High-resolution analysis of the human T-cell leukemia virus capsid protein reveals insights into immature particle morphology

Domain: physics
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
Infection with human T-cell leukemia virus type 1 (HTLV-1) can result in adult T-cell leukemia/lymphoma and HTLV-1 associated-myelopathy/tropical spastic paraparesis. The Gag polyprotein–the major structural protein–is crucial for driving virus particle assembly, with the capsid (CA) domain as the key determinant for Gag multimerization. Here, we characterize the immature CA lattice from immature virus particles by using cryo-electron microscopy and tomography (cryo-EM/ET). We report resolving the immature CA lattice to 3.4 Å resolution by single particle analysis (SPA). Our reconstruction reveals that the lattice is stabilized through a trimeric NTD inter-hexamer interface and a dimeric CTD inter-hexamer interface. Further analysis by cryo-ET reveals clear heterogeneity, notably the varying lattice curvatures and the varying distances from the CA layer to the membrane. Intriguingly, inositol hexakisphosphate (IP6) is dispensable for HTLV-1 immature particle assembly and proper immature lattice formation. These observations provide deeper insights into the molecular basis of HTLV-1 immature particle morphology as well as aid in revealing therapeutic targets.

Introduction
Human T-cell leukemia virus type 1 (HTLV-1) is the prototypical deltaretrovirus, and is the causative agent of an adult T-cell leukemia/lymphoma (ATLL) and HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP)1,2. About 2–6% individuals living with HTLV-1 will develop either disease3,4. Currently, there are no approved therapeutics to prevent or treat the infection. Like human immunodeficiency virus (HIV), HTLV-1 encodes the Gag structural polyprotein that is required for viral assembly5. The HTLV-1 Gag contains a matrix (MA), capsid (CA), and nucleocapsid (NC) domain. The MA domain is critical for targeting Gag to the plasma membrane, CA is required for Gag oligomerization by forming the immature CA lattice, and NC packages the single-stranded RNA viral genome6–8. Unlike HIV, the HTLV-1 Gag polyprotein does not contain the spacer peptide regions (SP1 and SP2) or the p6 domain.

The structure of CA proteins among several retroviruses has been studied, revealing a highly conserved tertiary structure despite a divergence in the amino acid sequence. The CA tertiary structure consists of two separate domains—a N-terminal domain (NTD) and a C-terminal domain (CTD), connected via a flexible linker9–13. Structural analysis of the immature HIV type 1 (HIV-1) lattice identified a wine-glass morphology, where the CA-NTD represents the cup of the glass and the CA-CTD and SP1 represent the stem11,12. The HIV-1 CA-NTD supports the hexameric lattice via a trimer interface from helix 2 and a dimer interface from helix 1, while helix 9 forms the dimer interface in the CA-CTD12. Similar arrangements for the immature lattice have been noted with HIV type 2 (HIV-2)14, equine infectious anemia virus (EIAV)15, murine leukemia virus (MLV)9, and human endogenous retrovirus K (HERV-K)16. These immature lattice structures also contain a ring of 6 helices on the C-terminal tail of CA or through a spacer peptide region, termed the 6-helix bundle (6HB)9,15,16. The HIV-1 6HB functions to stabilize the hexamer of the Gag lattice, can bind to the cofactor inositol hexakisphosphate (IP6), and serves as the binding site of HIV-1 maturation inhibitors11,14,15,17. Structural characterization of immature retroviral Gag lattices has largely relied on the use of cryo-electron tomography (cryo-ET)9,11,15,16,18 rather than single particle analysis (SPA) due to difficulties from the geometry of the lattice, the proximity to the membrane, and dampened lattice signal from other viral components. In situ single particle analysis of purified virus particles allows for improved resolution compared to cryo-ET and provides relevant structural information that may not be present from analysis of in vitro assemblies (e.g., presence of cellular co-factors)19.

Previous studies have indicated that HTLV-1 immature particles possess unique morphological features compared to that of retroviruses in other genera, implying that deltaretroviruses are distinct in regards to immature particle morphology. HTLV-1 immature particles often contain an incomplete Gag lattice with portions of the lattice deviating from the curvature of the viral membrane and appear to have a flattened lattice20,21. However, to date, a thorough analysis of the immature HTLV-1 lattice curvature has not been conducted, and it remains an open question if these observations are artifacts of two-dimensional imaging. It has been established that the primary driver for HIV-1 assembly is the CA-CTD and SP1, while the HIV-1 CA-NTD is dispensable for immature lattice formation22,23. In contrast, previous studies with HTLV-1 have indicated the opposite—i.e., the CA-NTD is required for proper lattice formation while mutations in the CA-CTD led to limited effects on immature lattice formation24–27. HTLV-1 CA-CTD is not completely dispensable for immature particle production, as has been observed with the HIV-1 CA-NTD6,25. Recent subtomogram averaging of HTLV-1-like particles and in vitro assembled helical tubes showed the CA-NTD forms the majority of the intra-hexamer and inter-hexamer interfaces, while the CA-CTD forms an inter-hexamer dimer interface and supplies no intra-hexamer interactions18. However, only the CA-NTD was resolved to high resolution from the in vitro assemblies, whereas the resolution in the CA-CTD was limited due to flexibility. Improving the resolution in the CA-CTD is critical for better understanding its role in HTLV-1 immature particle assembly, as is validating the in vitro CA-NTD structure in situ.

Depletion of IP6 from virus-producing cells drastically decreases viral infectivity for many lentiviruses28,29. Packaging of IP6 into the immature Gag lattice of HIV-1 particles is coordinated through electrostatic interactions between the highly negatively charged IP6 molecule and two lysine rings formed by K158 and K227 (based upon CA numbering)28,30,31. Upon virus maturation, IP6 binds to the central pore of the hexamer in the CA-NTD at the R18 and K25 rings32,33. IP6 promotes proper infectivity by strongly stabilizing the mature HIV-1 core and allowing for efficient reverse transcription32,34. The functional conservation of IP6 in virus assembly and maturation among retroviral genera has not been fully explored. Recently, it was shown that MLV, a gammaretrovirus, packages IP6 into the immature lattice, albeit at lower levels than HIV-1. However, unlike HIV-1, MLV infectivity was more sensitive to IP6 depletion in the target cell rather than the virus-producing cell29,35. To date, there is currently no knowledge regarding the role of IP6 in deltaretrovirus particle assembly and maturation. Such information can provide insights into the evolutionary conservation of IP6 in the viral lifecycle among retroviral genera.

In this study, we investigated HTLV-1 immature particle morphology through a combination of cryo-electron microscopy and tomography (cryo-EM/ET) to generate a reconstruction of the assembled immature lattice. The structure of the CA region in the Gag lattice was determined to 3.4 Å resolution using SPA, the highest resolution of HTLV-1 immature particles to date, highlighting SPA as a powerful tool for immature CA lattice determination. Tomography of HTLV-1 particles highlighted the morphological heterogeneity, corroborating previously observed phenotypes from two-dimensional analysis of HTLV-1 particles20,21. Furthermore, improved resolution in the CA-CTD and structure-guided mutagenesis studies allow for identification of residues in the CA-CTD dimer interface that have a critical role for particle assembly. Intriguingly, IP6 is found to be dispensable for immature particle production, despite IP6 enhancing HTLV-1 CA assembly in vitro and a weak unidentified density in the CA-NTD hexamer pore. Overall, our observations provide a clear structural basis for previous reports on the pleomorphic morphology of the HTLV-1 immature Gag lattice, further expand the understanding of these phenotypes, and provide evidence implicating that IP6 has a limited role in driving HTLV-1 immature particle assembly.

