Conserved assembly architecture of the essential herpesvirus packaging accessory factor

  1. Elizabeth J. Bailey
  2. Swapnil C. Devarkar
  3. Renata Szczepaniak
  4. Laura M. Meißner
  5. Xinyu Chen
  6. Chunxiang Wu
  7. Sandra K. Weller
  8. Yong Xiong
  9. Allison L. Didychuk

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Abstract

To create a new wave of infectious virions, all herpesviruses require an accessory factor of unknown function to package their viral genomes into nascent capsids. Here, we present cryo-EM structures of the packaging accessory factor from the α-herpesvirus herpes simplex virus type 1 (HSV-1, UL32) and the β-herpesvirus human cytomegalovirus (HCMV, UL52). Unlike homologs from the γ-herpesviruses, neither UL32 nor UL52 form stable homopentameric rings. UL52 forms incomplete pentameric rings lacking one or two protomers. UL32 does not form stable higher-order species, but stabilization through chemical crosslinking revealed a novel quaternary structure where three pentameric rings assemble into a “tripentamer.” Our results reveal that herpesvirus packaging accessory factors adopt distinct oligomeric states but are constrained to pentameric symmetry. Assembly of protomers into a ring creates a positively charged central channel that we show is critical for infectious virus production in HSV-1. Taken together, our study points to a structurally conserved, essential function of packaging accessory factors across the Herpesviridae .

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    Reply to the reviewers

    Reviewer #1

    *Minor comments

    1. The authors suggest that the weak 4th protomer in the HCMV UL52 3-mer map is a consequence of flexibility. This may be the case, but it may also be the case that the class is polluted with 4-mer particles leading to reduced occupancy. Erasing the weak density and running a multi-model 3D classification providing the erased 3-mer and a 4-mer starting map may separate these.*

    We performed additional analysis (i.e., 3-mer and 4-mer particles were combined into a multi-class ab initio reconstruction followed by multi-class heterogenous refinement) and found that the original 3-mer map was a mixture of 3-mer and 4-mer states.

    We have updated Fig. 2a, Supplementary Fig. 2, Supplementary Fig. 3, Supplementary Table 1, Supplementary Movie 1, and removed the discussion of the weak protomer in the 3-mer map from the results section. We have updated our EMDB and PDB depositions accordingly.

      1. I found the supplemental figure to show the DNA in the tripentamer map too small, this is an interesting finding and should be shown more clearly.*

    We have increased the size of Supplementary Fig. 6 and moved the figure caption to another page to accommodate this enlargement.

    Reviewer #2

    *Major issues

    1. There is a high probability that the tripentamer is an artifact of the cross-linking. Because of this, it'd be great to know more about the cross-linking reaction, ideally mass spec identification and quantification of cross-links. This would also address the authors' speculation of contacts that stabilize the tripentamer. *

    Crosslinking is a commonly used technique to stabilize complexes that are observed through other means but do not survive the cryo-EM vitrification process. In an EMSA experiment (Supplementary Fig. 4a), UL32 binds 30 bp DNA and migrates slower than when bound to a 10 bp probe, consistent with formation of a supra-pentameric complex. The samples in the EMSA gels are not crosslinked. Additionally, an SDS-PAGE gel of the crosslinked product used for cryo-EM showed tight bands at molecular weights expected for oligomers, supporting specific crosslinking (Supplementary Fig. 4b). These results suggest that crosslinking stabilizes a species that can form but is relatively unstable in solution.

    Moreover, the author's claim "However, mutation of K532A/C535A reduced infectious virion production by half (Fig. 4b), suggesting that the tripentamer interface may play a role in the viral life cycle." Seems to be an overreach. Perhaps this is semantics but the data just show that these residues play a role in viral replication (albeit not a huge role based on the modest effect).

    We have modified the title of the results section (Line 216-217) to state that "Residues at the tripentamer interfaces contribute to infectious virion production in HSV-1" as well as Line 234 and 241 to indicate that the residues play a role in the viral life cycle.

    2) The density for the potential DNA does not look very convincing, although it still remains the strongest hypothesis. The authors should try to strengthen their argument. Does this putative DNA contact residues that they show are necessary for viral replication? Showing seq conservation on the structure could help their argument for the shared function of DNA-binding.

