Cryogenic electron tomography reveals herpesvirus capsid assembly intermediates inside the cell nucleus

Domain: chemistry
Published: 2026-02-25
Editorial outcome: minor_revision

Abstract
Herpesviruses encapsulate their double-stranded DNA (dsDNA) genomes within an icosahedral nucleocapsid formed in the infected cell nucleus. Four biochemically purified nucleocapsids have been characterized, but their roles in herpesvirus replication remain controversial. The status of the capsid vertex-specific component (CVSC), essential for capsid stability and dsDNA packaging and retention, is also unclear. By integrating cryogenic focused ion beam milling with electron tomography and subtomogram averaging, we derive atomic models for all protein components, including the CVSC, across different herpesvirus capsid types within infected cell nuclei. Focused classification of pentonal vertex densities reveal differences in CVSC occupancy between genome-filled capsids and capsids lacking dsDNA, highlight structural heterogeneity and providing insight into distinct capsid assembly stages in situ. These intra-nuclear findings redefine the maturation model of herpesvirus capsid assembly, advancing the understanding of herpesvirus replication, and demonstrate the effectiveness of in situ electron imaging by studying virus assembly within host cells.

Introduction
The Herpesviridae encompass a large family of viruses comprised of alpha-, beta- and gamma subgenera that infect humans and most animal species with wide ranging health and economic consequences 1, 2, 3, 4, 5, 6, 7, 8. Varicella-zoster virus (VZV), a human alphaherpesvirus, causes varicella (chicken pox) as the primary infection, and zoster (shingles) after reactivation from latency in sensory ganglia1, and can be life threatening in immunocompromised patients 9, 10, 11, 12, 13, 14, 15. VZV vaccines have reduced morbidity substantially 16, 17, 18 and antiviral drugs are effective but neither prevent VZV latency in sensory neurons 19, 20 or post-herpetic neuralgia after zoster 21, 22, and antiviral resistance can emerge 23, 24. The other two human alphaherpesviruses, herpes simplex virus (HSV) 1 and 2 (HSVS-2), also create a significant disease burden 25, 26. HSV vaccines are not available and HSV persists in neurons despite antiviral treatment 8. Vulnerable populations need interventions for these viruses and against the betaherpesvirus, human cytomegalovirus 4, and the Epstein-Barr and Kaposi’s sarcoma gammaherpesviruses, which cause a range of cancers 2, 3.

Herpesviruses are classified by their distinct 4-layer morphology composed of a double stranded DNA (dsDNA) core, capsid, tegument, and envelope 27. These complex entities form through a convoluted morphogenesis process starting inside the infected cell nucleus. The dsDNA genome is packaged into newly synthesized capsids that exit the nucleus across nuclear membranes into the cytoplasm, where they traffic to sites of secondary envelopment, completing acquisition of tegument proteins and the lipid envelope containing virus glycoproteins. The nascent enveloped particles are transported in vesicular structures to the plasma membrane and released into the extracellular milieu. The glycoproteins protruding from the lipid membrane then mediate virus entry into neighboring cells and tegument proteins support the intracellular transport of capsids to nuclear pores where the dsDNA genome is injected; additional tegument proteins undergo nuclear translocation to initiate transcription factor functions. Since the herpesvirus capsid structure is essential for virus genome preservation during infection, high-resolution information about the progression of capsid assembly holds great potential to identify targets for next generation pharmaceutical interventions and vaccines.

Herpesvirus capsids are ~125nm in diameter and share a common icosahedral symmetry with a T=16 geometry made up of 60 asymmetric units. The capsid assembles in the nucleus from 161 capsomeres, composed of 150 hexons and 11 pentons that form an icosahedron with 12 vertices, including a portal vertex for the packaging of the dsDNA genome and its release through the host cell nuclear pore in newly infected cells. Capsids undergo several transformative stages starting with immature pro-capsids, which are thought to form as capsomeres coalesce with the scaffold protein to produce segments and subsequently spherical capsids 28, 29. Maturation of pro-capsids occurs as the scaffold protein is proteolytically digested by its companion protease, triggering a conformational change from a more spherical shape to their final icosahedral morphology, with 20 flat triangular faces and 30 edges. Mature capsids have been conventionally classified as A-, B-, and C-capsids, as they differ in their buoyant densities and morphologies by thin section electron microscopy (EM) 30. The cavities of C-capsids contain packaged dsDNA genomes, whereas B-capsids have persistent scaffold protein and its associated protease, and A-capsids are described as empty. While A- and B-capsids have been presumed to be dead-end assembly products 29, an alternative hypothesis is that one or both might represent assembly intermediates with the potential to encapsidate dsDNA genomes 31. Capsid purification can induce artefacts, such as loss of genomic DNA by disruption of the portal vertex cap, creating empty A-capsids from C-capsids 32. B-capsids accumulated when unit length genome formation was blocked by drug inhibition of the genome-transferase complex 31, suggesting that scaffold protein removal is linked to dsDNA insertion, and capsids with scaffold protein in the cavity might be capsid intermediates rather than representing a failure of genome encapsidation.

The proteins that assemble to create a mature herpesvirus capsid have orthologues in all Herpesviridae. These include the major capsid protein (MCP), the small capsomere interacting protein (SCP), and the capsid triplex proteins 1 (TRX1) and 2 (TRX2). The multimeric subunits formed by these proteins to produce fully mature capsids have been described at near atomic resolution for all three subfamilies of herpesviruses 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43. Briefly, the MCP forms the capsomeres of hexons and pentons of the icosahedral capsid. Six copies of the SCP decorate the tops of the hexons. Heterotrimeric triplexes (TRX1 monomer and TRX2 dimer) occupy the canyons between the capsomere towers and cover up the holes in the MCP floor to stabilize the capsid for dsDNA genome containment 33, 44. At each of the 12 capsid vertices, five copies of the capsid vertex specific component (CVSC) bind to triplexes adjacent to the pentons and the portal vertex 33. In HSV, the CVSC is a heteropentamer composed of orthologues shared with VZV; the capsid vertex-specific component 1 (CVC1) monomer, two copies of CVC2, and two copies of the protein designated VP1/2. CVSC binding has been associated with reinforcing capsid stability to withstand the very high packaging pressure of the dsDNA genome 45, 46, 47. At the portal vertex, a dodecamer (VZV ORF54; HSV-1 pUL6), replaces a penton with a portal and tentacle-like helices that ring the DNA translocation channel, and an ATP-driven terminase is recruited to cleave the herpesvirus dsDNA genome for packaging 29, 34, 35, 36, 38, 45, 48, 49, 50. The CVSC is expected to form part of the portal cap that retains the dsDNA genome in the capsid 29, 34, 35, 36, 38, 45, 48, 49, 50. The CVSC heteropentamer has also been called the capsid-associated tegument complex because its subunits include the large tegument protein (VP1/2), VZV ORF22 and HSV pUL36 51; in HSV and PRV, these orthologues have been identified as inner tegument proteins that function in microtubule-mediated capsid transport to cell nuclei 52, 53. The CVSC of CMV, a betaherpesvirus, has a non-conserved tegument subunit, pp150 42, 54, while gammaherpesvirus CVSCs are thought to resemble those of the alphaherpesviruses 51.

Critical information about the structural composition of herpesvirus capsids was gained using cryogenic-electron tomography (cryo-ETE) of purified particles but resolution was limited 55, 56, 57, 58, 59, 60, 61, 62, 63. The adaptation of single particle cryo-EM enabled capsid structures of most human herpesviruses to be determined at better than 4Å resolution 33, 35, 36, 37, 38, 39, 40, 41, 47. However, knowledge about capsid assembly as it occurs in situ within the infected cell nucleus remains limited. The CAVSC has been proposed to associate with the capsid inside the infected cell nucleus, but the structural organizations of their individual components have not been visualized directly in the nucleus of herpesvirus infected cells 64, 65. Whether the assembly of the VP1/2 subunit of the CVSC takes place in the nucleus or cytoplasm is unclear 64, 66, 67. Furthermore, despite previous high-resolution structures for VZV capsids, the CVSC and the portal vertex were not resolved in these studies 39, 41. Recent work with purified capsids partially resolved the CVSC and the portal vertex 68. To better understand capsid assembly and the relationship between capsids conventionally classified as A- and B-capsid types compared to the dsDNA containing C-capsids, we sought subnanometer resolution imaging of capsids within VZV infected cells. Imaging inside mammalian cells is challenging because cell thickness limits the penetration of the electron beam and reduces the image contrast due to inelastic scattering and multiple electron scattering 69. Even at high voltages (1MV), the electron beam can only penetrate 1µm; the nuclei of typical mammalian cells range in size from 5 to 10µm. To overcome this limitation, recent advances in cryogenic focused ion beam and scanning electron microscopy (cryo-FIB/SEM) have been introduced to mill away most of the thick and vitrified cell, leaving a thin lamella of 100 to 400 nm in thickness. When integrated with cryo-ET using a 300 keV electron microscope, 3D volumes can be produced at near-atomic resolution by imaging at different angles, cryo-ET reconstruction, and subtomogram averaging (STA) 70. We applied this approach to visualize VZV particles within infected cells in a frozen, hydrated state, without the use of contrast agents, and determined the structures of their component proteins.

Our findings reveal that the CVSC heteropentamer assembles on capsids in the nucleus of infected cells and that the definition of the asymmetric unit of the icosahedral VZV capsid should be expanded to incorporate the CVSC interactions with neighboring MCPs and triplexes. Focused classification, including of the portal vertex, indicates that genome-filled C-capsids have full CVSC occupancy, defined as the maximum of five heteropentamers. Among capsids lacking dsDNA, empty A-capsids are not detected within the infected cell nucleus whereas capsid assembly intermediates (CAIs) could be distinguished by differences in the number of CVSCs found at the vertices of the icosahedron, which correlate with levels of scaffold protein in the capsid cavity, suggesting a continuum along the assembly pathway. Notably, CAIs would be grouped together as B-capsids based on scaffold protein alone. These observations provide evidence that CVSC acquisition can serve as a temporal marker for the progression of intra-nuclear herpesvirus capsid assembly in situ, consistent with its biological importance for ensuring the stability of genome-containing capsids during nuclear egress and formation of complete virus particles in the cytoplasm.

Results
VZV morphogenesis visualized within infected cells in situ by cryo-FIB-SEM and cryo-ET.
To visualize herpesvirus morphogenesis in 3D-space, a workflow was developed to distribute VZV infected cells evenly on EM grids suitable for cryo-FIB milling. First, because VZV does not yield high titer cell-free virus particles 1, it was critical to optimize the passage of infected cells onto uninfected cells to initiate infection prior to vitrification. Infection was monitored by the fluorescence RFP signal produced by MeWo cells infected with a recombinant VZV (pOka-TK-RFP 71) expressing a thymidine kinase (TK) tagged with a monomeric red fluorescence protein (RFP; Fig. 1a [created in BioRender https://BioRender.com/c2ose1j] and Fig. 1b); the pOka-TK-RFP maintains the characteristics of the bacterial artificial chromosome derived WT pOka 71, 72, 73, 74. Second, to overcome the propensity of VZV infected cells to adhere to the grid bars when seeded on SiO EM grids, islands of extracellular matrix were micropatterned within the grid squares (Fig. 1b). With this technique, infected cells were positioned centrally within the grid squares after vitrification and ideal for cryo-FIB milling to produce lamella suitable for cryo-ET (Fig. 1c and 1d). Tomograms of cryo-FIB milled cells revealed the complex cellular environment in VZV infected cells (Fig. 1e; Suppl. Fig. 1 to 7; Suppl. Table 1 and 2). Cellular features were readily visible in medium magnification (16.9 to 39.1Å/pixel) montage maps used to facilitate cryo-ET tilt series data collection from cells thinned to a target thickness of ~175nm; the nuclear envelope with visible nuclear pores demarcated the intranuclear and cytoplasmic compartments (Fig. 1d; Suppl. Fig. 1a, 4a to 7a). A plethora of vesicular structures, mitochondria, and, within infected cells, VZV capsids and complete virus particles, was visible, facilitating the localization of regions of interest in a tilt series collection. The higher resolution tomographic reconstructions yielded substantial contrast enabling machine learning-based segmentation to identify ribosomes, microtubules, microfilaments, complex vesicular structures, and assembled capsids in the nucleus and sites of virus particle morphogenesis in the cytoplasm (Suppl. Fig. 7c).