Results
Cryo-EM reconstruction of the immature HTLV-1 Gag lattice
A SPA reconstruction of the HTLV-1 immature CA lattice was generated from purified HTLV-1 particles by co-expressing only the HTLV-1 structural proteins, Gag and envelope, in HEK293T cells (Fig. 1). As no HTLV-1 protease is present in the system, the Gag lattice in particles is trapped in their immature lattice morphology, and no mature cores are generated. A comparable methodology for SPA has been recently utilized for analysis of the immature Gag lattice of HIV-214. Particle picking was expanded to include all lattice regions below the viral membrane, rather than just the lattice regions tangent to the viral particle (Fig. 1a). This greatly enhanced the number of particles included in the reconstruction and increased the diversity of views of the lattice (Supplementary Fig. 1d), which improved the quality and resolution of the reconstruction. The reconstruction was generated by using six-fold symmetry centered on the central capsid hexamer and reached a global resolution of 3.4 Å (Supplementary Fig. 1a). Local resolution calculations revealed higher resolution in the CA-NTD compared to the CA-CTD, implicating flexibility in the CA-CTD (Fig. 1d). A 3DFlex refinement36 of the reconstruction depicts the flexibility in the CA-CTD relative to the CA-NTD that is caused by the varying curvatures in the immature lattice (Supplementary Movie 1). The resolution obtained allowed for the generation of a model of the in situ immature HTLV-1 CA lattice, comprising residues W15 to P207 (CA numbering) (Fig. 1e, f). At low resolution and low contour, a faint spherical density is observed in the central pore of the CA-NTD hexamer (Supplementary Fig. 1e–g).

Morphology of the HTLV-1 immature Gag lattice
The SPA cryo-EM reconstruction revealed the CA region of the Gag lattice was the best resolved Gag subdomain. The matrix and nucleocapsid had densities visible at low resolution within the CA reconstruction but were unable to be modeled (Supplementary Fig. 1e). The CA monomers consist of distinct CA-NTD and CA-CTD domains, connected through a flexible linker, like other described immature retroviral capsids9,12,15,16. Similar to these other retroviral lattices, the hexamer lattice is supported by a three-fold interface in the CA-NTD and a two-fold interface in the CA-CTD (Fig. 2b, d). However, the HTLV-1 Gag lattice does not adopt the typical wine-glass morphology that has been described for the other retroviral immature Gag lattices (Supplementary Movie 2). Rather, the CA-NTD forms a tight pore at the center of the hexamer, adopting a conformation similar to that of the CA-NTD seen in mature retroviral CA lattices (Supplementary Fig. 3a-f)37,38. The root mean square deviation of the backbone for the best aligned monomer within the central hexamer between the immature HTLV-1 CA-NTD and the mature bovine leukemia virus (BLV) or mature HIV-1 CA-NTD is 1.23 Å and 5.22 Å, respectively. For the HTLV-1 immature lattice, the CA-NTD provides most of the inter-hexamer and intra-hexamer interactions (Fig. 2). The CA-NTD trimerizes with a three-fold symmetric interface; one monomer from the central hexamer forms contacts with two other CA-NTD molecules, each from adjacent hexamers. This three-fold interface is composed of three copies of helix 4 packed together with helix 5, adding further interactions (Fig. 2b, c). The CA-CTD dimerizes with a two-fold symmetric interface with an adjacent hexamer’s CA-CTD, involving intermolecular contacts from residues on a 310 helix, helix 8, and helix 9 (Fig. 2d–h). Interestingly, there is no significant interface and limited intermolecular contacts between CA-CTD molecules within the same hexamer. Further, no analogous 6HB structure was resolved beyond the CTD (Fig. 1c and Supplementary Fig. 1e). Overall, the architecture of the immature Gag CA lattice from SPA agrees with recent tomography approaches using HTLV-1-like particles18.

Mutations in the CTD dimer interface are detrimental to particle production
Interactions between the CA-NTD trimer interface have been previously well described for the HTLV-1 immature Gag lattice18,24. However, flexibility in the CA-CTD has prevented high-resolution determination of the CA-CTD dimer interface. Furthermore, little is known regarding the role of the CA-CTD dimer interface in immature particle production. The total interface area between adjacent CA-CTD molecules is 1166 Å2. The orientation of the CA-CTD monomers reveals that the main interfaces consist of a 310 helix that interacts with helix 8 of the adjacent CA-CTD monomer, symmetric helix 8-helix 8 interactions, and symmetric helix 9-helix 9 interactions (Fig. 2e–h). The improved resolution in the CA-CTD region allowed for structure-guided alanine mutagenesis to probe specific residues important for maintaining this interface and to better understand the role of the CA-CTD in HTLV-1 assembly. Briefly, WT or mutated HTLV-1 Gag was co-transfected with HTLV-1 envelope into HEK293T cells. The assembly efficiency of the mutant was compared to WT by measuring the amount of virus released into the viral supernatant via an anti-HTLV-1 p24 immunoblot (Fig. 2i). Full length Gag is expected at 47 kDa, however, smaller bands are commonly seen from HTLV-1 Gag particles18,39, likely due to non-specific cleavage from host proteases as the HTLV-1 protease is not present in these experiments. Mutation of W133 (310 helix) and I168 (helix 8) severely decreased particle production, while mutation of L172 (helix 8) decreased particle production two-fold (Fig. 2i, j). These observations suggest a hydrophobic pocket forms between the 310 helix and the top of helix 8, analogous to the hydrophobic pocket in the HIV-1 CA-CTD11 (Fig. 2e, f). Further down along helix 8, mutation of S175 drastically diminished particle production while mutation of Y174 resulted in a two-fold reduction in immature particle production (Fig. 2i, j). Asymmetric copies of S175 form a network of hydrogen bonds with the backbone carbonyl of S175 and the hydroxyl of S171 on the adjacent CA monomer (Fig. 2h). Symmetric copies of Y174 on helix 8 (Fig. 2h) appear to form π-π stacking interactions to help stabilize the interface, as the Y174A mutation resulted in a two-fold reduction in immature particle production (Fig. 2i, j). The Q182A (helix 9) mutation resulted in greater than a two-fold reduction in particle production (Fig. 2i, j). Q182 appears to form hydrogen bonds with residues Y174 and Q186 on the adjacent monomer (Fig. 2h).

Other noted residues that are not included in the shown immunoblot and quantification (Fig. 2h–j) include S171 (helix 8), K179 (helix 9), and Q186 (helix 9). As previously mentioned, residue S171 is part of the extensive hydrogen bonding network with S175, supplying S171-S171 and S171-S175 interactions (Fig. 2h). Residue K179 is predicted to form hydrogen bonding to the backbone carbonyl of residue Q186 (Fig. 2h). Lastly, Q186 is predicted to form hydrogen bonding with the adjacent Q186 or Q182 residues (Fig. 2h). However, mutation at these residues did not significantly reduce particle production (Supplementary Fig. 4).