    The DNA likely contacts conserved residues at the base and midsection of the central channel (residues R302, R301, R293, K289, R580, R579, R572; see Fig. 6a). We have shown that these residues are important for the production of infectious virions (Fig. 6c): even a single point mutation (R572A) decreased production of infectious virus particles by more than 90%, and double and triple point mutants (R579A/R580A, K289A/R293A/R301A) eliminated production of infectious virus. Sequence conservation of these charged residues in the central channel regions is shown in Supplementary Fig. 1d, f.

    3) My last major issue is stylistic and concerns the descriptions of cryoEM structures. I found that the paper was a bit of challenge to read when the authors would introduce each structure. It was a bit of a slog to get through. Descriptions of the structures veered off into overly detailed comparisons that required constant comparison with the figure and didn't really advance my understanding past "the outer surfaces of the three orthologs are different." This masked the more interesting aspects of the authors' findings. Perhaps this could be summarized in supplementary figures or a table. Because this is a stylistic suggestion, the authors should feel free to ignore this request.

    We appreciate the reviewer's concerns about accessibility, but we are excited that these structures allowed us to thoroughly describe the convergent and divergent structural features across the *Herpesviridae *and hope that our in-depth analysis will allow for detailed mechanistic follow-up.

    *Minor comments

    1. The descriptions of structure determination in the text were often unclear. For example, "In the 3-mer map, a poorly-resolved fourth protomer is visible at low contour levels, suggesting that an additional protomer is present but highly flexible in this class (Supplementary Fig. 3a)." Alternatively, it could be that the classification algorithm wasn't able to fully separate particles that were 3-mers from the 4mers. *

    The reviewer is correct. As described above (Reviewer #1 comment 1), we performed additional analysis and found that the original 3-mer map was a mixture of 3-mer and 4-mer states. We have updated Fig. 2a, Supplementary Fig. 2, Supplementary Fig. 3, Supplementary Table 1, Supplementary Movie 1, the EMDB and PDB depositions, and removed the discussion of the weak protomer in the 3-mer map from the results section.

    *When describing the structure determination of the HSV1 accessory factor, the authors describe no other particles other than the tripentamer. Were there other particles observed? It'd be a bit surprising that all of the protein adopted the tripentamer state. *

    We agree that this result is striking. We picked particles using a 'blob picker' to avoid introducing template bias and found that the tripentamer is the predominant species. Below we show the results of 2D classification of blob picked particles (classes sorted by particle number; obvious junk classes excluded for clarity). There is one class that suggests a pentamer, but template picking with a pentamer template (based on ORF68) did not yield a pentamer class.

    Additionally, as we describe in the results section and show in Supplementary Fig. 6a, further processing of the consensus UL32 map showed that 60% of particles formed a complete tripentamer (i.e., 15-mer) while other the remaining 40% formed incomplete tripentamers, missing one or more protomers (e.g., 17% of particles formed a 14-mer).

    Was symmetry applied, particularly for the tripentamer that appears to have C-3 symmetry? This is in materials and methods but not clear why it isn't mentioned when describing the structure determination and results.

    No symmetry was applied in the reconstruction for either UL32 or UL52. While we previously noted this in the methods section and in Supplementary Table 1, we have added this information to the results section (Line 169-170), the Fig. 3 legend, and cryo-EM processing figures (Supplementary Figures 2, 5, 6) for clarity.

    2) Throughout the paper, the authors use the word "remodel" to describe structural differences between orthologs. However, this word usually carries the implication of conformational rearrangement within a protein, and not across orthologs. Please consider a different description.

    We agree with the reviewer and have removed the term "remodel" throughout the manuscript text (i.e., Lines 116, 118, 120, 122, 302, 306) and from Supplementary Figures 1, 3, and 5.

    3) Figure 2F is confusing and difficult to interpret. It seems that the main point is that these interfaces are conserved, which might be more easily displayed as a standard sequence conservation score mapped onto the structure. I'm also not sure that this figure is necessary as a main figure and could be supplemental.

    We agree that the conservation could also be shown this way and have added labels to universally conserved residues of the protomer interface to Supplementary Fig. 1b, c. We have also moved Fig. 2f to the supplement (now Supplementary Fig. 2g).

      1. The authors write "UL32 bound to the shortest probe tested (10 bp, Supplementary Fig. 4a)." This implies that ONLY the shortest probe is bound and that others are not bound. Consider rephrasing.*

    We have rephrased to clarify at all probes tested, included the shortest, bound DNA (Line 153).

      1. Frustum is misspellt. ;)*

    Thank you. Spelling has been corrected (Line 185).