Several critical steps in the herpesvirus replication cycle were captured within cryo-ET tomograms generated from cryo-FIB milled cells infected with VZV (Fig. 1e; Suppl. Fig. 1 to 8; Suppl. Movie 1 to 6). These included capsids docked at the nuclear pore (Fig. 1ei; Suppl. Fig. 1; Suppl. Movie 1), newly synthesized capsids in the nucleus (Fig. 1eii; Suppl. Fig. 2 and 3; Suppl. Movie 2), nuclear egress (Fig. 1eiii; Suppl. Fig. 4; Suppl. Movie 3), secondary envelopment (Fig. 1eiv; Suppl. Fig. 5; Suppl. Movie 4), VZV particle morphogenesis (Fig. 1ev; Suppl. Fig. 6 and 7; Suppl. Movie 5), and VZV particles bound to the extracellular plasma membrane surface (Fig. 1evi; Suppl. Fig. 8; Suppl. Movie 6), a typical feature of this cell associated virus 1. Within the infected cell nucleus, pro-capsids and A-, B-, and C-capsids based on conventional morphological features, were distinguishable in 3D-space at this resolution. Procapsids were spherical and contained a solid density of scaffold protein (Suppl. Fig. 2 and 3); capsids apparently devoid of scaffold protein or dsDNA (conventional A-capsids) were identified (Suppl. Fig. 3b); B-capsids were observed and defined by the presence of scaffold protein (Fig. 1eii; Suppl. Fig. 3c); and C-capsids were distinguishable by their cavities filled with dsDNA (Fig. 1eiii, 2A and 2B; Suppl. Fig. 4 to 8). Procapsid assembly precursors were identified within infected cell nuclei, appearing as angular segments of capsomeres associated with scaffold protein with a curvature similar to the procapsids (Suppl. Fig. 2). Importantly, high-resolution detail was evident by the visualization of individual capsid capsomeres (Fig. 1eii, Suppl. Fig. 3; Suppl. Movie 2), individual nuclear egress complexes (Fig. 1eiii, Suppl. Fig. 4; Suppl. Movie 3), and individual glycoproteins on enveloped VZV particles in the cytoplasm or bound to the plasma membrane (Fig. 1eiv and vi; Suppl. Fig. 5, 8 and 9; Suppl. Movie 5 and 6). Partially assembled virus particles were observed within cytoplasmic sites of VZV secondary envelopment and morphogenesis, including sites of tegument protein accumulation, based on the electron dense protein surrounding the capsids within the reconstructed tomograms (Fig. 1eiv and 1ev; Suppl. Fig. 5 and 6). For example, a VZV light particle, a glycoprotein-studded envelope containing tegument proteins but without a capsid, and a complete particle were observed in a vesicular structure, and in a neighboring vesicular structure, an elongated VZV particle contained a C-capsid and the accumulation of tegument protein associated with a membranous structure (Suppl. Fig. 7b). The virus envelope within these vesicular structures was detected by machine learning-based segmentation (Suppl. Fig. 7c). Collectively, these direct visualizations reveal details of the VZV replication cycle previously unobserved in its native hydrated state inside infected cells, particularly for nuclear egress, tegument acquisition during secondary envelopment, and virus glycoprotein interactions with both intra- and extracellular membranes.

Subnanometer reconstruction of VZV capsid proteins in infected cells.
From 194 tilt series, 1,084 capsids were identified and classified based on classical morphological features, the presence or absence of scaffold protein or the virus genome in the capsid cavity. A visual inspection of the image contrast in the cavity of capsids without the dsDNA genome revealed a distribution of scaffold protein density, filled to sparse, suggesting that completely empty A-capsids are absent or very rare within infected cell nuclei (Suppl. Fig. 10). Residual scaffold protein was limited enough in some capsids to be obscured by conventional EM techniques due to the plane of the cut in ultrathin sections in previous studies. The cellular distribution of the 1,084 VZV capsids was primarily localized inside the nucleus (n=980) with only 9.3% detected in the cytoplasm either as naked capsids, without tegument or envelope proteins (n=42), or as complete enveloped VZV particles (n=59) (Suppl. Table 3). The capsid distribution was typical for peak particle synthesis of VZV infected cells, which aligns with thymidine kinase (TK) expression, an early gene, as determined by the red fluorescence signal produced from the recombinant pOka-TK-RFP virus.

An initial 13Å cryo-ET map of the VZV capsid was generated by subtomogram averaging (STA) of 317 capsids (Fig. 2; Suppl. Fig. 11), containing variable quantities of scaffold protein or packaged DNA. This icosahedral symmetry-imposed map clearly shows the T=16 icosahedron, composed of 60 asymmetric units. Each asymmetric unit consists of a single pentonal MCP subunit (PP), one peripentonal hexon (P), one central hexon (C), and half of an edge hexon (E), along with interdigitating triplexes (Ta, Tb, Tc, Td, and Te) (Fig. 2c and 2d). Cryo-ET maps were produced for the vertex around the 5-fold axis (8.3Å) and the 3-fold symmetry axis (8.4Å) by subsequent symmetry expansion and focused refinement of the 317 capsids. (Fig. 2e and 2f; Suppl. Fig. 11). The 8.3Å vertex map had sufficient resolution to perform segmentation of individual proteins for the pentonal MCP (PP), six MCPs (P1-6) for the peripentonal hexon and the associated small capsomere interacting protein (SCP), triplex proteins TRX1 (monomer) and TRX2 (dimer) that form the α β heterotrimers of Ta and Tc, and the three proteins, CVC1 (ORF43 monomer), CVC2 (ORF34 dimer), and ORF22 dimer, that assemble to form the capsid vertex specific component (CVSC) heteropentamer (Fig. 3 and 4; Suppl. Table 4 and 5). Similar to the vertex map, individual proteins were segmented for the 8.4Å 3-fold symmetry axis cryo-ET map for the six MCPs (C1-6) for the central hexon and associated SCP in addition to the TRX1 and TRX2 proteins of the Te triplex (Suppl. Fig. 12; Suppl. Table 4). The densities in both 3- and 5-fold axes of symmetry were segmented using the structure derived from single particle cryo-EM of VZV capsids (7BW6)39. All of the features for the pentonal, peripentonal hexon, and central hexon MCPs were apparent, including the tower, consisting of the crown and buttress regions (Fig. 3a to c; Suppl. Fig. 12), and the HK97 phage capsid protein-like fold facing the interior chamber of the capsid (Fig. 3d), which is evolutionary conserved for herpesviruses and dsDNA bacteriophages 75. The resolution of these regions in the cryo-ET map was supported by the ability to observe densities for alpha helices in each of the MCP, TRX1 and TRX2 proteins and corresponding Map-Q scores (Fig. 3c; Suppl. Fig. 12c and e; Suppl. Fig. 13; Suppl. Movie 7). The classical HK97 phage capsid protein fold of the MCP was distinguishable with the long alpha helix (helix 4) interdigitating at the pseudo 3-fold axis of symmetry between the penton and neighboring peripentonal hexon MCPs (Fig. 3d). These data provide subnanometer resolution structural details of the MCP, SCP, TRX1 and TRX2 within the nucleus of a herpesvirus-infected cell, representing an unprecedented view of capsid architecture in situ.

The CVSC heteropentamer assembles as part of the capsid asymmetric unit in infected cell nuclei.
The CVSC status as a capsid component has been difficult to assess due to its propensity to disassociate from vertices during herpesvirus capsid purification, including VZV 45, 76,39, 41. In contrast, the CVSC density was apparent at all 12 vertices of the capsid structure in infected cell nuclei (Fig. 2c). Furthermore, the 8.3Å cryo-ET map of the VZV capsid vertex was of sufficient resolution to extract densities for the CVSC, demonstrating that the heteropentamer is a capsid component acquired independently of the full tegument layer of the virus particle (Fig.s. 2e, 4a and 4c). An AlphaFold model of the CVSC structure oriented the head group for each CVC2 (ORF34) molecule at a downward angle compared to the extracted CVSC cryo-ET map density (Suppl. Fig. 14a and 14b). The CVC2 head group AlphaFold models were fitted into the cryo-EM density with the aid of molecular dynamics flexible fitting (Suppl. Fig. 14c; Suppl. Movie 8), enabling segmentation of the cryo-EM density into the five individual subunits of the CVSC heteropentamer (Fig. 4a to 4c). This revealed that the CVC1 subunit (ORF43 monomer), which has a limited interaction with TRX1 from triplex Te, primarily associates with all three Ta triplex subunits along with MCP P1 and P2 of the peripentonal hexon from the neighboring asymmetric unit (Fig. 4b, 4d and 4e). A helical bundle forms between the CVC1 subunit, the two ORF22 proteins and the two CVC2 proteins, enabling the CVC2 head group to interact with the peripentonal hexon facing side of the pentonal MCP crown (Fig. 4a). Since almost all capsids analyzed were intra-nuclear, these observations definitively demonstrate that CVSC assembly at the capsid vertices occurs inside the nucleus. Furthermore, these findings enabled us to redefine the asymmetric unit of the mature C-capsid to be composed of six C hexon subunits (MCP and SCP), three E hexon subunits, and a pair of three peripentonal hexon subunits from neighboring hexons, one pentonal MCP, five interdigitating triplexes, and a single CVSC heteropentamer (Fig. 2d and 4b; Suppl. Fig. 15a). This arrangement differs from previous models of alphaherpesvirus asymmetric units, where the interaction between CVC1 with triplexes Ta and Tc and MCP P1 and P2 were not used to define the asymmetric unit, proteins were missing, or proteins from a neighboring asymmetric unit were included (Suppl. Fig. 15).

CVSC acquisition redefines capsid assembly intermediates formed during herpesvirus capsid assembly.
Given uncertainties about the stages of capsid assembly based on studies of purified capsids 45, 76, 77 31, 78, we investigated the distribution of CVSC heteropentamers at vertices of capsids in infected cells to determine whether the CVSC occupancy could be used to differentiate capsid types and achieve a more precise characterization for the stages of capsid assembly (Fig. 5). Uniform Manifold Approximation and Project (UMAP) is a dimension reduction technique that can be used for the visualization and categorization of complex data 79, including structural heterogeneity in single particle Cryo-EM 80, 81. Application of this unsupervised computational approach (Fig. 5a) using three-axis orthogonal projection in latent space classified the 317 capsids that were used to generate the 8.3Å vertex cryo-ET map (Fig. 2). Each capsid image was aligned to its symmetry axes, and icosahedral symmetry was applied to individual particles before the projections were generated to compensate for the missing wedge effect intrinsic to cryo-ET data 82. Three clusters were differentiated in latent space, revealing limited versus (i) extensive (ii) scaffold protein in the capsid cavity, and those with packaged DNA (iii) corresponding to C-capsids, as viewed along the 2-, 3-, and 5-fold axes of symmetry and the STA for each class (Fig. 5a to 5c). The detection of limited quantities of scaffold protein for class i capsids enabled the differentiation from conventionally designated B-capsids. The absence of any density inside the capsid cavity, previously established for classical empty A-capsids based on conventional negative stained images 30, was not detected within infected cell nuclei. Residual scaffold protein was visible in the native structures preserved by vitrification performed in this study (Suppl. Fig. 10). In addition to capsid cavity content differences, the three UMAP capsid clusters were found to differ in CVSC occupancy at the vertices (Fig. 5d to 5f). For each of the 317 capsids, sub-particles were extracted from the 12 vertices and aligned to the same C5 symmetry axis. These particles were initially subject to binary reference free focused classification, resulting in two classes reflecting vertices without or with CVSC occupancy (Fig. 5d and 5e). The CVSC was either absent or present, with the arms and lobes of the CVSC clearly visible in the C5 symmetry STA maps (Fig. 5d). Capsid vertices were then classified into those without and with the CVSC and by their UMAP cluster, i to iii (Fig. 5e). As expected, CVSC occupancy was highest on C-capsids in infected cells, as has been reported for purified capsids 33, 37, 40, 42, 47, and because they are considered complete and ready for egress from the nucleus for final tegumentation and envelopment in the cytoplasm 45. Persistent scaffold protein is the conventional marker for B-capsids by EM, resulting in greater buoyant density than empty A-capsids 30. Notably, capsids differed in CVSC occupancy depending on whether scaffold protein was limited or extensive within the capsid cavity, with the former having greater CVSC occupancy than the latter (Fig. 5b to 5f). Critically, for the 12 vertices of individual capsids, CVSC occupancy at each vertex increased with decreasing levels of scaffold protein density (Fig. 5f). Since C-capsids have the highest CVSC occupancy, these findings indicate that capsids with relatively more CVSC occupancy and less scaffold protein are capsid assembly intermediates (CAIs) further along the pathway of capsid maturation than those with less CVSC occupancy and more residual scaffold protein.

To further scrutinize the CVSC on capsids in infected cells, vertex particles were subjected to C5 symmetry expansion, generating five sub-particles for the asymmetric orientations. Additional focused classification using a mask covering a single CVSC site produced two classes of CVSC occupancy at each individual location, those with or without the heteropentamer. This procedure established the frequency of CVSC heteropentamers, producing vertex sub-particles with either zero copies, 1 to 5 copies, or 2 and 3 copies of non-consecutive CVSC distribution, which were used for STA without symmetry imposed (Fig. 5g and 5h). Each capsid has a potential maximum of 60 copies of the CVSC heteropentamer, as there are 12 capsid vertices and 5 CVSC per vertex, including the portal vertex 29, 34, 35, 36, 38, 45, 48, 49, 50. For the 317 capsids in the present study, there were 3,804 vertices. Frequencies ranged from 290 vertices without a CVSC to 1,198 vertices with a full complement of five copies of the CVSC (Fig. 5g). Additionally, alternative distributions of the CVSC were observed at vertices where 2 or 3 copies of the heteropentamer were not adjacent to one another, indicating a non-cooperative attachment of the heteropentamer to these capsid vertices (Fig. 5h). In contrast to purified VZV C-capsids 41, intranuclear C-capsids in infected cells had almost complete vertex occupancy (52; SEM = 0.861), demonstrating their maturity (Fig. 5i). CAIs with limited scaffold protein had greater CVSC occupancy (39; SEM = 0.859) compared to CAIs with extensive scaffold protein (32; SEM = 0.753), providing additional evidence that the former represent a stage closer to DNA encapsidation while the latter are at an earlier stage in nucleocapsid synthesis. Taken together, these subnanometer resolution map observations strongly support the concept that CVSC occupancy can be used as an indicator for the progression of herpesvirus capsid assembly in situ and identifies capsids with varying levels of residual scaffold protein as CAIs along a continuum, having the potential for genome packaging rather than failed assembly dead ends.