Cryo-ET reveals incomplete and heterogeneous immature Gag lattice morphologies
Previously, we observed HTLV-1 particles with a heterogeneous and incomplete Gag lattice based on transmission electron microscopy20,21. Specifically, it was noted that regions of the Gag lattice had varying curvatures within the same particle and often had flattened patches. Furthermore, these flattened regions were found to coincide with larger gaps between the viral membrane and the CA region of the Gag lattice20,21. To better understand these features of the HTLV-1 lattice in three-dimensions, we utilized cryo-ET to map individual CA hexamer positions within the Gag lattice (Fig. 3a–e). Iterative rounds of particle alignment and symmetry expansion were conducted to determine accurate hexamer positions, poorly aligned particles were removed after each round. The iterative approach provides us with good confidence that the gaps noted in the lattice are real. Based on the derived coordinates and angles of the CA hexamers, the local mean curvature was estimated based on neighboring hexamers. As expected, the local curvature estimates varied significantly, even within the same virus particle, with radii ranging from 28 nm to 75 nm (Fig. 3c, e). The average distance between individual hexamers to the membrane was determined to be 16.3 ± 2.49 nm. Measurements were conducted from the top of the CA-NTD to the outer layer of the viral membrane. The distribution was slightly skewed to the right, likely representing the patches of lattice that visibly appear further from the membrane (Fig. 3f). The identification of lattice positions allowed for a 7.9 Å resolution determination via subtomogram averaging (Figs. 3g and S1b). The subtomogram averaging reconstruction is similar to that of the SPA reconstruction (Fig. 3g), providing confidence in the accuracy of the curvature and the distance analysis.

IP6 is dispensable for immature HTLV-1 particle production and lattice formation
As noted, a faint spherical density was observed in the central pore of the CA-NTD hexamer, which is a similar location to the binding site of IP6 in the mature HIV-1 lattice (Fig. 4a). The density disappears following map sharpening and is only present in low pass filtered maps at low contour (Figs. 4a and S1e–g). The density is weak compared to the CA protein regions (Supplementary Fig. 1f, g) and to the IP6 density seen in other retroviral immature CA reconstructions from cell derived immature particles11,14. However, the density is present in adjacent hexamers off the symmetry axis, implying that it is not an artifact from the imposed C6 symmetry (Fig. 4a). Similarly, the density is present in a non-symmetric reconstruction and varies at different hexamer center positions, suggesting not every hexamer binding site is occupied by the density or there is flexibility in the location (Supplementary Fig. 1e). Given the density is near a ring of lysines, K18, and the established role for IP6 as an important co-factor for lentiviral particle assembly, we performed a molecular dynamics simulation of HTLV-1 in the presence of IP6 to better understand the capacity of HTLV-1 to interact with IP6 (Fig. 4b). Interestingly, we observed IP6 can be highly coordinated by R13 and K18 (Fig. 4b). Residue R13 had not been previously modeled from our reconstruction (Fig. 1e). To test the ability of HTLV-1 CA to utilize IP6 or other polyanions for assembly, we used an in vitro assembly assay of purified HTLV-1 CA39. IP6 and mellitic acid greatly enhanced CA assembly, as noted by the increasing proportion of CA in the pellet fraction at increasing concentrations of the polyanion (Fig. 4c). The CA protein in the pellet fraction was confirmed to form ordered assemblies via negative stain transmission electron microscopy, by either forming helical tubes with the addition of IP6 or crystal sheets with addition of mellitic acid (Fig. 4e). The proportion of CA in the pellet fraction did not increase with the addition of sodium phosphate or dNTPs, suggesting they are unable to stimulate HTLV-1 CA assembly in vitro (Fig. 4c). Mutagenesis of R13 and K18 was conducted to test their predicted role in coordinating IP6. Indeed, the amount of CA recovered in the pellet was greatly diminished for the R13A and K18A single mutations compared to WT and required higher concentration of IP6 (Fig. 4d). The R13A and K18A single mutants maintained their ability to assemble into ordered helical tubes, but only at 250 µM IP6 (Fig. 4e). Mutation of both R13A/K18A resulted in no CA recovered in the pellet for all IP6 concentrations tested (Fig. 4d). Furthermore, no ordered assemblies were seen for R13A/K18A via negative stain at 250 µM IP6, suggesting a loss of binding (Fig. 4e).

The heterogeneity of the helical tubes, specifically their preference to form multiple layers and aggregate together (Fig. 4e), prevented structure determination to validate the binding site of IP6. A low resolution subtomogram averaging reconstruction was conducted on a CA A94K mutation that preferentially formed microcrystals with addition of 250 µM IP6 (Supplementary Fig. 5). A strong density is apparent at the center of the CA-NTD pore, consistent with the predicted IP6 binding site (Supplementary Fig. 5c). The immature CA-NTD structure can fit well into this reconstruction, providing evidence that the in vitro assemblies form the immature lattice and that IP6 can bind to the immature structure (Supplementary Fig. 5c).

Given the role of IP6 in the HIV-1 lifecycle, the ability of IP6 to enhance HTLV-1 CA assembly in vitro, and the small density in the reconstruction, experiments were conducted to test if IP6 has a role in HTLV-1 immature particle assembly. To do this, a previously reported 293T cell line with knockout of either inositol pentakisphosphate 2-kinase (IPPK KO) or inositol polyphosphate kinase (IPMK KO) was used to produce particles28,29. IPPK is responsible for converting IP5 to IP6, while IPMK is responsible for converting IP4 to IP5 and IP5 to IP6. Transient overexpression of the multiple inositol polyphosphate 1 (MINPP1)—which converts IP6 to IP5, IP5 to IP4, and IP4 to IP3—enhances IP6 and IP5 depletion in both IPPK and IPMK KO cells40. HTLV-1 Gag and envelope were co-transfected with an empty vector into the WT control 293T cells or co-transfected with MINPP1 into either the IPPK or the IPMK KO cell lines. Co-transfection of the KO cell lines with HIV-1 Gag and envelope was selected to use as a control, based upon reports that IP6 depletion decreased immature particle production28,29,40. As expected, there was a reduction in HIV-1 immature particle production in both the IPPK KO and IPMK KO cells overexpressing MINPP1 compared to that of WT control 293T cells, measured by the amount of p24 capsid protein released into the cell culture supernatant (Fig. 4f, g). Intriguingly, there was no significant difference in the immature HTLV-1 particle production between the 293T WT cells and the IPPK KO and IPMK KO cells transiently overexpressing MINPP1 (Fig. 4f, g). This observation provides evidence in support of the conclusion that IP6 is not required for immature HTLV-1 particle assembly. To confirm whether HTLV-1 particles produced in IP6-depleted cells formed a WT particle morphology, particles were purified from IPPK KO cells overexpressing MINPP1 and then analyzed by cryo-EM (Fig. 4h). Analysis of particle morphology revealed a WT Gag lattice with no morphological defects, providing further support of the conclusion that IP6 depletion does not impact HTLV-1 particle assembly or Gag lattice morphology.

Methods
Immature viral particle production and purification
An expression plasmid encoding the HTLV-1 Gag gene (HTLV-1 Gag pN3 vector) was co-transfected with an expression plasmid for the HTLV-1 envelope gene into HEK293T/17 cells (ATCC-CRL3216) using GenJet Ver. II (SignaGen Laboratories, MD). The transfection ratio was at a 10:1 mass of the Gag:envelope expression plasmids. The supernatant was collected 48 h post-transfection, spun at 1000 RCF for 5 min., then passed through a 0.22 µm filter. The particles were purified following a previously described protocol14. Briefly, supernatant was pelleted over an 8% (v/v) Optiprep cushion in STE buffer (100 mM NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA). The sample was spun at 111,338 RCF for 100 min. in a 50.2 Ti rotor (Beckman Coulter, CA). The resulting pellet was resuspended in STE and loaded over a 10, 20, 30% (v/v) OptiPrep gradient for 3 h at 191,986 RCF in a SW55 Ti rotor (Beckman Coulter, CA). A band between the 20 and 30% (v/v) steps in the gradient was extracted and pelleted with a 1 h spin at 151,693 RCF and resuspended STE.