    6) In the discussion, the authors speculate that the variability of the outer surface is due to "virus- or host-specific interactions". I'm confused by "host-specific interactions", because the host is the same for all three viruses. Perhaps the authors mean that the different accessory factors could interact with different host factors? If so, are the authors making a Red Queen argument? If so, it'd be pretty cool to do dN/dS analysis to test that hypothesis.

    The reviewer is correct in that all three viruses (HSV-1, HCMV, KSHV) infect the same host; however, they replicate in different cell types, which could potentially express different host factors. We have no evidence to support this hypothesis and intended to propose that UL32 and UL52 may be interacting/co-evolving with other viral factors required for genome packaging. We have clarified Line 308 to generalize that "these regions are involved in virus-specific interactions".

    To me, this window into evolution of this factor is the biggest advance of the work, and tbh I felt that the authors could lean into this a bit more in the discussion section. Are there any differences in the packaging mechanisms of the different herpes families that can be related to their different behavior? Any other molecular evolution analyses (e.g. dN/dS ratio analysis) that could inform their study?

    We agree that understanding the evolution of the packaging accessory factor is an interesting future area of research. There are differences in capsid structure and occupancy of capsid-associated factors across the herpesvirus family (PMID: 34696343). However, we lack a mechanistic (or structural) understanding of viral genome packaging components across the herpesviruses, raising the possibility that there are differences in packaging mechanisms.

    Interestingly, the further diverged alloherpesviruses and malacoherpesviruses (other families in the order Herpesvirales) do not appear to encode a factor with similar predicted structure to the *Herpesviridae *packaging accessory factor (PMID: 41902279). It is unclear how the mechanism of packaging differs in the *Orthoherpesviridae *and whether replication in mammalian/avian/reptilian cells places additional evolutionary pressure on the viral genome packaging mechanism.

    Reviewer #3

    Major comments

    *1) [I]t is not clear whether the structures presented in the manuscript reflect those produced during HCMV or HSV-1 infection. *

    We agree with the reviewer that it is important to consider to what extent purified biomolecules resemble their *in vivo *counterparts. This criticism can be applied to any *ex situ *structural analysis. However, our experimental structures allowed us to make testable observations, including the correct assignment of structurally important zinc fingers and the identification of functionally important residues in the central channel.

    2) HCMV UL52 was presented to form two distinct structures, a 3-mer and a 4-mer (Fig. 2a). However, the authors acknowledge that the 3-mer is actually a 4-mer when the threshold for the cryo-EM map is lowered. The density is also visible in the PDB validation report for the 3-mer; EMD-74418.

    Reviewers #1 and #2 were also curious about the 3-mer. As described above, we performed additional analysis that showed that the original 3-mer map was a mixture of 3-mer and 4-mer states. We have updated Fig. 2a, Supplementary Fig. 2, Supplementary Fig. 3, Supplementary Table 1, Supplementary Movie 1, EMDB and PDB depositions, and removed the discussion of the weak protomer in the 3-mer map from the results section.

    *Given that ORF68, BFLF1, and UL32 (Didychuk et al., 2021) form complete pentamer rings, with BFLF1 forming stacked rings, it would seem odd for a protein with conserved function to deviate from a pentamer configuration, suggesting that the structures reported do not reflect the natively produced and functional protein. *

    We agree that this is a surprising finding; we initially anticipated that UL32 and UL52 would also form stable pentameric rings. While this study does not resolve a complete mechanism for this factor, it does provide the first structural evidence for the implications of their poor sequence conservation and lack of complementarity.

    Furthermore, this is not the first example of a conserved herpesvirus factor that possesses different oligomeric states across different subfamily homologs. As mentioned in the discussion, herpesvirus encode a sliding clamp processivity factor (HSV-1 UL42/HCMV UL44/KSHV ORF59) that shares a common PCNA-like fold, but which has varied oligomeric state across these herpesviruses.

    *3) Unlike ORF68 (Didychuk et al., 2021) and UL32 (Suppl. Fig. 4), dsDNA binding experiments were not performed with UL52. Could the partial pentamers simply be poorly formed due to expression in insect cells (mammalian cells were used for protein purification in Didychuk et al., 2021), absence of dsDNA, or inappropriate buffer conditions? Moreover, were the EM grid and vitrification parameters optimized? Grid geometries and chemistries can have profound effects of protein stability especially in the context of the air-water interface, leading to degradation of protein complexes (Glaeser, 2018; D'Imprima et al., 2019). Does UL52 form complexes with dsDNA? Data are shown for the HSV-1 packaging accessory factor. Perhaps dsDNA would stabilize the UL52 pentamer. *

    We have purified ORF68 and homologs from both human and insect cell expression systems, and do not observe changes in oligomeric behavior. We find that ORF68 purified as a stable pentamer from human cells (Didychuk eLife 2021) and from insect cells (this work). We have also recombinantly expressed and purified UL32 from human cells. UL32 was largely monomeric after strep affinity purification (chromatogram below, unpublished), as we report from insect cells (this work, Fig. 1c). We switched to insect cell expression systems because of the easier scalability.