Portal vertex architecture differences between CAIs and C-capsids.
In contrast to previous high-resolution single particle cryo-EM structures of biochemically purified herpesvirus capsids, the portal vertex for VZV capsids could not be reconstructed in previous reports 39, 41 but has been achieved by Cao et al 68; this was not performed in infected cells in situ. To address this issue, we developed a RELION pipeline to reconstruct the VZV portal vertex, which was successfully identified for CAIs without the packaged dsDNA genome, or C-capsids within a subset of cryo-FIB/ET tomograms using STA and 3D classification of capsid vertices (Fig. 6; Suppl. Fig. 16 to 18; Suppl. Table 4). An initial cryo-ET map (23.6Å) for the portal vertex revealed a density within the capsid cavity reminiscent of the herpesvirus portal 34, 38, 48, 49, 50, and an absence of density for penton proteins compared to the 8.3Å cryo-ET map of the capsid vertex (Fig. 3 to 5; Suppl. Fig. 17a). Further STA of the portal density generated a 22.9Å cryo-ET map of the VZV portal with C12 symmetry (Suppl. Fig. 17b). A molecular model based on an AlphaFold prediction of an individual VZV portal protein (pORF54; 769aa) and modelling of 12 subunits based on the entire HSV-1 portal was placed into the 22.9Å cryo-ET map of the VZV portal (Suppl. Fig. 17c and 17d). Additional densities in the portal cryo-ET map were attributed to poor modelling by AlphaFold of amino acids 1 to 64 and 656 to 769, which were disordered and removed from the model. Architectural differences between the portal vertices of CAIs and C-capsids were visible when cryo-ET map reconstructions were performed based on capsid type (Fig. 6; Suppl. Fig. 18). The portal cap was absent for CAIs compared to C-capsids, which was apparent when the entire capsid was reconstructed (Suppl. Fig. 18). The portal structure of C-capsids was confirmed as the portal density in the capsid cavity was only present at a single vertex (Suppl. Fig. 18). Further capsid-type specific architectural differences were evident from portal vertex cryo-ET maps and fitment of the molecular models for the peripentonal hexons, Ta and Tc triplexes, CVC1, ORF22 helices, and the portal (Fig. 6). As expected, densities for the pentonal MCP and alpha helices for the CVSC were absent and a channel for dsDNA translocation was visible through the center for the CAIs capsid portal vertex cryo-ET map (Fig. 6b and 6c). In contrast, the C-capsid cryo-ET map had a portal cap with densities for the CVSC, and which filled the portal vertex channel (Fig. 6e and 6f), presumably, tentacle helices and the dsDNA genome terminus as previously described for HSV-1 38. As expected from the VZV vertex focused classification analysis (Fig. 5g to 5i), occupancy of the CVSC at the non-portal vertices of the CAI was incomplete whereas each vertex for the C-capsid exhibited a full complement of the CVSC (Fig. 6a and 6d; Suppl. Fig. 16). These findings further support the CVSC as a defining element and thus a marker of herpesvirus capsid assembly and maturity inside the cell nucleus during replication.

Methods
Reagents, consumables, and resources. All reagents, consumables, and resources, including supplier information where applicable, are provided in Suppl. Table 6.

Cells lines. MeWo cells (HTB-65; ATCC) were propagated in minimal essential medium (Corning Cellgro) supplemented with 10% fetal bovine serum (FBS; Invitrogen), nonessential amino acids (100 μM; Corning Cellgro), antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml; Invitrogen) and the antifungal agent amphotericin B (250 µg/ml; Invitrogen) at 37°C in a humidified atmosphere with 5% CO2.

Viruses. The VZV parental Oka strain was originally cloned into a bacterial artificial chromosome (BAC) and designated pPOKA-BAC-DX 104. Recombinant viruses pOka-TK-GFP (pOka-rTK) and pOka-TK-RFP were generated in a previous study 71, 105.

ECM micropatterning of SiO EM grids. To position MeWo cells centrally within EM grid squares, 300 mesh gold grids coated with a thin perforated layer of SiO (Quantifoil R2/2) were subjected to micropatterning following the procedure available at protocols.io (https://www.protocols.io/view/micro-patterning-em-grids-for-cryo-electron-tomogra-rm7vz3wqrgx1/v1). Briefly, EM grids were exposed to atmospheric plasma, incubated in poly-l-lysine (0.01%; Sigma) and subsequent incubations with HEPES (0.1M; Sigma) + mPEGSuccinimidyl Valerate (PEG-SVA MW 5,000; 100 mg/ml; Laysan Bio Inc.) then PLPP gel (1:6 dilution in H2O; Alveole). The coated grids were exposed to UV light according to a digitally generated micropattern (30x30µm or 33x33µm to match the 300 mesh spacing) that covered the EM grid square using a Primo photo-micropatterning system (Alveole) to selectively degrade the PEG coating to remove the hydrophobic antifouling layer. An extracellular matrix (ECM) surrogate, Gelatin Oregon Green 488 (1 mg/ml; Invitrogen), was applied to the grids prior to cell seeding.

Vitrification of VZV Infected MeWo cells on ECM micropatterned grids. Cell free VZV cannot be purified to high titers making direct infection of cells grown on EM grids with high MOIs needed for productive infection impractical. Thus, infected cells were seeded directly onto ECM micropatterned EM grids. VZV (pOKA-TK-RFP) infected MeWo cells were passaged onto freshly seeded uninfected MeWo cells 24 hours prior to the day of seeding. On the day of seeding, the VZV infected MeWo cells were then passaged onto uninfected MeWo cells and incubated for 8 hours. The infected cells were then trypsinized, centrifuged at 400xg for 5 minutes then resuspended in maintenance media to yield one cell per grid square; determined empirically on the day of seeding. Infected and uninfected cell suspensions were applied to the ECM micropatterned grids in 35mm glass bottomed dishes (MatTech P35G-1.5-14-C). After 2 hours incubation at 37°C in a humidified atmosphere with 5% CO2, 3 ml of maintenance media was added and incubated for an additional 16 hours. Grids were removed from the maintenance media and plunge frozen into liquid ethane using an EM GP2 (Leica) with humidity at 99% and a blot time of 9 seconds.

Cryo-FIB/SEM of vitrified VZV infected MeWo cells. An Aquilos 2 cryo-FIB/SEM microscope (ThermoFisher) was used to generate lamella from VZV infected cells on micropatterned grids. Prior to milling, the grids were platinum coated. Microexpansion joints of 1µm width and 4µm between the milled area and the microexpansion joint were used to relieve tension on the final lamellae106. The primary milling step was performed at 1nA and 5µm height and 15µm width with two 1µm microexpansion joints milled either side at 4 µm from the lamella milling site. Successive milling steps of 3µm (300pA), 1µm (100pA), 500nm (50pA), and a final polishing step of 175nm (30pA) were performed to generate lamella with a target thickness of 175nm. Lamella were produced for 6 to 10 cells per grid.

Cryo-ET of whole or cryo-FIB/SEM milled VZV infected MeWo cells. Tilt series were either collected at the periphery of whole vitrified VZV infected MeWo cells on EM grids, using a 200kV Talos Arctica (FEI) or at cryo-FIB/SEM generated lamella, using a 300kV Titan Krios (FEI) controlled by SerialEM 107 to automate the data collection procedure using the parameters outlined in Suppl. Table 1. To identify areas of interest for tilt series collection, medium magnification (4,800-11,500x) montages were generated, and imaging sites were determined empirically by eye. Due to the large size of herpesvirus capsids, a 3.44Å/pixel size was chosen to accommodate areas where high frequencies of VZV capsids were present in the nucleus. A total of 399 tilt series were collected from cryo-FIB/SEM milled lamella across five, 3-day sessions on 300kV Titan Krios microscopes. A total of 194 tomograms were generated for VZV capsid quantification, analysis, and cryo-ET map reconstructions.

Cryo-ET image processing. Motion correction, damage compensation, CTF estimation, and tilt series generation was performed using Warp 108. Tomogram reconstruction for visualization was performed using the IMOD tomography GUI Etomo 109. Tomogram processing and associated movie files were generated using 3dmod 110. Segmentation of tomograms was performed using Dragonfly software (Version 2022.2 for [Windows]. Comet Technologies Canada Inc., Montreal, Canada; software available at https://www.theobjects.com/dragonfly). Movies were generated using FFmpeg (FFmpeg Developers. (2016). ffmpeg tool (Version be1d324) available from http://ffmpeg.org/).

Subnanometer resolution cryo-ET map reconstruction of the VZV capsid vertex. To reconstruct the VZV capsid vertex and 3-fold axis of symmetry a WARP-EMAN2 data processing strategy was used (Suppl. Fig. 11). Tilt series generated by WARP were imported into EMAN2 for tomogram alignment using patch tracking where handedness was checked and CTF measured. The convolutional neural network was employed to select particles (box size 512; n=1,084 capsids) from 194 tomograms and make sets (bin2). An initial model was generated from the sets using e2spt_sgd_new.py (--res=50.0; --niter=30; --shrink=4; --sym=icos) followed by subtomogram-subtilt refinement using e2spt_refine_new.py (--startres=30.0; --sym=icos; --iters=p3,t2,p,t,r,d; --keep=0.95; --tophat localwiener –goldstandard). Classification with icosahedral symmetry was performed on the output to remove poor quality particles that can negatively influence the subtomogram averaging; e2spt_refinemulti_new.py (--nref 3; --maxres 40; --loadali3d; --skipali; --sym icos; --maskalign spt_01/mask.hdf; --niter 20). Further refinement of 317 capsids produced a cryo-ET map of the entire VZV capsids of 13.0Å resolution. To generate a subnanometer resolution cryo-ET map for the VZV capsid vertex, symmetry expansion was performed with the 317 capsids using e2spt_extract.py (--postxf icos,0,0,110; --compressbits 8; --boxsz_unbin 192; --newlabel pent2; --mindist 500; --loadali2d) followed by building sets; e2spt_buildsets.py (--label=pent2; --allparticles; --spliteo). An average of the extracted particles was generated from the set using e2proc3d.py (--average; --averager mean.tomo; --avg_byxf). The average was then refined using e2spt_refine_new.py (--startres=30.0; --sym=c5; --iters=p3,t2,p,t,r,d; --keep=0.8,0.95,0.95; --tophat localwiener; --localrefine; --goldstandard) to achieve a cryo-ET map of the VZV capsid vertex at 8.3Å resolution. A similar procedure was used for the 3-fold axis of symmetry to achieve a cryo-ET map at 8.4Å resolution.

Segmentation of the 3-fold and 5-fold cryo-ET maps. The 3- and 5-fold cryo-ET maps of the VZV capsid were segmented based on the 7BW6 asymmetric unit model of the VZV A-capsid 39 using ChimeraX 111. To segment the CVSC density in the 5-fold axis cryo-ET map, AlphaFold 2 112 was used to model the heteropentamer based on the full-length proteins CVC1 (ORF43; monomer) and CVC2 (ORF34; dimer), and 45 C-terminal amino acids (2724-2768) of VP1/2 (ORF22). Molecular dynamics flexible fitting (MDFF) was performed to fit the two head groups of the CVC2 dimer into the cryo-ET map using NAMD 113 following the procedure outlined in the VMD 114 tutorial. Conversion of the cryo-EM map to an MDFF potential, rigid body docking of the CVSC, initial structure generation, and secondary structure restraint definitions were all performed using VMD. The output files were used as input to run NAMD with the following parameters; step 1 -gscale 0.3, -numsteps 50000; step 2 -gscale 10, -numsteps 100000. A model of the asymmetric unit of the VZV capsid vertex, including the CVSC, was refined using ISOLDE 115. All images and movie files were generated using ChimeraX. Movies were generated using FFmpeg.

Three axis orthogonal projection and uniform manifold approximation projection. The 3D VZV capsid particles were aligned to the symmetrized capsid reference from the EMAN2 icosahedral refinement. The subsequent 2D sub-tilt particles at the corresponding orientation were reconstructed with icosahedral symmetry to obtain symmetrized 3D particles free from missing wedge artifacts. Each particle was projected along three orthogonal axes of thin central slabs (72Å), using only one quarter of the volume closest to the center perpendicular to the corresponding axis. The resulting three 2D image projections per capsid were stacked and flattened into a 1D array that was used as input for uniform manifold approximation projection (UMAP). After manifold embedding, particles on the 2D UMAP space were clustered using a K-means algorithm to produce the individual classes.

