interesting researches to our attention. During\nthe revision of this manuscript, we carefully examined these highly relevant prior works\nand incorporated discussions comparing them with the findings of this study. In\naccordance with the reviewer’s suggestion, we have also included additional analyses\nof the structural features of the viral RNA, such as the maximum ladder distance (MLD),\nthe length distributions of hairpin structures, and the scalar order parameter S.\nFor the dsRNA structures, we analyzed the length distributions at varying salt\nconcentrations (Fig. R15a-c). The analysis shows that the dsRNA regions become\nshorter with increasing salt concentration. This observation suggests that the CTD-\nmediated structural organization of pgRNA can be modulated by ionic strength.\nFollowing the reviewer’s suggestion, we computed the largest eigenvalue (S) of the\nlocal Q-tensor to characterize the nematic alignment of neighboring dsRNA strands.\nSpecifically, S was evaluated for each spatially adjacent pair of dsRNA molecules and\nthen averaged across all pairs. Across different salt concentrations, the average value\nof S ranged between 0.3 and 0.5, which is characteristic of systems in the nematic\nphase (Fig. R15d). As expected, the average S value decreased with increasing salt\nconcentration, reflecting the enhanced flexibility of dsRNA under high-salt conditions.\nIn addition, our analysis revealed that the maximum ladder distance (MLD), which\nreflects the overall extension of RNA, was estimated to be between 110 and 120 under\nconditions where phase separation is prominent (Fig. R15e). This value is substantially\nlower than the MLD of a randomly permuted single-stranded RNA of comparable\nsequence length (~300 for a 3000-nt RNA), consistent with previous findings that viral\nRNAs adopt more compact conformations than their randomized counterparts (PNAS,\n2008, 105: 16153–16158; PLoS ONE, 2014, 9: e105875). Furthermore, we observed\na pronounced decrease in MLD at a high salt concentration, where phase separation\nis absent. This is likely because the suppression of phase separation impedes base\npairing as shown in Fig. 6c, thereby leading to a lower MLD.\nFigure R15. Structural characteristics of dsRNA. (a-c) Length distributions of dsRNA\n(L ) under varying salt concentrations. (d) Scalar order parameter S estimated at\ndsRNA\ndifferent salt concentrations. (e) MLD of pgRNA as a function of salt concentration.\nCorresponding changes:\n1. (Supplementary Material) We have added Fig. S22 in this revised manuscript.\n2. (page 12, paragraph 294) The following sentence was added:\n“Meanwhile, the dsRNA becomes shorter as the salt concentration increases (Fig.\nS22a-c).”\n3. (page 13, line 309) The following sentences were added to describe the dsRNA\nstructural characteristics:\n“To further characterize the structural organization of the pgRNA genome within the\nHBV capsid, we estimated the averaged maximum ladder distance (MLD) among the\nsampled structural ensemble of pgRNA in CG simulations, following previous works.73,\nseparation is prominent (Fig. S22d). This value is significantly lower than the MLD of\na randomly permuted single-stranded RNA of similar sequence length (∼300 for a\n3000-nt RNA), consistent with previous findings that viral RNAs are more compact than\ntheir randomized counterparts.74, 75.”\n4. (page 12, line 296) The following sentences were added in this revised manuscript:\n“To confirm this finding, we calculated the scalar order parameter S, defined as the\nlargest eigenvalue of the local Q-tensor. Specifically, S was evaluated for each spatially\nadjacent pair of dsRNA segments and then averaged across all pairs. Across different\nsalt concentrations, the average value of S ranged between 0.3 and 0.5 (Fig. 6i), which\nis characteristic of systems in the nematic phase.72 As expected, the average S value\ndecreased with increasing salt concentration, reflecting the enhanced flexibility of\ndsRNA under high-salt conditions.”\n5. Viruses tend to be overcharged, that is, there is more negative charge on the RNA\nthan needed to compensate for the positive charges on the ARMs in the virus. This\ncauses a Donnan equilibrium to establish itself for the mobile ions inside and outside\nthe virus, and between the empty core of the lumen of the capsid and the region where\nARMs and RNA interact. This equilibrium takes time to set up, and I wonder if the all-\natom simulations are sufficiently long to let this equilibrium establish itself. Also, the\nDebye-Hückel potential used by the authors for the coarse-grained simulations most\ncertainly cannot deal with this kind of phenomenology. It would be good to see if for\nphysiological conditions Donnan effects can indeed be neglected by calculating time\ndependent ion density distributions in the atomistic simulations.