Immature virus particle production, western blot, and quantification
To create the HTLV-1 CA-CTD site-directed mutants, mutations were engineered into the HTLV-1 Gag pN3 vector by using the Gibson Assembly® protocol (New England Biolabs, MA). Mutant or WT HTLV-1 or HIV-1 Gag expression plasmids were co-transfected with the corresponding HTLV-1 or HIV-1 envelope expression plasmids at a mass ratio of 10:1 (gag:env expression plasmids) into a single 10 cm plate of either WT HEK293T/17 (ATCC-CRL3216), IPPK, or IPMK cells. For the IP6 depletion assay, a MINPP1 expression or empty vector was co-transfected with the Gag:Env expression plasmids. The IPPK and IPMK KO cells, along with the MINPP1 expression vector, were kindly provided by Dr. Eric O. Freed (NCI-Frederick). The supernatant was collected 48 h post transfection, spun at 1000 RCF for 5 min. and filtered through a 0.22 µm filter. Virus particles were pelleted over an 8% (v/v) OptiPrep cushion in STE buffer at 111,338 RCF for 100 min. in a 50.2 Ti rotor (Beckman Coulter, CA). The pellet was resuspended in STE buffer, then pelleted once more at 16,000 RCF for 1 h and resuspended in 20 µL STE. The transfected HEK293T/17 cells were collected and lysed with CelLytic MTM (Sigma-Aldrich, MO) with the addition of a protease inhibitor cocktail (cOmpleteTM Proease Inhibitor Cocktail, Sigma-Aldrich, MO). The lysate was incubated at 4°C on a rocking platform and then centrifuged for 30 min at 16,000 RCF. The supernatant and lysate were run on an SDS-PAGE gel for 45 min. and then transferred to a nitrocellulose membrane for 1 h at 100 v. Membranes were blocked overnight with 5% milk (w/v) in TBST. Blots were incubated with 1:2000 anti-HTLV-1 p24 antibody (6G9, Santa Cruz) or anti-HIV-1 p24 antibody (24-4, Santa Cruz), washed 5x for 3 min. each in TBST, then incubated with 1:2000 anti-mouse IgG StarBrightTM Blue 700 (Bio-Rad, CA). Lysate loading was normalized to GAPDH by addition of 1:1000 anti-gapdh hFABTM rhodamine (Bio-Rad, CA). HTLV-1 p24 signal in the lysate and supernatant was quantified with Fiji ImageJ gel analysis. The supernatant signal was double normalized to the lysate p24 and GAPDH signal.

HTLV-1 CA purification
The HTLV-1 CA protein (Gag amino acids 132–349) was purified using the protocol previously described39. Mutations were engineered into the vector using the Gibson Assembly® protocol (New England Biolabs, MA). Briefly, transformed BL21 cells were grown overnight at 37°C in ZY auto-induction media. The cells were harvested by centrifugation and resuspended in lysis buffer (250 mM NaCl, 50 mM Tris, pH 8) and stored at −80°C until used. The resuspended cells were lysed by the addition of 10 mg of lysozyme and incubated on ice for 30 min. The lysate was sonicated for three, 10-min cycles at 40% amplitude (QSONICA, Q500). The lysate was clarified by centrifugation at 12,000 RCF for 30 min, and the supernatant was added to equilibrated NI-NTA beads (Ni Sepharose High Performance, Cytiva, MA). The lysate supernatant was incubated overnight at 4°C on a rocking platform. Unbound lysate was allowed to flow through the Ni-NTA beads. The beads were washed with 30 mL of lysate buffer followed by 30 mL of wash buffer 2 (250 mM NaCl, 50 mM Tris, 25 mM imidazole, pH 8). The bound protein was eluted by addition of 5 mL elution buffer (250 mM NaCl, 50 mM Tris, 500 mM imidazole, pH 8). The eluted protein was dialyzed overnight at 4°C in cleavage buffer (250 mM NaCl, 50 mM Tris, 1 mM DTT, pH 8) and the 6xHis SUMO tag was cleaved by addition of house made Ulp1 protease. The cleaved protein was run over a Ni column (HisTrap HP 1 mL, Cytiva, MA) to separate the 6xHis SUMO tag from the HTLV-1 CA. Fractions containing the purified CA protein were concentrated to 10 mg/mL and flash frozen until use.

HTLV-1 CA in vitro assembly
Purified protein was diluted to 100 μM and dialyzed (PierceTM Slide-A-Lyzer® Mini Dialysis Units, Thermo Fisher, MA) into assembly buffer (50 mM NaCl, 50 mM MES, pH 6.5) overnight at 4°C. Following dialysis, different concentrations of polyanions were added (IP6, mellitic acid, dNTP, or sodium phosphate) to initiate assembly. The protein was incubated at room temperature for 2 h and then spun at 10,000 RCF for 10 min to separate the assembled CA from the soluble CA. For the SDS-PAGE analysis, an aliquot was taken prior to centrifugation to serve as the total input. The pellet was resuspended in fresh assembly buffer with the corresponding polyanion.

Negative stain transmission electron microscopy
Carbon support film on square copper grids (CF300-CU-50, Electron Microscopy Sciences) was glow discharged for 30 s at 10 mA, and 3 µL of fresh HTLV-1 CA assemblies were applied. The grids were incubated for 2 min, blotted, washed 3 times in 2% uranyl acetate, blotted, incubated for 2 min. in 2% uranyl acetate, and blotted on once more. Grids were imaged at 120 keV in a FEI Tecnai Spirit Bio-Twin transmission electron microscope.

Transmission electron microscopy (TEM) preparation
Quantifoil® R2/1 200 mesh TEM grids were glow-discharged for 30 s at 10 mA. Frozen grids were prepared in a Leica EM GP2 automatic plunge freezer with 3.5 µL of concentrated sample. The sample was blotted at a range of different times with Whatman 1 filter paper (CAT# 1001-070 Cytiva, MA) and plunged into liquid ethane. Ice and sample quality were screened on a FEI TF30 transmission electron microscope (FEI Company, Hillsboro, OR).

HTLV-1 single particle analysis data collection and image reconstruction
Movies were collected at the National Cancer Institute Cryo-EM Facility at the Frederick National Laboratory on a Thermo Fisher Titan Krios and Gatan K2 camera (Supplementary Table 1). Movies were motion corrected and binned by 2 (1.32 Å/pixel) using RELION-4.0 implementation of MotionCor251. Contrast transfer function (CTF) information was determined using CTFFIND452. All processing was conducted using RELION-4.053,54 and RELION-5.055. See (Supplementary Fig. 2) for a detailed schematic of the processing pipeline. An initial subset of particles was picked from a randomly selected 50 micrographs. The extracted particles were subjected to 2D class averaging, and good classes were saved. The saved classes were used for Topaz auto-picking56, resulting in 2.5 million particles. Particles were extracted with a box size of 128 pixels, corresponding to 338 Å. The following particle list was aligned with C6 symmetry to an initial reference generated from the cryo-ET and subtomogram averaging procedure (Fig. 3g). Successive rounds of RELION-4.0 3D classification with image alignment resulted in a final particle set of 140,020. Two rounds of CTF correction were implemented in RELION-4.0 (anisotropic magnification, beam tilt, and per-particle defocus estimates), followed by 3D auto-refine. Next, two rounds of Bayesian polishing were conducted in RELION-4.0 along with new per-particle defocus estimates and a new 3D auto-refine. Finally, a 3D auto-refine in RELION-5.055 with blush regularization followed by a final Bayesian polishing step and per-particle defocus estimate resulted in the 3.4 Å reconstruction map. Finally, the map was locally sharpened using the high Res model from DeepEMhancer57.