Reconstruction of the VZV Portal Vertex. To reconstruct the VZV portal vertex a WARP-IMOD-EMAN2-Relion data processing strategy was used (Suppl. Fig. 16). Tilt series were aligned using IMOD and for the final aligned stack, unbinned, CTF estimation was performed using CTFplotter with the ‘Find astigmatism option’ checked. Tomograms for Relion were generated using relion_tomo_reconstruct_tomogram and used for particle picking of capsids in EMAN2 (n=471). Tomograms and particle coordinates were imported into Relion using Tomo Import. Make pseudo-subtomos was used to make subtomograms (bin 4; 13.76 Å/pixel) using a box size (pixels) of 360 and cropped box size (pixels) of 180. An initial model was generated using 3D initial model and a mask was generated using Dynamo 116 and Mask create. A 3D refinement was performed with 3D auto-refine with an initial low-pass filter of 50 Å and I3 symmetry. Particle reconstruction was performed (bin2; 6.88Å/pixel) with Tomo reconstruct particle with a box size of 440 and cropped box size of 220 with I3 symmetry, which was used to generate a new mask, followed by reconstruction of subtomograms using Make pseudo-subtomos. A 3D refinement was performed with 3D auto-refine with an initial low-pass filter of 30Å and I3 symmetry. The output was used for Tomo frame alignment, particle reconstruction (bin 1; 3.44Å/pixel; box size 440 pixels; I3 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 14Å and I3 symmetry, generating a 12.1Å cryo-ET map of the VZV capsid.

Symmetry expansion was performed using relion_particle_symmetry_expand with --sym I3. This process produces 5 rotations per vertex; redundant _rlnAngleRot values were removed to leave 12 unique vertices per capsid (n=5,652). Vertices beyond the boundaries of the tomograms were removed (n=5,318). The Z height was adjusted to center the vertices in the center of the box using relion_star_handler and --center_Z command. The output was used for particle reconstruction (bin 4; 13.76 Å/pixel; initial box size 120 pixels; cropped box size 60 pixels; C5 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 50Å and C5 symmetry. The output was used for particle reconstruction (bin 2; 6.88 Å/pixel; box size 240 pixels; cropped box size 120 pixels; C5 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 30Å and C5 symmetry. The output was used for particle reconstruction (bin 1; 3.44Å/pixel; box size 240 pixels; C5 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 15Å and C5 symmetry, generating a 10.3Å cryo-ET map of the VZV capsid vertex.

The output was used to generate a cryo-ET capsid vertex map at bin 4 using the above processing scheme then used to perform 3D classification (12 classes; 29 iterations). An initial 60 particles were selected based on the presence of a portal density and the absence of the pentonal major capsid protein then used to generate a cryo-ET portal vertex map at bin 4 using the above processing scheme. A focused cylindrical mask was generated and used for further 3D classification of the VZV capsid vertices yielding a total of 103 portal vertices. A final round of template-based 3D classification yielded a total of 109 portal vertices. The output was used for particle reconstruction (bin 2; 6.88Å/pixel; box size 240 pixels; cropped box size 120 pixels; C5 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 30Å and C5 symmetry, generating a 23.6Å cryo-ET map of the VZV capsid portal vertex. To reconstruct the portal, particle reconstruction (bin 2; 6.88Å/pixel; box size 90 pixels; cropped box size 30 pixels; C12 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 30Å, C12 symmetry, and C1 symmetry relaxation was performed, generating a 22.9Å cryo-ET map of the VZV portal.

To identify portal vertex specific features, the 109 portal vertices were divided into CAI (n=62) and C-capsids (n=47) based on morphological features; either scaffold protein (CAI) or genomic DNA (C-capsids) inside the capsid cavity. Cryo-ET maps for the portal vertices from CAI (23.8Å) and C-capsids (24.3Å) were generated as described above; particle reconstruction (bin 2; 6.88Å/pixel; box size 240 pixels; cropped box size 120 pixels; C5 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 30Å and C5 symmetry. Cryo-ET maps for the entire capsid from CAI (32.6Å) and C-capsids (33.6Å) were generated by first adjusting the Z-height of the particles, then performing particle reconstruction (bin 2; 6.88Å/pixel; box size 512 pixels; cropped box size 256 pixels; I3 symmetry), mask creation, reconstruction of subtomograms, and 3D refinement with an initial low-pass filter of 30Å, C5 symmetry, and C1 symmetry relaxation.

The resolution of all Relion generated cryo-ET maps were confirmed using post-processing with soft masks and assessed by Fourier Shell correlation curves with a cut-off at 0.143 from two independent half-sets of the pseudosubtomograms.