\nReply:\nWe thank the reviewer for the valuable comments and suggestion. By analyzing the\nion concentrations obtained from the all-atom MD simulations, we found that the\nnumber of mobile ions flowing in and out of the capsid becomes comparable within\n10ns (Fig. R14a). Moreover, time-dependent radial distributions of ion concentrations\nbecome converged within the simulation timescale (Fig. R14b,c). These results\nsuggest that the Donnan equilibrium can be established within the timescale of the all-\natom MD simulations. The converged radial distributions of ion concentrations further\nshowed that the relative ratio of the Na+ concentrations between the inner and outer\nsides of the capsid is ~2.3 (Fig. R14b), which is considerably smaller than the values\ntypical observed in other dsDNA viruses (>10) (Cosic et al, Nature, 2024, 627: 905-\n104). One possible reason of this smaller Donnan equilibrium effect is that at the\npgRNA intermediate stage of the HBV life cycle, only a single RNA strand is present.\nIn this case, the positive charges of the CTD segments (+3600 in total) and negatively\ncharged RNA (-3200 not including polyA tail) are approximately balanced. With the\nprogression of reverse transcription of pgRNA into dsDNA, the negative charges of the\ngenome will be roughly doubled and therefore will become much larger than the\npositive charges of the CTDs. Under such conditions, the Donnan equilibrium effect\nwould become more pronounced.\nAs commented by the reviewer, Debye-Hückel potential was used in the coarse-\ngrained model to account for ion effects. Therefore, the Donnan equilibrium cannot be\ncaptured within such an implicit solvent model. While neglecting this effect in the\ncoarse-grained model may limit the quantitative interpretation of the simulation results,\nthe major finding of CTD-pgRNA condensation and its impact is expected to be less\nsensitive to this limitation, as the positive charges of CTDs and negative charges of\npgRNA are roughly balanced.\nIn this revised manuscript, we have incorporated these additional results and\ndiscussions to illustrate the Donnan equilibrium effect.\nCorresponding changes:\n1. (Supplementary Material) Fig. S2 was added in the Supplementary Material of this\nrevised manuscript.\n2. (page 3, line 86) The following sentences were added to discuss the ion distribution\nand Donnan equilibrium effect:\n“By analyzing ion concentrations from the all-atom MD simulations, we found that the\nnumber of mobile ions flowing in and out of the capsid becomes comparable within 10\nns (Fig. S2a). Moreover, time-dependent radial distributions of ion concentrations\nbecome converge within the simulation timescale (Fig. S2b,c). These results suggest\nthat the Donnan equilibrium can be established within the timescale of the all-atom MD\nsimulations. The converged radial distributions further demonstrate90 that the\ndistribution of mobile ions inside the HBV capsid exhibits notable heterogeneity (Fig.\nS2d,e). In addition, the average concentration of Na+ inside and outside the capsid\nare 0.20 mM and 0.086 mM, respectively. The resulted relative ratio of the Na+\nconcentrations between the interior and exterior of the capsid (∼2.3) is considerably\nsmaller than the values reported for other dsDNA virus (>10)19. A likely explanation for\nthis weaker Donnan equilibrium effect is that only a single RNA strand is present at the\npgRNA intermediate stage of the HBV life cycle. In this case, the positive charges of\nthe CTD segments (+3600 in total) and the negative charges of pgRNA (-3200) are\napproximately balanced. With the progression of reverse transcription of pgRNA into\ndsDNA, the negative charges of the genome will significantly exceed the positive\ncharges of CTDs, which would lead to more pronounced Donnan equilibrium effect.”\n3. (page 16, line 458) The following sentences were added in the Methods section of\nthis revised manuscript:\n“The Debye-Hükel type electrostatic potential was used to characterize the salt-\nconcentration-dependent interactions of charged particles. It is worth mentioning that\nwith such an implicit solvent model, the Donnan equilibrium cannot be explicitly\ncaptured. While neglecting this effect in the coarse-grained model may limit the\nquantitative interpretation, the major results are expected to be less sensitive to this\nlimitation as the positive charges of CTDs and negative charges of pgRNA are roughly\nbalanced.”\nReviewer #3 (Remarks to the Author):\nBian et al computationally show that interaction of RNA with the basic C terminal\ndomain of the HBV core protein results in a condensed phase. The condensate\nresembles the hollow shell of density seen in image reconstructions of HBV with the\npregenome and with random RNA from the expression system. In the condensed phase\nthe RNA is able to diffuse. As one would expect, neutralisation of phosphates\nallows base pairing. Unsurprisingly, the dsRNA segments shows nematic organisation.