    Our SEC-MALS data (Fig. 1d) shows that purified UL52 does not oligomerize into a pentamer in solution, so the observed sub-pentameric (3-mer/4-mer) assemblies are unlikely to be an artifact of cryo-EM freezing conditions or the air-water interface. We have not tested if UL52 forms complexes with dsDNA, although it likely does; it is possible that this interaction would stabilize a pentamer.

    4) In Didychuk et al., 2021, HSV UL32 is shown to form pentameric rings; negative stained 2D class averages were generated from tagged protein (twin strep tag), produced in mammalian cells (HEK293T), and not purified using size exclusion chromatography. In the present study HSV UL32 was not observed to form pentameric complexes "We first attempted to visualize the pentameric species by negative stain electron microscopy but were unable to identify particles of the expected dimensions." However, it is not clear why this was the case. If the pentameric structures were readily produced in previous experiments, why was cross-linking needed in the current study? As such, the tripentamer complexes seem artifactual in nature.

    While a sufficient number of particles were observed in a pentameric state to do 2D class averages in the eLife paper, this was not the dominant state. The results we report in this work are consistent with those reported in the eLife paper. Reviewer #2 (comment #1) was also concerned about the possibility of a crosslinking artifact: we reproduce our response below:

    "Crosslinking is a commonly used technique to stabilize complexes that are observed through other means but do not survive the cryo-EM vitrification process. In an EMSA experiment (Supplementary Fig. 4a), UL32 binds 30 bp DNA and migrates slower than when bound to a 10 bp probe, consistent with formation of a supra-pentameric complex. The samples in the EMSA gels are not crosslinked. Additionally, an SDS-PAGE gel of the crosslinked product used for EM showed tight bands, supporting specific crosslinking (Supplementary Fig. 4b). These results suggest that crosslinking stabilizes a species that can form but is relatively unstable in solution."

    We have updated Line 148 to clarify this. We have also included a negative stain micrograph, below, in which UL32 pentamers (purified from insect cells) are visible in the absence of crosslinking.

    5) Although the data presented in Fig. 4b suggest that interface residues, K532 and C535, might play a role in the formation of the tripentamer and have a minor role in HSV-1 replication, these experiments are incomplete. Single mutations are needed for each residue to assess their individual contribution to tripentamer formation, evidence for a loss of tripentamer formation is needed, and evidence for protein expression is needed.

    We agree that we have not unambiguously defined the role of the tripentamer, the precise contributions of residues K532 and C535, or defined the contribution of the tripentamer to HSV-1 viral replication. We seek to report this novel structure to lay the basis for future mechanistic work. Reviewer #2 (comment 1) also questioned the role of these residues in HSV-1 replication, and we addressed this by modifying the title of the results section (Line 216) to state that "Residues at the tripentamer interfaces contribute to infectious virion production in HSV-1" as well as Line 246 and 253 to indicate that the residues play a role in the viral life cycle.

    Please refer to Supplementary Fig. 7e for a western blot showing that these mutants do not impact UL32 expression. We included explicit references to UL32 expression on Lines 239 and 288.

    *6) In the previous negative stain electron micrographs reported by Didychuk et al., 2021, were the higher order tripentamer complexes seen? *

    We did not observed tripentamers in the Didychuk et al. 2021 negative dataset. Tripentamer formation may be concentration dependent. Negative stain EM carried out at nanomolar concentrations would likely cause dissociation of tripentamers, but cryo-EM and EMSA in our work were carried out at micromolar concentrations and were able to capture the higher order tripentamer.

      1. Formation of disulphide bonds between cysteine residues in vitro is not indicative of complexes forming in vivo during replication. What evidence is there for disulphide bond formation between packaging accessory factor pentamers for KSHV, EBV, and LCMV? In the present study, the disulphide bond could form due to proximity as a result of the cross-linking and the presence of molecular oxygen rather than a bona fide enzyme catalysed reaction during herpesvirus replication to generate packaging accessory factor tripentamers. *

    We agree that it is unlikely that disulfide bonds form during infection and have removed this speculation from the manuscript (Line 343-346).