Real-space refinement of HTLV-1 Gag CA model based upon the cryo-EM density map
A specialized cryo-EM real-space refinement protocol was developed for the present work (Supplementary Fig. 6). First, we derived de-novo structures of the HTLV-1 Gag (HTLV-1 CA131-344)58 using alphaColab59,60. The alphaColab predicted CA-NTD, and obtained CA-CTD dimer18 were docked separately into the cryo-EM density map (Supplementary Fig. 7a) using ChimeraX61. Then, a model containing a CA hexamer and its immediate neighbors was achieved by rigid body docking of the model derived in the previous step (Supplementary Fig. 7b). Later, the CA-N terminus, CA-C terminus, and flexible linker were regenerated by homology modeling using Rosetta (Supplementary Fig. 7c, d)62–65. Local rebuilding procedures were performed using Cartesian Sampler in Rosetta to improve the quality of the model fitted to the density. Next, the model was iteratively refined by applying FastRelax66–68 protocol to the residues with low local-cross correlation (LCC), and poor MolProbity69–72 score utilizing supervised and unsupervised protein refinement tools, respectively (Supplementary Fig. 7e). A neutralized-solvated system, containing 18 HTLV-1 CA monomers, ions, and water molecules, was achieved following the procedure described in Perilla et al.73. Asymmetrical MDFF simulation, followed by minimization with conjugate gradient algorithm were performed using NAMD 3.0.174,75.

A separate model without IP6 was further refined by docking three CA monomers into the three-fold hexamer interface of the HTLV-1 CA lattice. One round of Phenix Real Space Refine was performed, followed by manual inspection and adjustments in the UCSF ChimeraX61 plugin ISOLDE76. This was followed by a final round of Phenix Real Space Refine.

Fitting of the resulting models to the cryo-EM density was examined quantitatively by calculating the local cross correlation between the simulated density of the model and the cryo-EM density map, and separately by calculating the per-atom Q-score (Supplementary Fig. 8 and Supplementary Fig. 9).

Tomography data collection, tomogram reconstruction, and subtomogram averaging
Samples were mixed with 10 nm gold beads prior to plunge freezing. The tilt series were collected at the National Cancer Institute Cryo-EM Facility at the Frederick National Laboratory on a Titan Krios using a K3 camera with a pixel size of 1.42 Å (Supplementary Table 2). Frames were motion corrected using MotionCor251. Tomogram reconstruction was performed in IMOD77, and CTF corrected using outputs from CTFFIND452. Reconstructed tomograms were imported to Dynamo78, and hexamer positions were identified following the described protocol for HIV-1 Gag lattice reconstruction79. Briefly, viral particles were estimated using a dipole model, and a coordinate was placed every ~300 Å. An initial reference was generated directly from 4x binned extracted subtomograms (64 pixel, 363 Å3) using orientations based on the spherical model. Subtomograms from one tomogram were aligned to the reference, and the six-fold (C6) symmetry of the central hexamer was manually determined. The entire subtomogram set was refined against the symmetrized reference using C6 symmetry. Following alignment, hexamer positions were filtered per tomogram based on the cross-correlation value in the Dynamo table file. New hexamer positions were added using symmetry expansion based on the parameters of the lattice. Iterations of particle refinement, particle filtering, and symmetry expansion were conducted until there was no improvement in the particle locations.

A custom MATLAB program was made to determine the principal local Gag curvatures and to determine the distance from the capsid layer to the membrane outer surface. Cross-correlation filtered coordinates were imported into RELION-4.054 for subtomogram averaging. Briefly, the imported coordinates were used to make an ab-initio model from 4x binned pseudo-subtomograms (64 pixel, 363 Å3) and used as a reference for an initial refinement. A 3D classification was performed with alignment, and the good classes were kept for refinement at 2x binning (128 pixels, 363 Å3) and finally 1x binning (256 pixels, 363 Å3).