\nUnsurprisingly, the heterogeneity of the layer follows the symmetry of the capsid.\nTo support their in silico studies, the authors demonstrated that isolated CTDs were\nable to form a phase when mixed with 15 nucleotide poly A oligomers.\nThere are several issues the authors need to address.\n1) The approach to adding pgRNA to a capsid is novel; do all trajectories lead to the\nsame result? How much do they differ?\nReply:\nWe thank the reviewer for the questions. For most cases, we carried out three\nindependent simulations. The analysis confirms that simulations under identical\nconditions yield comparable phase behavior. To quantitatively assess variability across\nMD simulation trajectories, we incorporated error bars (estimated from the three\nreplicates) into the distributions of key metrics, including the densities of the low- and\nhigh-density regions, the pgRNA mean squared displacement (MSD), and secondary\nstructure content. The results demonstrate consistent outcomes across independent\nsimulations under identical conditions. In this revised manuscript, we have also\nupdated the figure captions to explicitly highlight these error bars, and the related\ndiscussion has been added accordingly.\nCorresponding changes:\n1. Following the reviewer’s comment on the reproducibility of the simulations, the\nestimation of error bars from three independent replicates was highlighted in the\nrelated figure captions.\n2) While the peptide–RNA oligomer complex is experimentally shown to form a liquid-\nliquid phase separation, the two dimensional nature of the inner layer of the capsid\nstretches the definition of a phase separation. This needs to be addressed.\nReply:\nWe thank the reviewer for the valuable comments and question. The key feature of the\nCTD-RNA phase separation along the inner surface of HBV capsid is that one end of\nthe CTDs is anchored, effectively leading to a two-dimensional system. Although the\nCTD-RNA condensation of this two-dimensional system shares some important\nfeatures with typical macroscopic LLPS in free solution, such as coexistence of high-\nand low-density regions, liquid-like dynamics of the condensates, and high sensitivity\nto perturbation in temperature and salt concentration, it has the following differences.\nFirst, the CTD is anchored to the inner surface of capsid, and therefore it cannot diffuse\nfreely to form larger droplet by co-condensation with pgRNA. Second, the finite size of\nthe system makes the condensate structure distinct from the round-shaped droplets\ntypically observed in bulk-phase LLPS. To further characterize such two-dimensional\nphase separation for the CTD-RNA system, we performed atomic force microscope\n(AFM) characterization and observed morphological changes (Fig. R10). We further\nperformed additional all-atom MD simulations for the two-dimensional CTD-RNA\nsystem and observed the segregation of CTD-RNA into high- and low-density regions,\nsupporting the two-dimensional phase separation (Fig. R11). Following the reviewer’s\ncomments, we added the above results and discussion to address the differences of\ntwo-dimensional phase separation compared to the LLPS in free solute in this revised\nmanuscript.\nCorresponding changes:\n1. (page 15, line 371) The following paragraph was added in the main text of this\nrevised manuscript to address the features of two-dimensional phase separation:\n“It is also worth emphasizing that the above discussed phase separation of the pgRNA\ngenome within the HBV capsid differs from the macroscopic LLPS in free solution in\nthe following two important aspects: i) the CTD is anchored to the inner surface of\ncapsid and therefore cannot diffuse freely to form larger droplets through co-\ncondensation with pgRNA; and ii) the finite size of the system makes the condensate\nstructure distinct from bulk-phase LLPS, where low- and high-density phases are\ntypically well separated to form round-shaped droplets. At the same time, this CTD-\nRNA co-condensation shares key features with typical macroscopic LLPS, including\nthe coexistence of high- and low-density regions, liquid-like condensate dynamics, and\nsensitivity to temperature and salt concentrations.”\n3) The phase needs to be better described. Do the high RNA density clusters show a\ncharacteristic arginine-RNA ratio? How fluid are the CTD-RNA clusters? How fluid are\nthe dsRNA segments? If sufficiently stable, they will halt reverse transcription. A\nquantitative view of their stability will be appreciated.