    8) The DNA densities in Suppl. Fig. 6e to 6g are curious. As noted by the authors, the 30mer dsDNAs do not traverse through the central cavity of the pentamer. They appear to make contact with neighboring pentamers, again suggesting that these complexes are artefacts from cross-linking. This should be discussed more thoroughly.

    Please refer to above discussion of crosslinking and Supplementary Fig. 4.

    9) Previously proposed functional roles for ORF68 include a scaffold for terminase assembly, association of the terminase with the portal, generation of initial free ends, or coordination with other replication machinery (Didychuk et al., 2021). Presuming that the new structures for HCMV UL52 and HSV-1 UL32 occur naturally, how do they fit with the previously proposed functional roles of the herpesvirus packaging accessory factor? A more in-depth discussion of this would be valuable.

    The common core fold and pentamer/pentamer-like assemble are common features, as is the conserved, positively-charged central channel. We have added additional discussion of this.

    *Minor comments A lack of page numbers and line numbers made reviewing this manuscript more challenging than necessary. *

    We have included page numbers and line numbers in the revised manuscript.

    *As noted in the 'General comments' section above, ORF68 (3.37Å) and BFLF1 (3.60Å) both form pentamers (Didychuk et al., 2021) and were produced in mammalian systems HEK293T cells. Protein purification in the present study was performed in insect (SF9 or High Five) cells. Does this affect complex stability. Also, the tag was retained for UL32 in Didychuk et al., 2021; could this provide stability of the pentamer in the original studies? *

    As discussed above, we have no evidence to suggest that expression in human vs. insect cell expression systems dramatically changes oligomerization behavior (Reviewer #3, comment 3). N-terminal purification tags were also retained in this study for structural work but were removed for SEC-MALS, which shows that UL32 is likely in concentration dependent equilibrium between (unstable) pentamers and monomers.

    Suppl. Fig. 3 is missing.

    We apologize for this oversight and have included Supplementary Fig. 3.

    *"UL52 has two regions remodeled" The use of the word 'remodeled' is not appropriate in this context as it implies a single protein can form two shapes under different conditions rather than distinct structures between two disparate proteins; UL52 compared to ORF68. This should be rephrased. *

    This was also noted by reviewer 2, and we have removed the term "remodel" throughout the manuscript text (i.e., Lines 134, 138, 140, 337, 341) and from Supplementary Figures 1, 3, and 5.

    *What is the density in the central core of UL52 (Fig. 2a; Suppl. Fig. 2e)? Was any form of focused classification performed to establish the identity of the density within the central pseudocavity? *

    As noted in the manuscript, this density could be which could be attributed to co-purified protein or nucleic acid, or part of the unresolved, negatively charged loop (residues 82-181) interacting with the positively charged central channel. We have done additional analysis of the central channel density (3D classification with a focus mask) and do not resolve any distinct densities, suggesting that the density is very dynamic.

    *Does UL52 bind to dsDNA? To support the hypothesis that the herpesvirus packaging accessory factor has conserved functions across the three subfamilies dsDNA binding experiments should be performed. *

    We have not done this experiment. We think that demonstrating this finding for two of the three herpesvirus subfamilies is sufficient.

    There is no discussion about how these data relate to the previous functional model for ORF68 presented in Didychuk et al., 2021. Do the new data alter the previous functional models?

    The precise mechanistic contribution of the packaging accessory factor remains unknown, and our data do not delineate between the proposed potential roles described in Didychuk *et al. *2021. Importantly, our structural information, demonstration of pentameric ring formation, and significance of the positively charged central channel show that the core function of this factor is likely conserved across the virus family. This was not known before our work.

    *There are some interesting grammatical phrases; please address throughout the manuscript. One example - "...a notable shared aspiration..." Proteins do not have aspirations. Please use a more formal scientific statement. *

    We have updated the language on Line 327.

    *Fig. 4b - Statistical analyses missing. Please provide. *

    Fig. 6c - Statistical analyses are missing. Please provide. Protein folding/expression data missing; see Fig. 5C showing mutations that result in poor protein expression.

    Suppl. Fig. 7f - Statistical analyses absent.