Statistical analysis
Western blot quantification was performed by using Fiji ImageJ. Band intensity values were averaged, and the standard error of the mean was determined. Significance and p values were determined by using the calculated average and standard error of the mean using an unpaired two-sided t-test.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript provides a detailed structural analysis of the immature capsid lattice of HTLV-1. By cryoEM, the authors\nachieved a 3.4 Å resolution reconstruction of the immature CA lattice, resolving both the CA-NTD and CA-CTD domains.\nThe structure reveals stabilization through a trimeric NTD and a dimeric CTD interface. Complementary cryoET highlights\nheterogeneity in lattice curvature and variable spacing between the CA layer and the membrane. Notably, the study\ndemonstrates that inositol hexakisphosphate (IP6), while promoting CA assembly in vitro, is dispensable for lattice formation\nin situ, leaving an intriguing question for future inquiries. Nonetheless, we believe that these findings offer valuable insights\ninto HTLV-1 particle morphology, lattice organization, and the divergent role of IP6 in retroviral assembly. However, several\nareas require improvement to strengthen the manuscript, including several major issues and some minor points, as itemized\nbelow.\nMajor issues:\n1. Comparison with HIV. Since much is known for HIV concerning Gag assembly and capsid maturation, as well as the\nessential role of IP6 for HIV replication, the finding that IP6 is non-essential for HTLV-1 replication is significant. This point,\nhowever, is not described with sufficient clarity in the Discussion section. I suggest that the authors outline a detail\ncomparison between the two viruses, particularly, list all the conserved features, as well as the different features this study\nestablished in the current study. These can be done through a new Table.\n2. Insufficient Technical Detail for CryoEM and Subtomogram Averaging. The descriptions of single-particle cryoEM (SPA)\nand subtomogram averaging (StA) lack critical technical details. Please provide specifics on parameters such as box size,\napplied symmetry, refinement strategy, CTF refinement procedures, and particle orientation distributions. The reported\nresolution improvement from 4.3 Å to 3.4 Å following CTF refinement is significant. A detailed explanation of the methods\nused to achieve this enhancement, including any iterative refinement steps or software tools, is necessary to ensure\nreproducibility and transparency.\n3. Overly Lengthy Discussion. The Discussion section is protracted and lacks focus, obscuring the study’s key findings. A\nsubstantial revision is needed to streamline the content and highlight primary conclusions. Structuring the discussion into\nconcise paragraphs, each with a clear topic sentence, would enhance readability and emphasize the most significant\ninsights.\n4. Concerns with Fig. 3A Tomogram Processing. The tomograms in Fig. 3A appear overly smoothed after IsoNet processing,\nresulting in poor visibility of the lipid bilayer. To justify the use of IsoNet, please include a side-by-side comparison of\ntomograms before and after processing, showing both x–y and x–z planes. This comparison should demonstrate tangible\nimprovements in contrast or feature resolution to support the processing approach.\n5. Limited Novelty of Subtomogram Averaging Results. The subtomogram averaging results are of lower resolution\ncompared to previously published datasets. The manuscript should clearly articulate the unique biological insights gained\nfrom cryoET that could not be derived from the higher-resolution SPA reconstruction. A stronger justification of the cryoET\ncontribution is needed to underscore its value.\n6. Lack of Morphological Analysis for CTD Mutants. While the manuscript demonstrates the impact of CTD interface\nmutations on particle production, it does not assess the morphology of virus particles bearing these mutations. Have the\nauthors investigated whether these mutations disrupt lattice structure or curvature? Conducting cryoET analysis on these\nmutants would significantly bolster the conclusions regarding the functional role of the CTD interface.\n7. Issues with Fig. S9B Color Scheme and Validation. The color scheme in Fig. S9B is difficult to interpret due to low\ncontrast. A higher-contrast palette would improve clarity. Additionally, side chain fitting should be validated using\nquantitative metrics, such as Q-scores, for representative residues or key regions. Including these metrics would enhance\nthe robustness of the structural analysis.\n8. Improvements Needed for Fig. S3J Sequence Alignment. The sequence alignment in Fig. S3J lacks conservation\nindicators, limiting its interpretability. Please revise the figure to include a conservation bar, appropriate shading,\nsecondary structure annotations, and highlights for functionally critical residues (e.g., those at the CTD dimer interface). These\nadditions would significantly improve the figure’s utility.\n9. Validation of Side Chain Interactions. To support claims about side chain interactions, please provide a superposition of\nthe atomic model with the corresponding electron density. This is particularly crucial for validating hydrogen bonding and\nhydrophobic interactions at the dimer interface, ensuring the reliability of the structural interpretations.\n10. Discrepancy in MolProbity Scores. The MolProbity score for the 18-mer model (0.76) is unexpectedly better than that for\nthe 3-mer model (1.4), despite the former’s greater size and complexity. Please explain this discrepancy, clarifying whether\ndifferent refinement strategies or validation criteria were applied to the two models.\nMinor issues:\n1. Last sentence of abstract needs improvement.\n2. Some of the density maps are not appropriately displayed and appeared strange. For example, Fig. 1B, the density\nappeared disconnected yet there is no small noise present! Maybe this is due to inappropriate use of the “hide dust”\nfunctionality of UCSF Chimera.\n3. Grammar (Line 565): Revise “Differing concentrations of polyanions were added initiate assembly” to “Different\nconcentrations of polyanions were added to initiate assembly.”\n4. Movie Annotations: Movies S1 and S2 lack clear labels or contextual annotations. Adding captions or in-frame labels\nwould aid interpretation.\n5. Typographical Errors:\n  Line 615: Correct “preformed” to “performed.”\n  Line 608: Correct “molPorobity” to “MolProbity.”\n  Line 366: \"form the IP6-depleted cells\" should be \"from the IP6-depleted cells\" for correct preposition usage.\n  Line 616: \"preformed\" should be \"performed\" in the context of Phenix Real Space Refine.\n  Line 944: \"Author contributions\" section lacks consistent formatting; \"JR\" should be \"JRP\" to match initials used elsewhere.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Review of NatComms_611708_0: High-resolution in situ analysis of the human T-cell leukemia virus capsid protein reveals\ninsights into immature particle morphology.\nSummary\nThe manuscript describes the use of cryogenic electron microscopy (cryo-EM) to perform single particle reconstruction of the\ncapsid from immature particles for the deltaretrovirus Human T-cell Leukemia Virus (HTLV). Immature particles were\nproduced by transient transfection of HEK293T/17 cells with plasmids encoding gag and env genes and form due to the\nabsence of the virally encoded protease. The 3.4Å single particle cryo-EM map of the immature capsid revealed a\nhexameric lattice similar to other retroviruses with a notable difference that the capsid N terminal domain (CA-NTD) provides\nstabilizing interactions in contrast to other retroviruses. Critical residues at dimeric interfaces between neighboring CA-NTD\nwere identified by site directed mutagenesis with three mutants, W133A, I166A, and S175A, failing to produce immature\nparticles. Cryo-electron tomography (cryo-ET) and subtomogram averaging revealed heterogeneity and incomplete Gag\nlattice formation. In a further contrast to retroviruses, the inositol polyphosphate IP6 was not found to be required for\nimmature particle assembly despite its requirement for capsid lattice formation when expressed in vitro. Residues R13 and\nR18, which face inward into the CA-NTD central pore and predicted to interact with IP6, were critical for in vitro assembly of\nHTLV-1 CA.\nGeneral Comments\nThe manuscript is well written and provides interesting insights into the assembly of the immature HTLV-1 capsid. The\nfigures are well presented with sufficient detail to understand the concepts being conveyed. While the lack of stabilizing\nelements at the six-fold symmetry axis and requirement for the HTLV-1 CA-NTD to form critical lattice elements is not novel,\nas these were previously identified by Obr et al., 2024 (NSMB https://doi.org/10.1038/s41594-024-01390-8), the high\nresolution cryo-EM map (3.4Å) and mutagenesis data at the dimeric interfaces provide new knowledge about the formation\nof deltaretrovirus immature capsids.\nThe IP6 data were intriguing. However, the resolution is not sufficient to resolve the IP6 molecule in the immature capsids.\nAs this density was only seen in lowpass filtered maps and at low contour the authors note that not all CA-NTD central pores\nmight be occupied. Furthermore, the lack of IP6 requirement for immature HTLV-1 particle production suggests that another\nmolecule or molecules could be present. Given that mellitic acid can compensate for IP6, these data also imply other\nmolecules could be present in the CA-NTD central pores. Although IP6 was not a requirement for immature particle\nassembly, control experiments to establish the requirement of IP6 for mature particle assembly were not performed to rule\nout its role in the production of infectious virus. Would IP6 be needed for the production of infectious HTLV-1?