\nReply:\nWe thank the reviewer for the insightful comments and suggestion. In this revised\nmanuscript, we performed a more thorough characterization of the condensate phase\nand analyzed the stability of dsRNA segments. The results reveal that within the dense\nphase, the arginine-RNA ratio stabilizes at a characteristic value of 1.05~1.11 across\nvarying salt concentrations where phase separation is prominent (Fig. R16c). This\nvalue is lower than the overall system average among all the CTDs and pgRNA (~1.13),\nimplying a fractionation effect that is typical of multicomponent phase separation\nsystems. In addition, the CTD-RNA interactions are highly dynamic and flexible, as\nevidenced by substantial variations in the intermolecular contacts for a chosen pair of\nCTD and pgRNA segments (Fig. R16d). While the mobility of the CTDs themselves is\nconstrained by their grafting to the inner capsid surface, the encapsulated RNA exhibits\nconsiderable fluidity, which is clearly demonstrated by the significant mean squared\ndisplacement (MSD, Fig. 2k). We also found that the dynamics are different in the\nssRNA region and dsRNA region (Fig. R16e). Compared to the ssRNA region, the\nfluidity of the dsRNA region is much suppressed. In addition, dsRNA segments,\nincluding the typical protein binding motifs can dynamically fold and unfold dynamically\n(Fig. R1b-d), demonstrating the flexibility of dsRNA segments. We also observed a\nlength dependence of the dsRNA stability as quantified by their lifetime (Figure R1f),\nwhich were estimated using a maximum-likelihood method.\nFigure R16. Interaction modes between CTD and dsRNA from CG simulations of the\npgRNA-containing HBV capsid. (a,b) A representative conformation showing the\ninteractions between CTD (blue) and dsRNA (wheat) as viewed from the exterior (a)\nand interior (b) of HBV capsid, respectively. (c) Ratio of the numbers of arginine in\nCTDs and the nucleotides in RNA within high-density regions under different salt\nconcentrations where phase separation is prominent. (d) The contact map between a\nrandomly selected CTD segment and a specified pgRNA segment at two different\nsimulation snapshots. The large change of the contact maps suggests the dynamic\nnature of the base pairs. (e) MSD of dsRNA and ssRNA within a short lag time.\nCorresponding changes:\n1. (Supplementary Material) Fig. S20 was added in the Supplementary Material of\nthis revised manuscript.\n2. (page 13, line 316) The following sentences were added in the main text of this\nrevised manuscript:\n“Notably, the CTD-RNA interactions are also highly dynamic, as evidenced by\nsubstantial variations in the number of CTD-RNA contacts within the clusters (Fig.\nS20d). More detailed analysis revealed that the arginine-RNA ratio in the dense phase\nstabilizes at a characteristic value of approximately 1.05~1.11 across varying salt\nconcentrations (Fig. S20c). This value is lower than the overall system average of 1.13,\nindicating a fractionation effect typical of multicomponent phase separation system56\n3. (page 12, line 263) The following sentences were added in the main text of this\nrevised manuscript:\n“The formed base-pairs exhibit pronounced dynamics in the CG simulations.\nUnwinding events of dsRNA can be clearly observed in the simulation trajectories (Fig.\nS17b and Supplementary Movie 11). Moreover, substantial variation in the number of\nformed dsRNA segments during the CG MD simulations supports the highly dynamic\nnature of the dsRNA conformation (Fig. S17e). The stability of dsRNA is length-\ndependent. Segments containing more base pairs exhibit longer lifetimes (Fig. S17f),\nwhich were estimated using a maximum-likelihood method. While the mobility of the\nCTDs is constrained by their anchoring to the inner capsid surface, the encapsulated\nRNA displays considerable fluidity, as evidenced by dynamic changes in local density\nat specified capsid site (Fig. 2j) and by its significant mean squared displacement (Fig.\n2k). The dsRNA segments also exhibit pronounced mobility, although markedly\nreduced compared to the ssRNA region (Fig. S20e). This structural plasticity is\nfunctionally important for HBV pgRNA, as overly stable or rigid secondary structures\ncould interfere with subsequent reverse transcription.”\n4) One of the advantages of molecular dynamics, even with coarse graining, is the\nability to visualize molecular contacts. How do the CTDs interact with the RNA? Do\nthey interdigitate between strands? Do the fit in major grooves of dsRNA?\nReply:\nWe thank the reviewer for the suggestions. In this revised manuscript, we added\nvisualization analysis to the results of molecular dynamics simulations. As shown in a\nrepresentative region containing dsRNA, CTDs tend to be embedded between\nadjacent dsRNAs, acting as bridges that connect neighboring dsRNA strands (Fig.