Statistics and Reproducibility. Data were generated from 399 tilt series that were collected from cryo-FIB/SEM milled lamella across five, 3-day, cryo-ET data acquisition sessions. Fig. 1b to 1d are representative images of 35 grids (1b), 265 lamella (1c), and 134 montages (1d). Fig. 2a and 2b are representative images of 73 tomograms containing capsids in the nucleus. Quantitative data (Fig. 5i) were analyzed by two-way ANOVA with Tukey’s multiple comparison test to determine statistical significance using Prism (GraphPad Software).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The work presented here aim to elucidate the protein structure of Herpesvirus via in situ tomography in the aim to propose a\nviral maturation model. The manuscript is well written but suffer from numerous typos, missing scale bars… The science is\nsound and the data interpretation to my knowledge is done well.\nIf the author could respond to some points, this article should be published in Nature communication as this work represents\nthe main benefit of in situ structural biology.\nMajor:\nCould you please define TK in the results as soon as mentioned and explain the rational allowing you to use a tagged\nprotein while aiming for structural determination (aka how you know it won’t disrupt the native structure of interest) or the life\ncycle (the presence of absence of certain forms). Also, could you demonstrate or cite references to demonstrate that the RFP\nvirion as the same IFU and life cycle.\nWhat is the pattern used (size compare the cell size) and shape. As micropatterning affect the cell organisation, could you\nplease explain the rational in the choice of the micropattern with regards the infection?\nCould you please deposit the segmentation, training data, manual segmentation to train the model and model on EMPIAR?\n‘details of the VZV replication cycle previously unobserved’ By no mean I am a virus expert\nhowever this type of sentence should be rephrased to take into account what has been published\nbefore (for example J Virol\n. 2014 Dec 3;89(4):2436–2441. doi: 10.1128/JVI.03215-14)\nThe first result section (VZV morphogenesis visualized within infected cells in situ by cryo-FIB-SEM and cryo-ET) does not\nbring enough metrics, it is descriptive and could be anecdotal. For example, A-capsids not detected: is it because you do not\nhave enough data? As the authors claim to have trained model, could the authors provide some metrics on some relevant\nviral/host state? Alternatively, adjust the claims and include the description in shorter version to the second paragraph to\nexplain how you decide which particle was what and which one have been push to STA. There is also something left\nunexplored which is the A capsids which are not found in the nucleus but in the cytoplasm, again so quantification would\nhelp with significance of this data.\nI found the application of UMAP interesting, and this type of analysis is relevant for in situ cryo ET.\n‘to address this issue, we developed a new RELION pipeline ‘ could you please explain what is new and why this novelty\nwas required?\nminor:\nauthors’ affiliation not in order (1,2, 6…)\n‘3D volumes are produced at near-atomic resolution’ is miss leading if we consider the parameters used in this paper (and\nmany others). I would re-phrase as the near atomic resolution in the STA comes from averaging.\nI think it is good to give success rate for the milling, how the percentage of lamella who have been considered as usable for\ncryo-ET would be amazing, with a short description of the criteria used.\nGive the parameters for the molecular dynamics flexible fitting please.\n‘First, because VZV does not yield high titer cell-free virus particles, it was critical to optimize the passage of infected cells\nonto uninfected cells to initiate infection prior to vitrification.’ Is it a well-known fact that VZV does not produce virion in high\nyield? In this case could you put a reference. If it is in your hand, please indicate.\nFigure 1, no point to add the orange box as it calls for another figures, make sure you mentioned the supp figure in the text\nand that would do. Can not really see the scale bar in panel B and C and not sure we need the microscope bars t the bottom.\nPanel D could do with a scale bar. Panel E, cannot read easily the number at the top of the scale bars, remove and put in\nlegend. Could you also please used different colours for the different event you would use to observe as it is confusing.\nCould you please state where in the cells the tomograms are (nucleus vs cytoplasm) as it seems important for the\ncomprehension of the different viral event. The figure legend can do with way less. Not sure we need panel A to C.\nFigure 2: watch the scale bars please, they are not consistent and place randomly. Panel A also have a scale bar and 3\naxes, and for some reason the contrast is inverted.\nFigure legend 6 is all over the place.\nFigure 6C do you have hypothesis of what are the density which are not in the map on the ‘inner’ part of the portal?", "reviewer_2": "This study employs an integrated cryo-FIB/cryo-ET approach to achieve subnanometer-resolution visualization of\nintranuclear VZV capsid assembly intermediates in-situ within infected cells, overcoming limitations inherent in studying\npurified particles. Key findings redefine herpesvirus capsid maturation, establishing the Capsid Vertex-Specific Component\n(CVSC) as both a nuclear assembly marker and an intrinsic capsid component. Furthermore, CVSC occupancy is utilized to\ncharacterize Capsid Assembly Intermediates (CAIs). Collectively, this work provides unprecedented subnanometer-\nresolution structural insights into VZV capsid assembly within its native nuclear environment and systematically details\nthese in situ structures. However, several issues require clarification before publication in Nature Communication.\nMajor comment:\n1. The study redefines Capsid Assembly Intermediates (CAIs) by correlating CVSC occupancy levels with scaffold protein\ndensity within the capsid cavity. Scaffold protein removal precedes dsDNA packaging to form intranuclear C-capsids. While\nCVSC assembly initiates prior to DNA packaging, their data reveal that CVSC occupancy increases progressively as\nscaffold density decreases (Fig. 5F-I). Crucially, CVSC acquisition correlates with scaffold reduction across intermediate\nstates, supporting its utility as a temporal marker for CAI classification. However, there is a gap between the “so-called” A\nparticle (no visible scaffold inside) and viral genome packaging. If the CVSC copies will reach up to 11 prior to DNA\npackaging or the CVSC copies will increase during DNA packaging needs to be further discussed.\n2. Among the 1,084 identified VZV capsids, the majority (n=980) localized within the nucleus. However, only a subset of\nthese particles (n=317) underwent subsequent subtomogram averaging to generate high-resolution reconstructions and\nsubsequent analysis. What is the standard for particle selection strategy, which may cause potential artifacts or not?\n3. While this paper redefines herpesvirus capsid assembly intermediates (CAIs) within the infected cell nucleus using cryo-\nET, demonstrating a continuum of maturation based on CVSC occupancy rather than the traditional A/B/C classification, it\ndoes not explicitly discuss how these new findings revise the current model of capsid assembly. A more detailed description\nof the proposed refined assembly model, incorporating the role of CVSC acquisition as a temporal marker and the\nsignificance of the observed heterogeneity, should be included in the Discussion section.\nMinor issues:\nFigure 1E: To improve interpretability, distinct labeling of intranuclear versus cytoplasmic compartments and key nuclear\ncomponents (e.g., DNA) is essential. Additionally, identical arrowhead colors must consistently represent the same structure\nacross all panels. For examples, the blue arrowhead denotes nuclear pore in i, 3-fold symmetry axes in ii, NEC in iii and\ntegument layers in iv, v, vi.\nLine 174: The \"(\" is missing before the referenced term.\nFigure 1Eiv-vi: Zoom-in views of the glycoprotein might be provided, as the authors describe them as typical observable\nfeatures. However, the claimed structures remain indiscernible in the current presentation.\nLines 224-244: While the authors analyze subnanometer-resolution structural details of MCP, TRX1, and TRX2 within the\ninfected cell nucleus, two critical aspects require attention: the comparison between in situ structures and existing high-\nresolution purified structures should be substantively addressed rather than emphasizing novelty claims, and priously\nreported structures (8X9Z or others) should be fitted into the current in situ reconstructions for biological context before\nstructural predictions.\nLine 264: The redefinition of the mature C-capsid's asymmetric unit requires justification, as identical asymmetric units have\nbeen reported in Nature Communications (2018), Protein & Cell (2020), and hlife (2024).\nLine 292: While the authors' UMAP classification of 317 capsids linking CVSC acquisition to assembly intermediates is\ninnovative, relax the C5 symmetry to reconstruct vertices and quantify CVSC-positive versus CVSC-negative vertices\nrelative to scaffold density remains insufficient; rigorous validation requires measuring both the CVSC/penton density ratio at\neach vertex and the scaffold/capsid density ratio, followed by linear regression between these ratio values to establish\ncorrelation.\nLine 336: Reconstruction of the portal vertex in VZV capsids has been achieved by Cao et al. (hlife 2023).\nLines 377-391: These paragraphs appear unrelated to CVSC and CAIs. The authors should explicitly establish connections\nbetween these concepts and the presented content.", "reviewer_3": "The authors report on cryo-electron tomography (cryoET) of cells infected by the Varicella-Zoster virus. This is an important\nexamination of parts of the life cycle of the virus in vivo. The visualization of individual particles enriches knowledge about\nthe different states of assembly, partial and full capsids, as well as the attachment of tegument proteins. The manuscript is\nworth publishing, but with some major issues.\nThe authors identify known phases in virus assembly and maturation, highlighting the addition of the capsid vertex-specific\ncomponent (CVSC) during capsid maturation and DNA encapsidation. However, they use the term \"capsid assembly\nintermediate (CAI)\" for a stage that is really during capsid maturation. One could call the addition of the CVSC assembly, but\nthat confuses it with the assembly of the procapsid, which they don't study in any detail.\nOne serious omission from the cited references is to other tomographic studies of herpesviruses.\nLine 73:\nThere is no evidence that capsomers are assembly intermediates. Also, the major capsid protein (MCP) associates with\nscaffold before assembly, and are incorporate as MCP-scaffold assembly units. The text here makes it sound like the\nscaffold aggregates first with capsomers added onto it. This is not what the evidence shows and is not expressed this way in\nmost papers.\n(Also stated around line 376)\nActually, lines 394-397 better state the evidence-based situation and contradict the previous statements.\nLine 92:\nThis should be subfamilies rather than subgenera. This is an important distinction in taxonomy.\nLine 135 and Figure 4:\nThe asymmetric unit is defined as a geometric part that constitutes one unique element of an icosahedron. It does not refer to\nthe contents and thus a \"redefinition\" to just include more proteins is nonsensical. I'm not aware of any other paper that\nclaims this when adding tegument proteins to the capsid.\nLine 352:\nHow sure are the authors that there is only ever one portal vertex per capsid? I don't see an analysis\nthat would reveal multiple or no portals in individual capsids. How does this compare to portal\nquantitation in HSV1?\nLines 376-387:\nThis part is a duplication of introductory material and does not address the results in this manuscript.\nLines 398-406:\nThe original distinction between A-, B- and C-capsids were based on density, not whether they contain scaffold or DNA.\nThus capsids with a little scaffold qualifies as A-capsids because they would occur in the A-capsid band on a density\ngradient. Capsids with a lot of scaffold would likewise appear in the B-capsid band.\nLine 438:\nThere is no mention or reference to the primary enveloped virion that is pertinent to the discussion here of nuclear egress.\nLine 575:\nHow were the 2D images \"flattened\" into a 1D array? Were the rows just concatenated? The UMAP algorithm requires\nvectorization of the data. How was this done here? Please add more detail to clarify how it was done."}, "author_response": "Author’s Response to the Reviewers’ Comments.\nOur responses to the reviewers’ comments are provided in-line with each comment in blue text.\nAll line numbers are referenced in the context of the revised manuscript.\nReviewer #1 (Remarks to the Author):\nThe work presented here aim to elucidate the protein structure of Herpesvirus via in situ\ntomography in the aim to propose a viral maturation model. The manuscript is well written but\nsuffer from numerous typos, missing scale bars… The science is sound and the data interpretation\nto my knowledge is done well.\nIf the author could respond to some points, this article should be published in Nature\ncommunication as this work represents the main benefit of in situ structural biology.\nReply: We very much appreciate and thank the reviewer for their overall positive assessment of\nour manuscript.\nMajor:\nCould you please define TK in the results as soon as mentioned and explain the rational allowing\nyou to use a tagged protein while aiming for structural determination (aka how you know it won’t\ndisrupt the native structure of interest) or the life cycle (the presence of absence of certain forms).\nAlso, could you demonstrate or cite references to demonstrate that the RFP virion as the same IFU\nand life cycle.\nReply: The pOka-TK-RFP virus used in this study was engineered to produce a thymidine kinase\n(TK) with a monomeric red fluorescence protein (RFP) tag 1. We have used and extensively\ncharacterized this virus in previously published studies1-4, demonstrating that pOka-TK-RFP\nreplicates similarly to the wild type pOka strain with similar kinetics and plaque sizes. In addition,\nthe thymidine kinase is not a structural component. We have amended the results section to include\nthis information and define TK (lines 158 to 160)…\n“a thymidine kinase (TK) tagged with a monomeric red fluorescence protein (RFP; Fig. 1); the\npOka-TK-RFP maintains the characteristics of the bacterial artificial chromosome derived WT\npOka 71-74.”\nWhat is the pattern used (size compare the cell size) and shape. As micropatterning affect the cell\norganisation, could you please explain the rational in the choice of the micropattern with regards\nthe infection?\nReply: The pattern used was a square to fit inside the grid squares of a 300 mesh grid. The\ndimensions of the individual squares were 30 x 30 or 33 x 33µm. The shape was chosen to prevent\nthe cells from adhering to the grid bars and remain centrally located to the grid squares. The\nmaterials and methods have been updated (line 521) to provide the dimensions of the individual\nsquares of the micropatterning…\n“…(30x30µm or 33x33µm to match the 300 mesh spacing) that…”\nCould you please deposit the segmentation, training data, manual segmentation to train the model\nand model on EMPIAR?\nReply: At the request of the reviewer, the Dragonfly training data, segmentation, mrc, and\nChimeraX files have been deposited into EMPIAR under accession number EMPIAR-12464.\nPage 1 of 14\n‘details of the VZV replication cycle previously unobserved’ By no mean I am a virus expert\nhowever this type of sentence should be rephrased to take into account what has been published\nbefore (for example J Virol. 2014 Dec 3;89(4):2436–2441. doi: 10.1038/s41564-024-01716-8)\nReply: Thank you for your comment. The Field’s Virology chapter on VZV 5 was cited as a\nsource that summarizes and cites the previous work on VZV capsid assembly performed over the\npast decades and the proteins involved in nuclear egress. The J Virol. 2014 reference that you\nkindly provided is included in the Field’s Virology chapter and the 10.1038/s41564-024-01716-8\nreference is cited in the revised discussion (line 455).\nThe first result section (VZV morphogenesis visualized within infected cells in situ by cryo-FIB-\nSEM and cryo-ET) does not bring enough metrics, it is descriptive and could be anecdotal. For\nexample, A-capsids not detected: is it because you do not have enough data?\nReply: Thank you for raising this point and we agree that metrics are needed to provide information\nbeyond anecdotal observations. To this end, we provided metrics for the frequency of capsid types and\ntheir cellular location in our original manuscript in Suppl. Table 3. The purpose of the first section was to\nhighlight the different stages of VZV morphogenesis and provide 3D information in the form of tomograms\n(Suppl. Movies 1 to 6) that correspond to previously published 2D observations. Our 3D observations both\nconfirm and support previous 2D data demonstrating capsid docking at the nuclear pore, the assembly of\nthe capsid in the nucleus, nuclear egress, secondary envelopment, and transport of nascent virus particles\nto the extracellular environment. However, the focus of this study was on the relationship between the\nCVSC and capsid assembly where we provide detailed 3D and statistical analyses to provide metrics to\nsupport our conclusions.