\nAs there is uncertainty about the identity of the molecule in the CA-NTD central pores, the molecular dynamics simulation,\nalthough useful for modelling molecules into map densities, does not add to the manuscript on this occasion and should be\nomitted. It is unsurprising that mutagenesis of R13 and K18 disrupted the in vitro assembly of HTLV-1 CA. This did not\nrequire molecular dynamics modelling and could have been inferred from the positively charge nature of these residues and\nthe interaction between the negatively charged IP6.\nSpecific Comments\nRemove in situ from the title. The data are not generated in situ, where the original process occurs inside an HTLV infected\ncell. The cryo-EM and cryo-ET data are generated from artificially produced immature particles generated by transfection.\nThe capsid structures are certainly intra particle, yes, but not in situ.\nThe tomographic analysis and subtomogram averaging of the immature HTLV-1 Gag lattice reveal incomplete and\nheterogenous lattice morphologies. Could these differences arise from the centrifugation steps used to purify the immature\ncapsid containing particles?\nLine 285 Fig. 3 figure legend and Fig. S1B states 7.9Å resolution for the subtomogram averaging where in the text it is\nreported as 7.8Å. Please correct.\nIn the materials and methods, there are inconsistencies describing the centrifugation steps. In some cases ‘rpm’ is used\nrather than ‘x g’. Replace both with RCF.\nLines 587 and 621 “Frederick” is spelt incorrectly.\nLines 631 to 634, please provide a more detailed description of the Relion-4.0 refinement. There are a lot of details missing\nand it would not be possible to repeat the reconstruction using the details provided."}, "author_response": "July 30, 2025\nRe: Revision of Nature Communications manuscript NCOMMS-25-35579\nBelow, we have provided our point-by-point responses to each of the reviewers’ comments.\nIn response to the comments of Reviewer’s 1 & 2:\nComment: 1. Comparison with HIV. Since much is known for HIV concerning Gag assembly and capsid\nmaturation, as well as the essential role of IP6 for HIV replication, the finding that IP6 is non-essential for\nHTLV-1 replication is significant. This point, however, is not described with sufficient clarity in the Discussion\nsection. I suggest that the authors outline a detail comparison between the two viruses, particularly, list all the\nconserved features, as well as the different features this study established in the current study. These can be\ndone through a new Table.\nResponse: A paragraph has been added to the discussion (instead of a table) to the discussion (lines 396-\n467) to indicate the main differences between the HIV-1 and HTLV-1 immature lattice organization, conserved\nfeatures, and differences in IP6 binding. Comparisons are also made in other paragraphs in the discussion,\nparticularly in regard to IP6 incorporation.\nComment: 2. Insufficient Technical Detail for CryoEM and Subtomogram Averaging. The descriptions of\nsingle-particle cryoEM (SPA) and subtomogram averaging (StA) lack critical technical details. Please provide\nspecifics on parameters such as box size, applied symmetry, refinement strategy, CTF refinement procedures,\nand particle orientation distributions. The reported resolution improvement from 4.3 Å to 3.4 Å following CTF\nrefinement is significant. A detailed explanation of the methods used to achieve this enhancement, including\nany iterative refinement steps or software tools, is necessary to ensure reproducibility and transparency.\nResponse: Extra detail has now been added in the Materials and Methods section for both the SPA and StA\nexperiments. Box size, applied symmetry, refinement tools, CTF refinement steps, and Bayesian polishing\nsteps are now described in detail. All software tools used are described. These details have also been updated\nin Fig. S2 (i.e., SPA workflow). The particle orientation distribution in the final reconstruction is now described\nin Fig. S1. Details regarding the box size and applied symmetry for the StA have now been included in the\nMaterials and Methods section, as well as a more thorough description of the processing in Dynamo.\nComment: 3. Overly Lengthy Discussion. The Discussion section is protracted and lacks focus, obscuring the\nstudy’s key findings. A substantial revision is needed to streamline the content and highlight primary\nconclusions. Structuring the discussion into concise paragraphs, each with a clear topic sentence, would\nenhance readability and emphasize the most significant insights.\nResponse: The discussion has now been streamlined and revised in order to emphasize (1) comparisons\nbetween HIV-1 and HTLV-1, (2) the role of IP6, (3) HTLV-1 particle heterogeneity, (4) the role of the HTLV-1\nCA-CTD, and (5) implications for maturation.\nComment: 4. Concerns with Fig. 3A Tomogram Processing. The tomograms in Fig. 3A appear overly\nsmoothed after IsoNet processing, resulting in poor visibility of the lipid bilayer. To justify the use of IsoNet,\nplease include a side-by-side comparison of tomograms before and after processing, showing both x–y and x–\nz planes. This comparison should demonstrate tangible improvements in contrast or feature resolution to\nsupport the processing approach.\nResponse: The tomograms in Fig. 3A were originally IsoNet-corrected for visualization purposes only. These\nimages have now been replaced with the non-corrected tomogram slices.\nComment: 5. Limited Novelty of Subtomogram Averaging Results. The subtomogram averaging results are of\nlower resolution compared to previously published datasets. The manuscript should clearly articulate the\nunique biological insights gained from cryoET that could not be derived from the higher-resolution SPA\nreconstruction. A stronger justification of the cryoET contribution is needed to underscore its value.\nResponse: We previously observed distinct curvature changes in HTLV-1 CA lattices by cryoEM. CryoET and\nSTA allowed us to analyze the curvatures of the lattice in 3D, as well as the distance distribution between the\ninner leaflet of the viral membrane and the Gag lattice. Although the STA results are of lower resolution\ncompared to the SPA, the similarities of the STA reconstruction to the SPA reconstruction provides validation\nto the accuracy of the curvature and CA to membrane distance analysis. This reasoning was clarified in the\nresults section.\nComment: 6. Lack of Morphological Analysis for CTD Mutants. While the manuscript demonstrates the impact\nof CTD interface mutations on particle production, it does not assess the morphology of virus particles bearing\nthese mutations. Have the authors investigated whether these mutations disrupt lattice structure or curvature?\nConducting cryoET analysis on these mutants would significantly bolster the conclusions regarding the\nfunctional role of the CTD interface.\nResponse: We agree with Reviewer 1 that a morphological analysis of the CTD mutants would be an\nimportant next step. We believe this analysis – and a thorough analysis of the lattice structure or curvature that\nresult from these mutations – is beyond the scope of the current manuscript. We also note that some mutants\n(e.g., W133A, I168A, and S175A) do not produce enough particles to readily allow for high resolution cryoEM\nor cryoET analysis.\nComment: 7. Issues with Fig. S9B Color Scheme and Validation. The color scheme in Fig. S9B is difficult to\ninterpret due to low contrast. A higher-contrast palette would improve clarity. Additionally, side chain fitting\nshould be validated using quantitative metrics, such as Q-scores, for representative residues or key regions.\nIncluding these metrics would enhance the robustness of the structural analysis.\nResponse: We thank the reviewer for this insightful comment. We have re-rendered the local correlation figure\nusing a more contrasting color palette for improved visual clarity. In addition, we have calculated the Q-score\nfor all atoms and visualized them in Fig. S8b,c. At the calculated model-to-map resolution (4.0 Å), our 18-mer\nmodel matches the density with good accuracy. The average Q-score for all residues has a mean of 0.37 and\nstandard deviation of 0.15, which is within the expected Q-score (0.41) for models at 4.0 Å resolution. In\naddition, we have included Fig. S9, which visualizes the specific fitting of residues involved in interactions at\nthe dimer interface and IP6 coordination. Local cross-correlation values for residues at the dimer interface\nvaried between 0.4 to 0.9 with a mean of 0.8, indicating good agreement with the density. Side chain Q-scores\nfor the hydrophobic interface are 0.5 (W133), 0.46 (I168), and 0.45 (L172). Residues S171, S175 and Y174 at\nthe dimer interface have decent Q-score values around 0.3, while K179 and Q182 exhibit lower Q-score\nvalues, which is expected due to the flexibility of their sidechains. For residues involved in IP6 coordination,\nK18 shows good fitting to the density as indicated by its average LCC (0.75) and Q-score (0.34) values. R13,\nhowever, has a poorer representation in the density (avg. LCC 0.43, avg Q-score 0.04 across all chains). R13\nis the uppermost residue for which there is resolvable backbone density, however, there is no strong density to\nconfidently place the sidechain, which accounts for the low LCC and Q-score values. Despite this, we believe\nthat the R13 side chain orientation in the model is plausible, as it results from the physics-informed refinement\nprotocol performed on the model, which optimizes the backbone and sidechain conformations to minimize the\ninteraction energy between surrounding residues and ligands. We have added this analysis to the SI of the\nmanuscript.