\nR16a). Although we can also observe that CTDs fit to the major or minor grooves of\ndsRNA, the occurrences of such specific interaction modes are not dominant.\nCorresponding changes:\n1. (Supplementary Material) Fig. S20 was added in the Supplementary Material of this\nrevised manuscript.\n2. (page 13, line 314) The following sentences were added in the main text of this\nrevised manuscript:\n“We also analyzed the interaction characteristics between RNA and CTD. Structural\nvisualization showed that CTDs tend to be embedded between adjacent dsRNA\nsegments, acting as bridges (Fig. S20a,b). Although CTDs can also fit into the major\nor minor grooves of dsRNA, such specific interaction modes are not dominant.”\n5) The authors appear to use the HBV pgRNA in their study but do not identify the\nsequence used. It is not clear if they have a polyA tail.\nReply:\nWe thank the reviewer for the comments. In our study, the RNA sequence is a wild-\ntype sequence without the polyA tail derived from the complete genome sequence\n(GenBank accession number V00866.1). The 5’- and 3’-termini of the pgRNA were\ndetermined by referring to the information documented in a previous study (J. Mol. Biol.\n2007, 370: 471–480). Following the reviewer’s comments, we have added the\ndescription of the HBV pgRNA sequence to improve clarity.\nCorresponding changes:\n1. (page 16, line 418) The following sentences were added to this revised manuscript:\n“The initial structure of the 3200-nt pgRNA was generated using the Nucleic Acids\nBuilder (NAB) script in Ambertools101 with the wild-type pgRNA sequence. The pgRNA\nsequence without the polyA tail was retrieved from GenBank (accession number\nV00866.1). The 5’- and 3’- termini of the pgRNA sequence were identified by referring\nto the information documented in a previous study102.”\n6) The means of adjusting ionic strength are not clearly defined. Are the authors\nholding the capsid interior to the stated bulk ionic strength? is the exterior held at the\nstated ionic strength? If the latter, what is the interior ionic strength or total number of\nions?\nReply:\nWe thank the reviewer for the comments. In the all-atom MD simulations, the average\nion strength is fixed. The mobile ions inside the capsid are freely exchangeable with\nthose of exterior environment through the capsid cavities, leading to uneven ion\ndistribution between the inside and outside of capsid. To quantify the spatial distribution\nof ionic strength, we calculated the radial distributions of Na+ and Cl- concentrations\n(Fig. R14). The results revealed a highly inhomogeneous distribution of the ionic\nstrength. The average concentration of Na+ (Cl-) inside and outside the capsid are 0.20\nmM/mL and 0.086 mM/mL, respectively. In this revised manuscript, we added the\nabove results and discussion to address the spatial distribution feature of ionic strength.\nCorresponding changes:\n1. (Supplementary Material) Fig. S2 was added in the Supplementary Material of this\nrevised manuscript.\n2. (page 16, line 429) The following sentence was added in the Methods section of this\nrevised manuscript:\n“Here the salt concentration corresponds to the average salt concentration of the whole\nsystem.”\n3. (page 5, line 89) The following sentence was added in the Methods section of this\nrevised manuscript:\n“The converged radial distributions further demonstrate that the distribution of mobile\nions inside the HBV capsid exhibits notable heterogeneity (Fig. S2d,e). In addition, the\naverage concentration of Na+ inside and outside the capsid are 0.20 mM and 0.086\nmM, respectively.”\n7) There are several papers that are germane to this study that ought to be discussed\nto provide deeper context. Most importantly, Harati Taji et al (DOI:\n10.1021/jacs.1c12439) showed that HBV-RNA interactions were highly fluid in NMR\nstudies. How does your study conform to Harati Taraji predictions? Chu et al (DOI:\n10.1128/JVI.03235-13) showed that HBV CTDs could act as RNA chaperones. Is this\nconsistent with your results? Do dsRNA duplexes readily break in your simulations?\nPatel et al (DOI: 10.1038/nmicrobiol.2017.98) identify putative packing sites; are they\nfolded in your study and how would a stable fold affect your predictions. Abraham and\nLob (DOI: 10.1128/JVI.80.9.4380-4387.2006) predict long-range base pairing ; how\nwould such base pairing bias the packaging of RNA.