\nAs the authors claim to have trained model, could the authors provide some metrics on some\nrelevant viral/host state? Alternatively, adjust the claims and include the description in shorter\nversion to the second paragraph to explain how you decide which particle was what and which one\nhave been push to STA. There is also something left unexplored which is the A capsids which are\nnot found in the nucleus but in the cytoplasm, again so quantification would help with significance\nof this data.\nReply: It is not clear what the reviewer means by the phrase ‘…trained model…’ and the\nrelevance to viral/host state. In the original manuscript, Supplementary Table 3 provided a\nbreakdown of the statistics for the 1,084 capsids, including their types and their cellular\nlocations across the 194 tilt series. Of the 1,084 capsids, there were a total of 214 classically\ndefined (appeared empty in a single plane) A-capsids; 204 in the nucleus, 8 free in the\ncytoplasm, and 2 contained in virus particles; the 10 A-capsids in the cytoplasm and particles\nsignificantly fewer than the 72 classically defined (DNA filled) C-capsids in the cytoplasm\nand particles.\nI found the application of UMAP interesting, and this type of analysis is relevant for in situ cryo\nET.\nReply: We thank the reviewer for noting that UMAP was an interesting and relevant analysis for\nin situ cryo-ET.\n‘to address this issue, we developed a new RELION pipeline ‘ could you please explain what is\nnew and why this novelty was required?\nReply: The RELION pipeline was a new approach compared to EMAN2. The word ‘new’ has\nbeen removed to eliminate any suggestion of novelty as per Nature Communications guidelines.\nPage 2 of 14\nHowever, the RELION pipeline was rather complex and required several software packages\nWARP (tilt stack generation), IMOD (manual alignment of the tilt stacks), RELION (tomogram\ngeneration and subtomogram averaging), EMAN2 (particle picking), and DYNAMO (mask\ngeneration) to process the data. Initial attempts to isolate the portal vertex solely using EMAN2\nresulted in limited success. We surmise that in the RELION pipeline, the manual alignment of the\ntilt stacks using IMOD generated high quality tomograms for particle picking and the subsequent\nsubtomogram averaging. Due to the incomplete angular sampling leading to the missing wedge\nartefact and the low signal to noise ratio in the tomograms, the portal vertex is very likely obscured\nor masked if the tomograms are poorly aligned.\nminor:\nauthors’ affiliation not in order (1,2, 6…)\nReply: Thank you for highlighting the out of order author affiliations. These have now been\ncorrected.\n‘3D volumes are produced at near-atomic resolution’ is miss leading if we consider the parameters\nused in this paper (and many others). I would re-phrase as the near atomic resolution in the STA\ncomes from averaging.\nReply: The statement “3D volumes are produced at near-atomic resolution” has been revised to\n“3D volumes can be produced at near-atomic resolution by imaging at different tilted angles, cryo-\ntomographic reconstruction, and subtomogram averaging” (lines 133 and 134).\nI think it is good to give success rate for the milling, how the percentage of lamella who have been\nconsidered as usable for cryo-ET would be amazing, with a short description of the criteria used.\nReply: We agree that this is a useful statistic. However, this aspect is not straightforward. Firstly,\nbecause the quality of the lamella are judged by eye with the thickness estimated using the SEM\nwhile retaining enough platinum at the edge of the lamella to prevent its destruction. Secondly,\nsome lamella can contain small holes, which could be deemed as poor quality, but they are still\nuseable for cryo-ET and subsequent subtomogram averaging. What is clear from our experience\nis that the largest ‘loss’ of useable lamella was due to two factors; ice contamination, large crystals\nobscuring regions of interest, or, a premature end to tilt series collection caused by failures in\nautomated alignment or focus to the end of the tilt series collection. Thus, although the percentage\nof cryo-ET usable lamella was high, this did not correlate with the frequency of lamella that\ngenerated high quality tilt series data needed for tomogram reconstruction or subtomogram\naveraging.\nGive the parameters for the molecular dynamics flexible fitting please.\nReply: We have included the parameters for the molecular dynamics flexible fitting as the NAMD\nparameters in the Source Data files and updated the Materials and Methods (lines 593 to 598)…\n“Molecular dynamics flexible fitting (MDFF) was performed to fit the two head groups of the\nCVC2 dimer into the cryo-ET map using NAMD 110 following the procedure outlined in the VMD\ntutorial. Conversion of the cryo-EM map to an MDFF potential, rigid body docking of the CVSC,\ninitial structure generation, and secondary structure restraint definitions were all performed using\nVMD. The output files were used as input to run NAMD with the following parameters; step 1 -\ngscale 0.3, -numsteps 50000; step 2 -gscale 10, -numsteps 100000.”\nPage 3 of 14\n‘First, because VZV does not yield high titer cell-free virus particles, it was critical to optimize the\npassage of infected cells onto uninfected cells to initiate infection prior to vitrification.’ Is it a well-\nknown fact that VZV does not produce virion in high yield? In this case could you put a reference.\nIf it is in your hand, please indicate.\nReply: Yes, this is a well-known phenomenon for VZV and is described in the Field’s Virology\nchapter on VZV 5. We have included a citation to the Field’s chapter on VZV at the end of this\nstatement (lines 154 to 155).\n“First, because VZV does not yield high titer cell-free virus particles 1…) \nFigure 1, no point to add the orange box as it calls for another figures, make sure you mentioned\nthe supp figure in the text and that would do. Can not really see the scale bar in panel B and C and\nnot sure we need the microscope bars t the bottom. Panel D could do with a scale bar. Panel E,\ncannot read easily the number at the top of the scale bars, remove and put in legend. Could you\nalso please used different colours for the different event you would use to observe as it is confusing.\nCould you please state where in the cells the tomograms are (nucleus vs cytoplasm) as it seems\nimportant for the comprehension of the different viral event. The figure legend can do with way\nless. Not sure we need panel A to C.\nReply: The purpose of this figure is to both highlight the technological approaches used to capture\nthe specific stages of the VZV replication cycle and represent them in 3D-space. This figure will\nbe especially important for the virologist readers and also provides a simplified overview of the\nherpesvirus replication cycle suitable for virologists and non-virologists alike. To address the\nconcerns of the reviewer, the scale bars have been added to panels B, C, and D. We have retained\nthe orange box as it highlights the tilt series collection site for panel Ev; we have removed reference\nto the supplemental figure to eliminate any confusion. For further clarity, we have also used\ndifferent colors to identify specific features as requested by Reviewers #1 and #2. Furthermore,\ncellular locations have been added to improve clarity and context. We feel that panels A to C are\nbeneficial to the readers as they provide context for both the stages of the replication cycle and the\ntechnical aspects of the data collection, including correlative fluorescence cryo-FIB/cryo-ET, to\ngenerate the images in Panel E.\nFigure 2: watch the scale bars please, they are not consistent and place randomly. Panel A also\nhave a scale bar and 3 axes, and for some reason the contrast is inverted.\nReply: For panel A the contrast was inverted to improve the visibility of the capsids in a 3D\nvolume; the 3-axes represent the directionality of the 3D-volume. Although the scale bars\nrepresent the same lengths, we have now edited the scales bars to be uniform.\nFigure legend 6 is all over the place.\nReply: We have revised the Fig. 6 legend carefully to be sure that each panel (A to F) is clearly\ndefined in the figure legend and the panels are organize by concept; cryo-ET of the complete\ncapsids (A and D), portal vertices (B and E), and docking of molecular models (C and F), relating\nto capsid assembly intermediates (A to C) and C-capsids (D to F).\nFigure 6C do you have hypothesis of what are the density which are not in the map on the ‘inner’\npart of the portal?\nPage 4 of 14\nReply: The remaining density for the portal in Fig. 6C is likely to be a portion of the dsDNA\ngenome. However, due to the low resolution (32.6Å) of the reconstructions it is currently not\npossible to make an informed statement about the additional density.\nReviewer #2 (Remarks to the Author):\nThis study employs an integrated cryo-FIB/cryo-ET approach to achieve subnanometer-resolution\nvisualization of intranuclear VZV capsid assembly intermediates in-situ within infected cells,\novercoming limitations inherent in studying purified particles. Key findings redefine herpesvirus\ncapsid maturation, establishing the Capsid Vertex-Specific Component (CVSC) as both a nuclear\nassembly marker and an intrinsic capsid component. Furthermore, CVSC occupancy is utilized to\ncharacterize Capsid Assembly Intermediates (CAIs). Collectively, this work provides\nunprecedented subnanometer-resolution structural insights into VZV capsid assembly within its\nnative nuclear environment and systematically details these in situ structures. However, several\nissues require clarification before publication in Nature Communication.\nReply: We very much appreciate and thank the reviewer for their very positive assessment of our\nmanuscript.\nMajor comment:\n1. The study redefines Capsid Assembly Intermediates (CAIs) by correlating CVSC occupancy\nlevels with scaffold protein density within the capsid cavity. Scaffold protein removal precedes\ndsDNA packaging to form intranuclear C-capsids. While CVSC assembly initiates prior to DNA\npackaging, their data reveal that CVSC occupancy increases progressively as scaffold density\ndecreases (Fig. 5F-I). Crucially, CVSC acquisition correlates with scaffold reduction across\nintermediate states, supporting its utility as a temporal marker for CAI classification. However,\nthere is a gap between the “so-called” A particle (no visible scaffold inside) and viral genome\npackaging. If the CVSC copies will reach up to 11 prior to DNA packaging or the CVSC copies\nwill increase during DNA packaging needs to be further discussed.\nReply: Thank you for the overview and request for clarification. Our new high-resolution\nobservations necessitate the adjustment of the current interpretations of herpesvirus capsid\nassembly. Our proposed model, for VZV, is that CVSC addition, predominantly, occurs before\ngenome packaging, and should be viewed as a dynamic process. These conclusions, as noted by\nthe reviewer, are based on the observations of decreasing scaffold content and increasing levels of\nCVSC. We find that true A-capsids are extremely rare events, which is supported by our 3D\nanalysis of capsids within the nucleus. There is also additional evidence that A-capsids are\nartefacts of capsid purification; see Stevens et al., 2024 6. What we do not currently understand is\nwhether the CVSC is acquired fully (five copies) at one vertex first or if multiple vertices partially\nacquire their CVSC simultaneously. These data are lacking, as the portal vertex would need to be\nidentified for all the capsids used in the subnanometer reconstruction, an approach that will be\nused in future studies.\n2. Among the 1,084 identified VZV capsids, the majority (n=980) localized within the nucleus.\nHowever, only a subset of these particles (n=317) underwent subsequent subtomogram averaging\nto generate high-resolution reconstructions and subsequent analysis. What is the standard for\nparticle selection strategy, which may cause potential artifacts or not?\nReply: Thank you for your insightful question. We provided an outline for the selection of capsids\nthat were used in the subnanometer reconstructions in the materials and methods section\nPage 5 of 14\nSubnanometer resolution cryo-ET map reconstruction of the VZV capsid vertex. “Classification\nwith icosahedral symmetry was performed on the output to remove poor quality particles that can\nnegatively influence the subtomogram averaging…” (lines 574 to 576).\nThe selection was dependent on the ‘quality’ of each individual capsid. Much like single particle\ncryo-EM, some particles will contribute more to the final structure based on certain metrics relating\nto the ice thickness (lamella thickness in our case), signal to noise ratio, conformational\nheterogeneity, and contrast transfer function. For a typical single particle analysis, up to 30% of\nthe particles will make it into the final high-resolution structure; 317 of 1,084 particles made it\ninto the vertex and 3-fold cryo-ET maps, which is in-line with a single particle workflow.\n3. While this paper redefines herpesvirus capsid assembly intermediates (CAIs) within the infected\ncell nucleus using cryo-ET, demonstrating a continuum of maturation based on CVSC occupancy\nrather than the traditional A/B/C classification, it does not explicitly discuss how these new\nfindings revise the current model of capsid assembly. A more detailed description of the proposed\nrefined assembly model, incorporating the role of CVSC acquisition as a temporal marker and the\nsignificance of the observed heterogeneity, should be included in the Discussion section.\nReply: Thank you for suggesting that a more detailed description of our proposed model was\nneeded. We have now included the following in the final paragraph of the revised discussion (lines\n485 to 494)…\n“Rather than discrete A-, B-, and C-capsid categories, we propose a model of herpesvirus capsid\nassembly where CVSC acquisition serves as a temporal marker towards completion; CAIs\nprogress along a continuum defined by decreasing scaffold protein and increasing CVSC\noccupancy prior to genome packaging. Early intermediates retain abundant scaffold and have few\nCVSCs, whereas later intermediates contain limited scaffold with more extensive CVSC\noccupancy at the vertices, culminating in C-capsids with full vertex occupancy and packaged\ngenomes. This model integrates structural heterogeneity into a dynamic pathway, resolves the\nambiguity surrounding A- and B-capsids, and highlights CVSC acquisition as a critical\ndeterminant of capsid stability, genome retention, and readiness for nuclear egress.”\nMinor issues:\nFigure 1E: To improve interpretability, distinct labeling of intranuclear versus cytoplasmic\ncompartments and key nuclear components (e.g., DNA) is essential. Additionally, identical\narrowhead colors must consistently represent the same structure across all panels. For examples,\nthe blue arrowhead denotes nuclear pore in i, 3-fold symmetry axes in ii, NEC in iii and\ntegument layers in iv, v, vi.\nExtra labels added to more clearly define cellular compartments and arrowhead colours have been\nrevised so that each colour represents the same structure.\nReply: Reviewer 1 had similar comments and we have now added extra labels for the cellular\ncompartments and unified the arrowheads to show one color per structural feature.\nLine 174: The \"(\" is missing before the referenced term.\nReply: This has now been corrected (line 179).\nFigure 1Eiv-vi: Zoom-in views of the glycoprotein might be provided, as the authors describe them\nas typical observable features. However, the claimed structures remain indiscernible in the current\npresentation.\nPage 6 of 14\nReply: We acknowledge that the glycoproteins are somewhat difficult to see in Fig. 1 E iv and vi.\nWe have now included an additional supplementary figure (Suppl. Fig. 9) that provides zoomed\nin views with arrowheads to highlight the glycoproteins associated with membranes and the VZV\nenvelope.\nLines 224-244: While the authors analyze subnanometer-resolution structural details of MCP,\nTRX1, and TRX2 within the infected cell nucleus, two critical aspects require attention: the\ncomparison between in situ structures and existing high-resolution purified structures should be\nsubstantively addressed rather than emphasizing novelty claims, and priously\nreported structures (8X9Z or others) should be fitted into the current in situ reconstructions for biological context\nbefore structural predictions.\nAt the time of writing our response to the review comments, 8X9Z was still not released from\nthe RSCB. However, we did use the previously published 7BW6 VZV structure to segment\nour cryo-ET maps in the original manuscript as outlined in our Materials and Methods (lines\n589 to 590). Furthermore, previous publications of VZV capsids, including Cao et al., 2024\ndo not provide atomic models for the complete CVSC and portal hence our use of AlphaFold\nto predicted models for these multimeric proteins.