\nComment: 8. Improvements Needed for Fig. S3J Sequence Alignment. The sequence alignment in Fig. S3J\nlacks conservation indicators, limiting its interpretability. Please revise the figure to include a conservation bar,\nappropriate shading, secondary structure annotations, and highlights for functionally critical residues (e.g.,\nthose at the CTD dimer interface). These additions would significantly improve the figure’s utility.\nResponse: We have updated the sequence alignment and have included shading for conserved residues,\nsecondary structure annotations, and now indicate the residues important for IP6 binding and those residues\ninvolved in the CTD dimer interface.\nComment: 9. Validation of Side Chain Interactions. To support claims about side chain interactions, please\nprovide a superposition of the atomic model with the corresponding electron density. This is particularly crucial\nfor validating hydrogen bonding and hydrophobic interactions at the dimer interface, ensuring the reliability of\nthe structural interpretations.\nResponse: The superpositions of the side chains with the electron density have now been added to Figure S4.\nWe have also added supplemental Figure S9 visualizing the interacting residues at the dimer interface as well\nas residues interacting with IP6 in context with the cryo-EM density and local cross-correlation and Q-score\nmetrics.\nComment: 10. Discrepancy in MolProbity Scores. The MolProbity score for the 18-mer model (0.76) is\nunexpectedly better than that for the 3-mer model (1.4), despite the former’s greater size and complexity.\nPlease explain this discrepancy, clarifying whether different refinement strategies or validation criteria were\napplied to the two models.\nResponse: The same validation score was used for both models. However, the 18-mer model with IP6 was\ngenerated using only the molecular dynamics flexible fitting (MDFF) methodology. The 3-mer was initially\ngenerated using MDFF, but was also ran through several rounds of traditional Phenix refine for optimal side\nchain fitting. The discrepancy between the MolProbity scores is likely due to the different refinement strategies\nused.\nComment: 1. Last sentence of abstract needs improvement.\nResponse: The last sentence of the abstract has been edited to enhance clarity.\nComment: 2. Some of the density maps are not appropriately displayed and appeared strange. For example,\nFig. 1B, the density appeared disconnected yet there is no small noise present! Maybe this is due to\ninappropriate use of the “hide dust” functionality of UCSF Chimera.\nResponse: The “hide dust” feature was not used on any maps presented in the figures. The map previously\nshown in Figure 1b and 1c was the filtered unsharpened map directly from the RELION-5.0 refinement. Figure\n1B and 1C has now been updated to the final sharpened map (i.e., sharpened with deepEMhancer, which\nlimits noise) to match what is shown in other figure panels (i.e., Figure 1d, 2a, 2b, 2d).\nComment: 3. Grammar (Line 565): Revise “Differing concentrations of polyanions were added initiate\nassembly” to “Different concentrations of polyanions were added to initiate assembly.”\nResponse: This has been corrected (see lines 1124-1125).\nComment: 4. Movie Annotations: Movies S1 and S2 lack clear labels or contextual annotations. Adding\ncaptions or in-frame labels would aid interpretation.\nResponse: Annotations have now been added to both movies to indicate the different features of the models\nand maps.\nComment: 5. Typographical Errors:\nLine 615: Correct “preformed” to “performed.”\nLine 608: Correct “molPorobity” to “MolProbity.”\nLine 366: \"form the IP6-depleted cells\" should be \"from the IP6-depleted cells\" for correct preposition\nusage.\nLine 616: \"preformed\" should be \"performed\" in the context of Phenix Real Space Refine.\nLine 944: \"Author contributions\" section lacks consistent formatting; \"JR\" should be \"JRP\" to match\ninitials used elsewhere.\nResponse: All of these errors have been corrected in the revised manuscript.\nIn response to the comments of Reviewer 3:\nComment: The IP6 data were intriguing. However, the resolution is not sufficient to resolve the IP6\nmolecule in the immature capsids. As this density was only seen in lowpass filtered maps and at low\ncontour the authors note that not all CA-NTD central pores might be occupied. Furthermore, the lack\nof IP6 requirement for immature HTLV-1 particle production suggests that another molecule or\nmolecules could be present. Given that mellitic acid can compensate for IP6, these data also imply\nother molecules could be present in the CA-NTD central pores. Although IP6 was not a requirement\nfor immature particle assembly, control experiments to establish the requirement of IP6 for mature\nparticle assembly were not performed to rule out its role in the production of infectious virus. Would\nIP6 be needed for the production of infectious HTLV-1?\nResponse: We agree with Reviewer 3 that there remains uncertainty regarding the requirement for IP6 in\norder to produce infectious HTLV-1 particles. We now discuss the requirement of IP6 in the context of\ninfectious HTLV-1 particles in the revised discussion section (lines 698-716).\nComment: As there is uncertainty about the identity of the molecule in the CA-NTD central pores, the\nmolecular dynamics simulation, although useful for modelling molecules into map densities, does not\nadd to the manuscript on this occasion and should be omitted. It is unsurprising that mutagenesis of\nR13 and K18 disrupted the in vitro assembly of HTLV-1 CA. This did not require molecular dynamics\nmodelling and could have been inferred from the positively charge nature of these residues and the\ninteraction between the negatively charged IP6.\nResponse: We thank Reviewer 3 for this comment. We believe that the molecular dynamics is important for\nthis study and provides additional insights into the interpretation of the data present. As the density in the\ncenter of the CA-NTD pore is weak, it is unclear if this could be a polyanion. While it is true that the\ninvolvement of positively charged residues such as R13 and K18 in binding negatively charged ligands like IP6\ncan be inferred from basic electrostatics, the molecular dynamics simulations go beyond inference by providing\natomic-level insight into the stability and specificity of these interactions within the CA-NTD central pore. Given\nthe critically important role of IP6 in the lentiviral lifecycle, the observation that IP6 enhances assembly in vitro,\nand the observation of a strong density in our A94K CA reconstruction that was assembled with IP6 (Fig. S5),\nwe believe that there is strong justification to model IP6 into the density. Given the current uncertainty\nsurrounding the identity of the molecule within the pore, the MD simulations offer a mechanistic framework that\nsupports our mutagenesis studies of R13 and K18 and complements the cryo-EM data. As there are no space\nlimitations at this stage, we believe that retaining the simulation results enhances the completeness and\ninterpretive strength of the manuscript.\nComment: Remove in situ from the title. The data are not generated in situ, where the original\nprocess occurs inside an HTLV infected cell. The cryo-EM and cryo-ET data are generated from\nartificially produced immature particles generated by transfection. The capsid structures are certainly\nintra particle, yes, but not in situ.\nResponse: The term in situ has been removed from the manuscript title.\nComment: The tomographic analysis and subtomogram averaging of the immature HTLV-1 Gag\nlattice reveal incomplete and heterogenous lattice morphologies. Could these differences arise from\nthe centrifugation steps used to purify the immature capsid containing particles?\nResponse: We agree with Reviewer 3 that centrifugation could potentially cause morphological alterations to\nthe immature particles during purification. However, this is a common method to purify immature particles for\nimage analysis. Similar purification methods and subsequent morphological analysis has been conducted on\nHIV-1 (DOI: 10.1126/science.aaf9620), HIV-2 (DOI: 10.1016/j.jmb.2023.168143), MLV (DOI:\n10.1073/pnas.1811580115), and M-PMV (DOI: 10.1038/nature13838). We note that by using the same\npurification method, HTLV-1 is clearly distinct in the heterogenous morphology observed with immature\nparticles.\nComment: Line 285 Fig. 3 figure legend and Fig. S1B states 7.9Å resolution for the subtomogram\naveraging where in the text it is reported as 7.8Å. Please correct.\nResponse: This has been corrected.\nComment: In the materials and methods, there are inconsistencies describing the centrifugation\nsteps. In some cases ‘rpm’ is used rather than ‘x g’. Replace both with RCF.\nResponse: This has been corrected.\nComment: Lines 587 and 621 “Frederick” is spelt incorrectly.\nResponse: This has been corrected.\nComment: Lines 631 to 634, please provide a more detailed description of the Relion-4.0 refinement.\nThere are a lot of details missing and it would not be possible to repeat the reconstruction using the\ndetails provided.\nResponse: Extra details have now been added. Please also see our response to comment # 2 from Reviewer\n1."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The authors have answered all of my questions. The additional edits to the text and new figures very much help with\nunderstanding the data processing and the interpretation of the data."}, "author_response": ""}]

Decision letter