\nReply:\nWe thank the reviewer for bring these important studies into our attention. As\ncommented by the reviewer, these previous papers are highly relevant to the findings\nof this study. For example, the highly fluid feature of the HBV-RNA interactions\nrevealed by Taji et al by NMR studies is in line with the liquid-like dynamics of the CTD-\nRNA condensate formed by LLPS as discussed in this work. The observed role of\nLLPS in regulating the pgRNA base pairing support the chaperone activity of ARMs\nsuggested in previous work by Chu et al. Interestingly, we can also observe the\nbreaking events of dsRNA duplexes in the coarse-grained MD simulations. Moreover,\nas highlighted by Patel et al., RNA secondary structures, particularly those involving\npackaging signals, are important for HBV virion assembly. Here, we find that secondary\nstructures formed by local sequence segments (e.g., the cis‑acting element ε, capsid\nprotein packaging site PS3) frequently form and break during simulations. This\ndynamic behavior appears functionally important, as excessively stable or rigid\nstructures could interfere with subsequent reverse transcription. The biological\nrelevance of the phase separation enhanced formation of long-range base pairing can\nalso be supported by the study of Abraham and coworkers on the role of long-range\nbase pairs for template switching involved in the reverse transcription. In this revised\nmanuscript, we have refined the discussion to better highlight the connections between\nthese previous studies and our current work, providing a more comprehensive context\nfor the present study.\nCorresponding changes:\n1. (page 7, line 195) The following sentences were added in this revised manuscript:\n“Interestingly, the highly fluid feature of HBV-RNA interactions within the capsid has\nbeen observed by Taji et al through nuclear magnetic resonance (NMR) studies,60\nconsistent with the liquid-like dynamics of the CTD-RNA condensate described in this\nwork.”\n2. (page 12, line 296) The following sentences were added in this revised manuscript:\n“Interestingly, the arginine-rich CTD has been suggested to act as a specific nucleic\nacid chaperone to facilitate the structural organization of the viral genome.29, 71 The\nCTD-induced pgRNA condensation and base-pairing observed in this study is in line\nwith the chaperone role of CTDs as proposed in previous studies.\n3. (page 12, line 270) The following sentences were added in this revised manuscript:\n“This structural plasticity is functionally important for HBV pgRNA, as overly stable or\nrigid secondary structures could interfere with subsequent reverse transcription.\nFurthermore, the formation of secondary structures through local base-paring in\npgRNA, particularly sites involving packaging signals, has been proposed to play a\ncritical role in the assembly of the HBV capsid.67–70 Notably, Patel et al. demonstrated\nthat folding of packaging sites into stem-loop structures promotes capsid assembly67.\nIn line with this, our CG simulations showed that the segment corresponding to a\npotential packaging site (PS3) can spontaneously form stem-loop-like structure (Fig.\nS17d). In addition, another important protein recognition motif, the cis-acting element\nε, can also fold to stem-loop-like structure, although it involves frequent folding and\nunfolding transitions (Fig. S17c).”\n4. (page 13, line 334) The following sentences were added in this revised manuscript:\n“Notably, Abraham et al. demonstrated that interactions between the cis-acting\nelement ε and the distal φ site facilitate template switching from ε to DR1,69 highlighting\nthe importance of long-range base pairing in reverse transcription. Here, we can\nobserve an increase of long-range base pairing (Fig. 7d and Fig. S23), which is\nessential for enabling the template switches as discussed above. In this study, the\nbase pairs with sequence separation larger than 100 are classified as long-range\nbase pairs.”\nResponses to Reviewer's Questions and list of changes\nReviewer #1 (Remarks to the Author):\nThe authors have done an incredible job with addressing all comments from the\nprevious round of review. I recommend publication in its present form.\nReply:\nWe thank the reviewer for the kind comments and for recommending its publication.\nReviewer #2 (Remarks to the Author):\nI am satisfied with the extensive responses of the authors to the queries and comments\nof all three reviewers, and with the substantial revision of the manuscript that includes\na discussion of additional studies that makes it an outstanding one.\nReply:\nWe thank the reviewer for the kind comments and the positive assessment.\nReviewer #3 (Remarks to the Author):\nThis revised manuscript addresses this reviewer’s questions. With this revised ms, I\nbetter understand the effect of phase separation on polymerase location, the molecular\ninteraction between CTDs and RNA, and the effect of CTDs on RNA mobility. The\nagreement between computation and experimental observation is convincing, notably\nthe asymmetric reconstruction in reference 66, and will lead to a far better\nunderstanding of the process of in-capsid reverse transcription peculiar to HBV. This\nmechanism of RNA packaging is also likely relevant to many other small RNA viruses.