\nLine 264: The redefinition of the mature C-capsid's asymmetric unit requires justification, as\nidentical asymmetric units have been reported in Nature Communications (2018), Protein &\nCell (2020), and hlife (2024).\nReply: We thank the reviewer for the opportunity to clarify our justification for redefining the\nasymmetric unit. Firstly, all previous studies were based on defining the herpesvirus\nasymmetric unit using reconstructions from purified capsids or virus particles without taking\ninto consideration the intranuclear assembly of herpesvirus capsids. Secondly, our asymmetric\nunit differs from Sun et al., proposed for VZV 7 and each of the references provided by the\nreviewer 8-10.\nWang et al., 2018 provide an alternative asymmetric unit compared to the original proposed for\nHSV-1 that does include the CVSC interaction with the clockwise peripentonal MCPs at P1 and\nP2 9,11. They use the complete pentonal hexon (P-hexon) clockwise of the CVSC, complete\ncentral hexon (C-hexon), and subunits 1-3 of the edge hexon (E-hexon) clockwise of the C-\nhexon. The triplex order was revised to Ta (pentonal), and Te, Tb, Tc, Td around the C-hexon.\nEMD-6976 and PDB 5ZZ8: MCP(UL19), SCP(UL35), TRX1(UL38), TRX2(UL18),\nCVC1(UL17), CVC2(UL25), and VP1/2(UL36). Our proposed asymmetric unit differs as it\nincludes the clockwise and anticlockwise peripentonal interactions of triplexes Ta and Tc and\nthe CVSC. Wang N. et al., 2020 used cryo-EM to reconstruct HSV-2 B- and C-capsids but the\nCVSC was not used to define the asymmetric unit and the placement of the triplexes deviate\nsignificantly from the original HSV-1 assembly 10,12, which includes two Tc triplexes.\nFurthermore, the data and structures are not available in EMDB or PDB. Cao et al., 2024 was\nunfortunately omitted from our original manuscript as it is not referenced in PubMed. However,\ntheir structure data are now available in the PDB (8XA0) and EMDB (38190). Their proposed\nasymmetric unit is based on Wang, J. et al., 2018 and includes the CVC1 of the CVSC but omits\nthe remaining proteins of the heteropentamer and excludes the Ta and Tc triplex interactions with\nthe anticlockwise P-hexon 8,9.\nPage 7 of 14\nWe have included a new supplementary figure to provide comparisons between the different\nhuman alphaherpesvirus asymmetric units in the cited references (Suppl. Fig. 15) and updated\nthe results section (lines 273 to 278)…\n“Furthermore, these findings enabled us to redefine the asymmetric unit of the mature C-capsid to\nbe composed of six C hexon subunits (MCP and SCP), three E hexon subunits, and a pair of three\nperipentonal hexon subunits from neighboring hexons, one pentonal MCP, five interdigitating\ntriplexes, and a single CVSC heteropentamer (Fig. 2d and 4b; Suppl. Fig. 15a). This arrangement\ndiffers from previous models of alphaherpesvirus asymmetric units, where the interaction between\nCVC1 with triplexes Ta and Tc and MCP P1 and P2 were not used to define the asymmetric unit,\nproteins were missing, or proteins from a neighboring asymmetric unit were included (Suppl. Fig.\n15).”\nLine 292: While the authors' UMAP classification of 317 capsids linking CVSC acquisition to\nassembly intermediates is innovative, relax the C5 symmetry to reconstruct vertices and quantify\nCVSC-positive versus CVSC-negative vertices relative to scaffold density remains insufficient;\nrigorous validation requires measuring both the CVSC/penton density ratio at each vertex and the\nscaffold/capsid density ratio, followed by linear regression between these ratio values to establish\ncorrelation.\nReply: While a more quantitative validation would be useful, the reviewer’s proposed method\nwould be difficult to implement. The density ratio in cryo-EM maps is generally a poor metric for\nquantification, because it is affected by the sample and imaging conditions as well as the structural\nflexibility of the target protein. Additionally, the “scaffold density” here is hard to measure since\nthere is not a good way to distinguish whether a piece of density in a particle corresponds to\nscaffolds or DNA. To characterize maturation of virus at the penton level would also be\nchallenging, since events like DNA insertion only happens at one site per capsid.\nAs an alternative validation, we keep the three classes from UMAP, and test the correlation\nbetween the number of CVSC per capsid (0-60) and the stage of virus maturation. The p-value of\nt-test between CVSC number distribution in any of the two classes pairs is smaller than 10-5,\nindicating clear correlation between the CVSC acquisition and the structural difference of the\ncontent inside capsids.\nLine 336: Reconstruction of the portal vertex in VZV capsids has been achieved by Cao et al. (hlife\n2023).\nReply: Thank you for bringing our attention to this paper. We would like to note that,\nalthough this paper is stated to be peer reviewed it is not found in PubMed. The VZV portal\nvertex reconstruction by Cao et al., published in 2024 was based on purified particles rather\nthan in situ in infected cells as accomplished in our study. To address the reviewer’s\ncomment, we have now acknowledged this work in the introduction and the results section\n(lines 122 to 123 and 345)…\n“Furthermore, despite previous high-resolution structures for VZV capsids, the CVSC and the\nportal vertex were not resolved in these studies 39,41. Recent work with purified capsids\npartially resolved the CVSC and the portal vertex 68.”\n“…but has been achieved by Cao et al 68; this was not performed in infected cells in situ.”\nLines 377-391: These paragraphs appear unrelated to CVSC and CAIs. The authors should\nexplicitly establish connections between these concepts and the presented content.\nPage 8 of 14\nReply: Prior to discussing the importance of the CVSC as it relates to CAIs, we first wanted to\ndiscuss, in this paragraph, our finding that VZV produces segmented structures that are capsid\nassembly precursors to procapsids, a form of CAI. However, in light of the reviewers comment,\nwe have revised this paragraph to bring our observations of pre-procapsid segments to the\nbeginning of the paragraph to focus on our findings and how they relate to the current knowledge\nof procapsid assembly (lines 386 to 395)…\n“Herpesviruses are thought to assemble in a similar fashion to dsDNA bacteriophages where the\ncapsomeres coalesce with the scaffold protein to generate procapsids 28,29. The high-resolution\nimaging inside the nucleus of VZV infected cells in the present study revealed angular segment\nstructures where capsomeres, formed by the MCP and SCP, were associated with electron dense\nmaterial presumed to be scaffold protein (Suppl. Fig. 2). This interpretation is supported by cell\nfree systems that recapitulate herpesvirus capsid assembly by expression of the MCP, TRX1,\nTRX2 and VP22a proteins 83. VP22a is the scaffold protein expressed from UL26.5 (VZV\nORF33.5) cleaved by the VP24 protease during maturation 84,85. VP24 is a cleavage product\nderived from UL26 (VZV ORF33), where the protease autocatalytically cleaves the UL26 gene\nproduct into VP24 and VP21.”\nReviewer #3 (Remarks to the Author):\nThe authors report on cryo-electron tomography (cryoET) of cells infected by the Varicella-Zoster\nvirus. This is an important examination of parts of the life cycle of the virus in vivo. The\nvisualization of individual particles enriches knowledge about the different states of assembly,\npartial and full capsids, as well as the attachment of tegument proteins. The manuscript is worth\npublishing, but with some major issues.\nThe authors identify known phases in virus assembly and maturation, highlighting the addition of\nthe capsid vertex-specific component (CVSC) during capsid maturation and DNA encapsidation.\nHowever, they use the term \"capsid assembly intermediate (CAI)\" for a stage that is really during\ncapsid maturation. One could call the addition of the CVSC assembly, but that confuses it with the\nassembly of the procapsid, which they don't study in any detail.\nReply: We appreciate the reviewer’s stance on capsid assembly and recognize how the classical\ninterpretation of capsid assembly might be grounded in the procapsid. However, the reviewer does\nagree that we could interpret the addition of the CVSC as ‘assembly’, which is our position given\nthe new observations of VZV capsid assembly and how it is completed inside the infected cell\nnucleus. Although our work does not study procapsid assembly in detail, we do make significant\nobservations about the formation of procapsid segments that come together to form procapsids,\nand to our knowledge, a phenomenon previously only observed in biochemical experiments due\nto limitations of earlier electron microscopy technologies. We argue that clarity of terms is\nimproved by using maturation to relate to either conformation or biochemical changes to capsid\ncomponents whereas the addition of proteins to the capsid is further capsid assembly. We propose\nthat capsids lacking all or some of the CVSC at the vertices are assembly intermediates, which can\nonly be captured by performing high-resolution imaging and data processing studies in situ to\neliminate purification, fixation, and staining artefacts.\nOne serious omission from the cited references is to other tomographic studies of herpesviruses.\nReply: To our knowledge, there have been a limited number of studies that use tomography to\ninvestigate herpesviruses in situ. The application of tomography in most prior studies have done\nPage 9 of 14\nso using purified virus particles from infected cells 13-19. Some herpesvirus structure studies have\nused the term ‘in situ’ but that refers to the capsid inside virus particles and not inside infected\ncells 14,20. There are four examples of electron tomography used to examine herpesviruses inside\ninfected cells. The most recent study used a similar procedure to our approach, cryo-FIB/cryo-\nET, but the focus of this work was on the plasticity of the nuclear egress complex (NEC) using\nherpesviruses mutants 21. A second study used cryo-ET to examine capsids in neurons infected\nwith HSV-1 with low-resolution (56 to 97Å) icosahedral capsid reconstructions 22. The remaining\ntwo studies used fixation of herpesvirus infected cells followed by sectioning then performed either\ncryo-ET 23 or electron tomography on resin embedded contrast stained cells 24. As our studies are\na departure from each of these tomography approaches they were not cited in the original\nmanuscript for brevity. We have now cited several of these studies in the context of the broader\ntopic of electron tomography and our work (lines 112, 113, 453, and 454).\nLine 73:\nThere is no evidence that capsomers are assembly intermediates. Also, the major capsid protein\n(MCP) associates with scaffold before assembly, and are incorporate as MCP-scaffold assembly\nunits. The text here makes it sound like the scaffold aggregates first with capsomers added onto it.\nThis is not what the evidence shows and is not expressed this way in most papers.\n(Also stated around line 376)\nActually, lines 394-397 better state the evidence-based situation and contradict the previous\nstatements.\nReply: We thank the reviewer for their insight and agree that the MCP associates with the scaffold\nprotein to form assembly units. We have edited the text to better reflect this statement (lines 73 to\n74 and 401 to 404)…\n“…which are thought to form as capsomeres coalesce with the scaffold protein to produce\nsegments and subsequently spherical capsids 28,29.”\n“Further characterization of HSV procapsid formation in cell free expression systems noted the\nfragility and production of angular segments, leading to a proposed model where capsomeres and\nscaffold protein aggregate to form angular segments which in turn form spherical procapsids 92.”\nLine 92:\nThis should be subfamilies rather than subgenera. This is an important distinction in taxonomy.\nReply: Thanks for spotting the error. We have substituted ‘subgenera’ for ‘subfamilies’ (line 93).\nLine 135 and Figure 4:\nThe asymmetric unit is defined as a geometric part that constitutes one unique element of an\nicosahedron. It does not refer to the contents and thus a \"redefinition\" to just include more proteins\nis nonsensical. I'm not aware of any other paper that claims this when adding tegument proteins to\nthe capsid.\nReply: We agree with the reviewer that the definition of an asymmetric unit is a geometric\npart that constitutes one unique element of an icosahedron. However, we argue that the CVSC\nforms part of the completed icosahedron, as it makes contact with the underlying protein shell,\nand should be a defining factor of the asymmetric unit. Our data provide two critical aspects\ntowards defining the herpesvirus asymmetric unit. Firstly, the CVSC is not a tegument protein\nPage 10 of 14\nas its incorporation into the capsid occurs in the nucleus. Secondly, the CVC1 interacts with\ntriplexes Ta and Tc and makes contacts with the clockwise P-hexon, not the anticlockwise P\nhexon; this architecture creates a nucleation point for CVSC acquisition into the asymmetric\nunit. Furthermore, incorporation of the CVC1 is conserved amongst all herpesviruses. Thus,\nthe CVSC is a critical component of capsid assembly, especially as all 11 pentons and the\nportal vertex are fully occupied with the 60 CVSC heteropentamer, finalizing assembly of the\nherpesvirus capsid icosahedron. Please also review our justification in response to Reviewer\n#1.\nLine 352:\nHow sure are the authors that there is only ever one portal vertex per capsid? I don't see an analysis\nthat would reveal multiple or no portals in individual capsids. How does this compare to portal\nquantitation in HSV1?\nReply: The reviewer poses an astute question that has not been addressed in the current study.\nGiven the prior literature, it would be unlikely that multiple portals are present in a single capsid.\nSeveral studies have structurally defined the portal vertex at high resolution using tens of\nthousands of particles 8,20,25-28. None of those specifically addressed multiple copies of portal\nvertices. It should be noted that in all of these single particle studies, fewer portal vertices were\nidentified than the total frequency of capsids used for the reconstruction of the portal vertex: Liu\net al., 2019, of 45,445 HSV-1 particles, 42,857 were used for portal reconstruction; Gong et al.,\n2019, of 44,328 KSHV particles, 39,773 were used for portal reconstruction; Li et al., 2021, of\n26,050 HCMV particles, 23,136 were used for portal reconstruction; Li et al., 2023, of 25,502 (A-\ncapsid) and 31,265 (B-capsid) HCMV particles, 22,059 and 26,181 were used for portal\nreconstruction respectively; Li et al., 2020, of 32,721 EBV particles, 28,639 were used for portal\nreconstruction; Cao et al., 2024, of 57,722 VZV particles, 34,822 were used for blocked-based\nreconstruction of the portal vertex. These data imply that only a single portal was present for\nherpesvirus capsids. However, these studies did not perform quantitative analyses to define portal\nfrequency per capsid. To our knowledge, there have not been published reports of multiple portals\nfor herpesvirus capsids.\nWe can extrapolate that all DNA filled capsids must have a portal vertex for genome packaging to\noccur. However, given the data in this study, establishing the absence of portals in capsid assembly\nintermediates is more challenging. Due to incomplete angular sampling, in situ cryo-ET suffers\nfrom a low signal to noise ratio and the missing wedge artefact, leaving gaps in Fourier space,\npotentially obscuring the portal if the capsid is at a non-optimal orientation in the tilt series. This\nis less of an issue with the projection images used in the single particle approaches in prior studies\nof herpesvirus portal reconstructions 8,20,25-28. Although capsids can form in the absence of the\nportal 29, whether this occurs in situ, inside the cell nucleus, remains an open question.\nLines 376-387:\nThis part is a duplication of introductory material and does not address the results in this\nmanuscript.\nReply: Thank you for this comment. We have now better integrated our findings with the\npreviously publish literature about the formation of procapsids from segments of capsomeres and\nscaffold (lines 386 to 392)…\nPage 11 of 14\n“Herpesviruses are thought to assemble in a similar fashion to dsDNA bacteriophages where the\ncapsomeres coalesce with the scaffold protein to generate procapsids 28,29. The high-resolution\nimaging inside the nucleus of VZV infected cells in the present study revealed angular segment\nstructures where capsomeres, formed by the MCP and SCP, were associated with electron dense\nmaterial presumed to be scaffold protein (Suppl. Fig. 2). This interpretation is supported by cell\nfree systems that recapitulate herpesvirus capsid assembly by expression of the MCP, TRX1,\nTRX2 and VP22a proteins 83.”\nLines 398-406:\nThe original distinction between A-, B- and C-capsids were based on density, not whether they\ncontain scaffold or DNA.\nThus capsids with a little scaffold qualifies as A-capsids because they would occur in the A-capsid band on a density\ngradient. Capsids with a lot of scaffold would likewise appear in the B-capsid band.\nReply: We agree that the original distinction between A-, B-, and C-capsids was based on\ndensity and that capsids with little scaffold would previously be classified as A-capsids.\nHowever, the original designation for A-capsids using 2D transmission electron microscopy\nis that they were empty 30, which we demonstrate by 3D analysis of capsids inside the nucleus\nis not the case. Moreover, true A-capsids, completely devoid of scaffold and lacking the\ndsDNA genome likely arises due to purification, as was discussed in the original manuscript\n(lines 472 and 477). Our stance, based on the 3D cryo-ET observations of capsids inside the\nnucleus of vitrified cells, in the absence of purification, fixation and staining artefacts, is that\nherpesvirus capsids present a continuum of scaffold protein dependent on their stages of\nassembly.\nLine 438:\nThere is no mention or reference to the primary enveloped virion that is pertinent to the discussion\nhere of nuclear egress.\nReply: Thank you for raising this point. A statement about the budding of capsids through the\nnuclear envelope, interaction with the nuclear egress complex (NEC), and acquisition of a primary\nenvelope has been incorporated into the discussion (lines 451 to 457).\n“The process of nuclear egress is thought to be initiated by capsids budding at the nuclear envelope\ninto the perinuclear space through interactions of CVC2 head groups with the nuclear egress\ncomplex (NEC) embedded in the INM, leading to acquisition of a primary envelope 95,97. Although\nthe NEC has been shown to induce curvature in the absence of CVC2 via a mechanism that\ninvolves a random lattice formation 98, these studies were performed using a US3 mutant that\ncauses capsids to become trapped within the perinuclear space 99.”\nLine 575:\nHow were the 2D images \"flattened\" into a 1D array? Were the rows just concatenated? The\nUMAP algorithm requires vectorization of the data. How was this done here? Please add more\ndetail to clarify how it was done.\nReply: We thank the reviewer’s suggestion to add more detail to clarify the procedure. We have\nupdated the materials and methods section (lines 602 to 611)…\n“Three axis orthogonal projection and uniform manifold approximation projection. The 3D\nVZV capsid particles were aligned to the symmetrized capsid reference from the EMAN2\nPage 12 of 14\nicosahedral refinement. The subsequent 2D sub-tilt particles at the corresponding orientation were\nreconstructed with icosahedral symmetry to obtain symmetrized 3D particles free from missing\nwedge artifacts. Each particle was projected along three orthogonal axes of thin central slabs\n(72Å), using only one quarter of the volume closest to the center perpendicular to the\ncorresponding axis. The resulting three 2D image projections per capsid were stacked and\nflattened into a 1D array that was used as input for uniform manifold approximation projection\n(UMAP). After manifold embedding, particles on the 2D UMAP space were clustered using a K-\nmeans algorithm to produce the individual classes.”\nReferences.\nFusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection. J\nVirol 91, (2017).\nrole in fusion. PLoS Pathog 17, e1008961, (2021).\nregulates Varicella-Zoster Virus induced cell-cell fusion. PLoS Pathog 16, e1009022,\n(2020).\ncritical domain for herpesvirus fusion initiation. Nat Commun 11, 4141, (2020).\nDavid M. Knipe, Blossom A. Damania, & Jeffrey I. Cohen) 0 (Lippincott Williams &\nWilkins, a Wolters Kluwer business, 2022).\nmutagenesis targeting interactions between pp150 tegument protein and small capsid\nprotein identify five lethal and two live-attenuated HCMV mutants. Virology 596, 110115,\n(2024).\n4795, (2020).\natomic resolution structures. hLife 2, 64-74, (2024).\nspecific component. Nat Commun 9, 3668, (2018).\nfor Herpesvirus assembly and maturation. Protein Cell 11, 366-373, (2020).\ntegument protein complexes. Science 360, (2018).\nKSHV replication. Nature 553, 521-525, (2018).\nElectron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with\nthe Scaffold and a Shift on Maturation. mBio 12, (2021).\ntomography. Virology 361, 426-434, (2007).\nportal in the herpesvirus capsid. J Virol 81, 2065-2068, (2007).\nKaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures\nessential to capsid assembly and maturation. J Struct Biol 161, 419-427, (2008).\nelectron tomography. Science 302, 1396-1398, (2003).\nRole in Nuclear Egress. mBio 8, (2017).\ncytomegalovirus by cryo electron tomography with Volta phase plate. PLoS Pathog 14,\ne1007452, (2018).\ndevoid capsids of human cytomegalovirus. Nat Commun 14, 2025, (2023).\nMicrobiol 9, 1842-1855, (2024).\nand secondary envelopment in primary neurons. PLoS Pathog 7, e1002406, (2011).\nvirus capsids within cell nuclei by correlative light and cryo-electron tomography. Sci Rep\n10, 17596, (2020).\ngammaherpesvirus life cycle in host cells by electron tomography. Structure 18, 47-58,\n(2010).\nvirus type 1 portal vertex and packaged genome. Nature 570, 257-261, (2019).\nTumor Herpesvirus KSHV. Cell 178, 1329-1343 e1312, (2019).\nejection in human cytomegalovirus. Nat Commun 12, 4538, (2021).\n873-884, (2020).\nof the herpes simplex virus capsid. J Virol 68, 6059-6063, (1994).\nand composition of multiple capsid forms of subtypes 1 and 2. J Virol 10, 1044-1052,\n(1972).\nPage 14 of 14\nAuthor’s Response to the Reviewers’ Comments.\nOur responses to reviewers #3’s comments are provided in-line with each comment in blue text.\nAll line numbers are referenced in the context of the revised manuscript.\nReviewer #3 (Remarks to the Author):\nI commend the authors for detailed consideration of the comments and their revisions. I support\naccepting the manuscript with only a few more comments on the issues I raised.\nReply: We thank the reviewer for their positive evaluation of our revisions and for the opportunity\nto provide further clarification for the following three points raised.\nThe delineation of the asymmetric unit is arbitrary from an image processing point of view, which\nwas the intent of my original statement. The delineation advocated here is interpretative to\nhighlight the interaction of CVSC around the vertices. It is still an opinion and thus arbitrary.\nReply: We acknowledge the intent of the reviewer’s original statement. However, the asymmetric\nunit of an icosahedral virus particle is meant to be the smallest number of molecular components\nthat has 60 identical copies making up the particle 1,2. In the context of VZV capsid with T=16,\nan asymmetric unit was thought to be composed of the pentonal major capsid protein (PP), the\nthree types of hexon (peripentonal (P1-P6), edge (E1-E3), and center (C1-C6)), and the five\ntriplexes (Ta, Tb, Tc, Td, and Te). In our work, we propose that an asymmetric unit of VZV capsid\nfound in the nucleus of an infected cell should also include the CVSC, a heteropentamer complex.\nBecause of the complexity of the molecular components in VZV, we believe that this is an effective\nway to communicate with the readers in terms of an asymmetric unit as shown in Figure 2, 3 and\n4.\nThe insistence that only one vertex of a capsid is ever occupied by a portal is unjustified. In\nasymmetric single particle reconstructions revealing the portal, any portal at another vertex is\nwashed out by the averaging. A portal at another random vertex simply cannot compete with 11\ncopies of a penton. This is the same reason why the portal is washed out in full icosahedral\nreconstructions. The single particle reconstructions can therefore not be used as justification. The\nevidence for variable portal numbers is threefold: 1) The original quantitation of portals per vertex\nalways resulted in more than one; 2) Portal incorporation is not required for assembly, resulting in\nsome capsids without portals; 3) A tomographic study quantified the number of portals from a set\nof individual capsids, concluding with a distribution with 0,1,2 and 3 portals per capsid, and in\nagreement with earlier quantitative studies.\nReply: We thank the reviewer for further clarification about using single particle data to justify a\nsingle herpesvirus portal vertex. We acknowledge that HSV-1 capsids were previously proposed\nto have a distribution of portals per capsid by biochemical and low resolution cryo-EM and cryo-\nET methodologies 3-7. These interesting observations with highly purified viral mutants do not\nnecessarily reflect the natural assemblies of herpesvirus capsids inside the infected cell nucleus.\nOur revised text now emphasizes that our observations do not contradict prior reports of variable\nPage 1 of 3\nportal numbers in purified capsids. We have amended the discussion with an additional paragraph\nto acknowledge these studies (lines 451 to 463)…\n“Biochemical and cryo-ET studies with HSV-1 have demonstrated either the absence or presence\nof multiple vertices containing portal densities using highly purified HSV-1 procapsids from a\nprotease deficient virus (m100) or A-capsids and pUL6 labelled B-capsids from wild type virus,\nor a UL25 null virus (KUL25NS) 55,56,95-97. Our in situ study of naturally synthesized capsids did\nnot quantify the frequency of portals per capsid because portal structures were not observed for all\ndsDNA genome containing C-capsids. Critically, a portal is required for genome packaging. The\nlack of at least one portal per C-capsid was attributable to a combination of incomplete angular\nsampling, the low signal to noise ratio of in situ cryo-ET, and the missing wedge artefact, leaving\ngaps in Fourier space, potentially obscuring the portal if the capsid is at a non-optimal orientation\nin the tilt series. Thus, any estimate of portal frequency per capsid would be grossly\nunderestimated. Therefore, our data do not undermine previous portal distribution estimates. To\naddress the observations of the previous HSV-1 studies 55,56,95-97, future work must be performed\nto establish whether herpesvirus capsids form inside the nucleus without or with multiple portals.”\nWe also appreciate the reviewer’s reminder that icosahedral-averaged single-particle structures\ncannot exclude the presence of additional portals, hence the application of the Relion-based data\nprocessing pipeline outlined in the Materials and Methods, which started with an icosahedral\nrefinement. Subsequently, to identify vertices containing a portal, symmetry expansion was\nperformed to generate 5,318 unique vertices from the 471 capsids, which were used for C5\nsymmetry reconstruction, and followed by an initial 3D classification to identify and extract portal\ndensities to generate a template for further classification. Even with a portal template, this pipeline\nwas only able to extract 109 portals from the 471 capsids; in situ cryo-ET suffers from a low signal\nto noise ratio combined with the missing wedge artefact, potentially obscuring the portal if the\ncapsid inside the cell is at a non-optimal orientation in the tilt series. Importantly, a portal structure\nin the capsid is required for genome packaging but our data processing pipeline was unable to\nextract portal densities from all dsDNA genome containing capsids; only 47 of the 114 C-capsids.\nGiven this poor recovery rate (41%) of portals from capsids that must contain a minimum of one\nportal, the portal frequency per capsid was not investigated. However, the hypothesis ‘capsids\ncontain multiple portals when assembled in the cell nucleus’ will be an exciting one to test in situ\nusing improved cryo-FIB/cryo-ET and computational approaches.\nI don't think there was ever a problem with different amounts of scaffold per capsid. What is clear\nfrom numerous density centrifugation experiments is that there are two distinct peaks associated\nwith A- and B-capsids, indicating that the transition from mostly scaffold-containing capsids to\nmostly empty capsids should be relatively rapid. To describe it as a continuum without clarifying\nthe distribution makes it sound as if the different amounts from none to full are equally represented.\nReply: We thank the reviewer for bringing attention to how our wording could imply equal\nrepresentation along the scaffold-removal trajectory. We have performed additional edits to clarify\nthe text in accordance with the reviewer’s assertion (lines 210 to 213; 426 to 428)…\nPage 2 of 3\n“A visual inspection of the image contrast in the cavity of capsids without the dsDNA genome\nrevealed a distribution of scaffold protein density, filled to sparse, suggesting that completely\nempty A-capsids are absent or very rare within infected cell nuclei (Suppl. Fig. 10).”\n“Although the 3-axis orthogonal projection clusters capsids into three classes, our observations\nindicate that the conventionally designated A- and B-capsids (class i and ii) represent a continuum\nof CAIs that can potentially progress towards DNA encapsidation and formation of mature capsids\nready for nuclear egress.”\nWe also agree that current biochemical data support the concept that the conversion of B-capsids\nto packaging competent capsids should be relatively rapid. It was not our intention to imply that\nthe different amounts of scaffold from none to full are equally represented. The current\ncomputational classification procedure is non-optimal as the manifold cannot fully distinguish the\ncontinuum. However, the subsequent analysis of CVSC occupancy versus capsid cavity scaffold\ndensity provides an alternative metric and supports the concept of a continuum based on direct\nstructural information inside the capsid cavity of intranuclear capsids rather than biochemically\nderived capsids that could undergo maturation during centrifugation. We acknowledge that\nimprovements are needed in the 3-axis orthogonal projection to computationally define the\nscaffold protein continuum, work that is planned for the future. Furthermore, our studies\ndemonstrate that the temporal nature of this continuum is still open for debate. Future in situ\nstudies of intranuclear capsids, in the absence of fixation and purification artefacts, with cryo-\nFIB/cryo-ET and herpesvirus replication molecular clocks will be needed to precisely define the\nrelationship between the structures of assembly intermediates and genome packaging competent\ncapsids.\nReferences.\n1573–1586 (Academic Press, 1994).\nof biological macromolecules. (Oxford University Press, 2007).\ntomography. Virology 361, 426-434, (2007).\nformation and viral growth. J Virol 68, 3702-3712, (1994).\nherpes simplex virus capsid. J Virol 75, 10923-10932, (2001).\nfor encapsidation but not for cleavage of replicated viral DNA. J Virol 72, 1060-1070,\n(1998).\nElectron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with\nthe Scaffold and a Shift on Maturation. mBio 12, (2021).\nPage 3 of 3"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for taking time to review my comments, sorry I have missed supp table 3 for some reasons.\nGood for publication for me.\nMaud", "reviewer_2": "Sorry to reply late. The authors have addressed all my concerns and the revised manuscript has significantly been\nimproved. It's suitable for publication now.", "reviewer_3": "I commend the authors for detailed consideration of the comments and their revisions. I support accepting the manuscript\nwith only a few more comments on the issues I raised.\nThe delineation of the asymmetric unit is arbitrary from an image processing point of view, which was the intent of my\noriginal statement. The delineation advocated here is interpretative to highlight the interaction of CVSC around the vertices.\nIt is still an opinion and thus arbitrary.\nThe insistence that only one vertex of a capsid is ever occupied by a portal is unjustified. In asymmetric single particle\nreconstructions revealing the portal, any portal at another vertex is washed out by the averaging. A portal at another random\nvertex simply cannot compete with 11 copies of a penton. This is the same reason why the portal is washed out in full\nicosahedral reconstructions. The single particle reconstructions can therefore not be used as justification. The evidence for\nvariable portal numbers is threefold: 1) The original quantitation of portals per vertex always resulted in more than one; 2)\nPortal incorporation is not required for assembly, resulting in some capsids without portals; 3) A tomographic study\nquantified the number of portals from a set of individual capsids, concluding with a distribution with 0,1,2 and 3 portals per\ncapsid, and in agreement with earlier quantitative studies.\nI don't think there was ever a problem with different amounts of scaffold per capsid. What is clear from numerous density\ncentrifugation experiments is that there are two distinct peaks associated with A- and B-capsids, indicating that the transition\nfrom mostly scaffold-containing capsids to mostly empty capsids should be relatively rapid. To describe it as a continuum\nwithout clarifying the distribution makes it sound as if the different amounts from none to full are equally represented."}, "author_response": ""}]

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