\nReply:\nWe thank the reviewer for the careful review and kind comments on this manuscript.\nThere are several minor typos and clarifications to address:\n15 “rich in” instead of “rich of”\n40 some HBV serotypes have a 183 residue length\n75 notably\n88 delete “become”\n91 are the Na+ concentrations in units of M or mM?\n221 “AFM morphology” or “atomic force microscopy (AFM)”\n235, 237, etc there are no sixfold axes. There are quasi-sixfold vertices\n298 define Q-tensor\n304 provide units for “100”. Is it Å, bp?\n312 provide units for ladder length (Å or bp)\n337 units\nReply and corresponding changes:\nWe thank the reviewer for suggesting the changes and for pointing out the typos. In\nthis revised manuscript, we made the suggested modifications and corrected the\nrelated typos.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReply:\nWe thank the reviewer for the carefully reviewing of this manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done an incredible job with addressing all comments from the previous round of review. I recommend publication in its present form.", "reviewer_2": "I am satisfied with the extensive responses of the authors to the queries and comments of all three reviewers, and with the substantial revision of the manuscript that includes a discussion of additional studies that makes it an outstanding one.", "reviewer_3": "This revised manuscript addresses this reviewer’s questions. With this revised ms, I better understand the effect of phase separation on polymerase location, the molecular interaction between CTDs and RNA, and the effect of CTDs on RNA mobility. The agreement between computation and experimental observation is convincing, notably the asymmetric reconstruction in reference 66, and will lead to a far better understanding of the process of in-capsid reverse transcription peculiar to HBV. This mechanism of RNA packaging is also likely relevant to many other small RNA viruses.\nThere are several minor typos and clarifications to address:\n15 “rich in” instead of “rich of”\n40 some HBV serotypes have a 183 residue length\n75 notably\n88 delete “become”\n91 are the Na+ concentrations in units of M or mM?\n221 “AFM morphology” or “atomic force microscopy (AFM)”\n235, 237, etc there are no sixfold axes. There are quasi-sixfold vertices\n298 define Q-tensor\n304 provide units for “100”. Is it Å, bp?\n312 provide units for ladder length (Å or bp)\n337 units", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to Reviewer's Questions and list of changes\nReviewer #1 (Remarks to the Author):\nThe authors have done an incredible job with addressing all comments from the\nprevious round of review. I recommend publication in its present form.\nReply:\nWe thank the reviewer for the kind comments and for recommending its publication.\nReviewer #2 (Remarks to the Author):\nI am satisfied with the extensive responses of the authors to the queries and comments\nof all three reviewers, and with the substantial revision of the manuscript that includes\na discussion of additional studies that makes it an outstanding one.\nReply:\nWe thank the reviewer for the kind comments and the positive assessment.\nReviewer #3 (Remarks to the Author):\nThis revised manuscript addresses this reviewer’s questions. With this revised ms, I\nbetter understand the effect of phase separation on polymerase location, the molecular\ninteraction between CTDs and RNA, and the effect of CTDs on RNA mobility. The\nagreement between computation and experimental observation is convincing, notably\nthe asymmetric reconstruction in reference 66, and will lead to a far better\nunderstanding of the process of in-capsid reverse transcription peculiar to HBV. This\nmechanism of RNA packaging is also likely relevant to many other small RNA viruses.\nReply:\nWe thank the reviewer for the careful review and kind comments on this manuscript.\nThere are several minor typos and clarifications to address:\n15 “rich in” instead of “rich of”\n40 some HBV serotypes have a 183 residue length\n75 notably\n88 delete “become”\n91 are the Na+ concentrations in units of M or mM?\n221 “AFM morphology” or “atomic force microscopy (AFM)”\n235, 237, etc there are no sixfold axes. There are quasi-sixfold vertices\n298 define Q-tensor\n304 provide units for “100”. Is it Å, bp?\n312 provide units for ladder length (Å or bp)\n337 units\nReply and corresponding changes:\nWe thank the reviewer for suggesting the changes and for pointing out the typos. In\nthis revised manuscript, we made the suggested modifications and corrected the\nrelated typos.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReply:\nWe thank the reviewer for the carefully reviewing of this manuscript."}]

Decision letter