    Statistical analysis of the viral complementation in Figs. 4b and 6c has been included. Note that the viral yields reported in Supplementary Fig. 7f were used to calculate complementation efficiency in Figs. 4b and 6c. Protein expression of mutants shown in Fig. 6c was previously included in Supplementary Fig. 7e and is referenced on Lines 288 and 293.

    *Suppl. Fig. 2 and 5 - FSC curves have oddities, especially in the corrected curves. The cryo-EM resolution estimates calculated by CryoSPARC for the UL52 '3-mer' and 4-mer, and UL32 tripentamer are likely overestimated. In the PDB validation files each of the deposited structures has a warning for the resolution estimate "The value from deposited half-maps intersecting FSC 0.143 CUT-OFF 4.31 differs from the reported value 3.32 by more than 10 %", suggesting that the resolution estimates are inaccurate. The authors should provide a resolution estimate using loose masks and generate FSC curves using another software program such as RELION's postprocess to provide resolution estimates. *

    Thank you for bringing this to our attention. The differences in the resolution estimates are a known issue and are highly influenced by the tightness of the mask. In the revised manuscript we have updated the FSC curves to not include auto-tightened masks and revised our resolution estimates. This slightly changed the resolution to 3.29 Å for both UL52 3-mer and 4-mer and to 3.09 Å for the UL32 consensus map. Please also see the local resolution estimation maps in Supplementary Figures 2e and 5e for an illustration of the range of resolutions in each map.

    Suppl. Fig. 6f and 6g - Is there any visible density that might resemble the EGS crosslinking reagent?

    We do not expect to observe density for EGS due to the long flexible linker (~16 Å) between the two reactive groups.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary.

    The manuscript describes the cryo-EM structures of a conserved, necessary, herpesvirus genome packaging accessory factor for human cytomegalovirus (HCMV), UL52, and herpes simplex virus type-1 (HSV-1), UL32. Herpesvirus packaging accessory factors have unknown function but bind dsDNA. The UL52 and UL32 structures revealed a 5-fold symmetry similar to the previous X-ray crystallography structure for Kaposi's Sarcoma-associated herpesvirus (KSHV) ORF68 and the cryo-EM structure of Epstein-Barr virus (EBV) BFLF1. However, HCMV UL52 was reported to form two structures, a 3-mer and 4-mer whereas, HSV UL32 formed a supercomplex of trimeric pentamers (tripentamer) produced by dsDNA binding and crosslinking. Similar to previous studies with ORF68, mutagenesis of HSV-1 UL32 demonstrated the importance of zinc finger residues C297, C308, C544, and H581 for core fold stability and positively charged residues H563, R572 in the central channel in the pentamer for HSV-1 recovery in virus complementation assays. In addition, mutagenesis of K532 and C535 at the tripentamer interface helix reduced virus complementation by 50%. These findings have significant overlap and similarities to previously published experiments and confirm the properties of ORF68 and BFLF1, demonstrating the conserved nature of the required packaging accessory factor for herpesviruses.

    Major comments.

    The manuscript is generally well written with beautifully presented cryo-EM figures. Unfortunately, the new data seem to muddy the water rather than provide clarification about the role or function of the herpesvirus packaging accessory factor. Furthermore, it is not clear whether the structures presented in the manuscript reflect those produced during HCMV or HSV-1 infection. HCMV UL52 was presented to form two distinct structures, a 3-mer and a 4-mer (Fig. 2a). However, the authors acknowledge that the 3-mer is actually a 4-mer when the threshold for the cryo-EM map is lowered. The density is also visible in the PDB validation report for the 3-mer; EMD-74418. Given that ORF68, BFLF1, and UL32 (Didychuk et al., 2021) form complete pentamer rings, with BFLF1 forming stacked rings, it would seem odd for a protein with conserved function to deviate from a pentamer configuration, suggesting that the structures reported do not reflect the natively produced and functional protein. Unlike ORF68 (Didychuk et al., 2021) and UL32 (Suppl. Fig. 4), dsDNA binding experiments were not performed with UL52. Could the partial pentamers simply be poorly formed due to expression in insect cells (mammalian cells were used for protein purification in Didychuk et al., 2021), absence of dsDNA, or inappropriate buffer conditions? Moreover, were the EM grid and vitrification parameters optimized? Grid geometries and chemistries can have profound effects of protein stability especially in the context of the air-water interface, leading to degradation of protein complexes (Glaeser, 2018; D'Imprima et al., 2019). Does UL52 form complexes with dsDNA? Data are shown for the HSV-1 packaging accessory factor. Perhaps dsDNA would stabilize the UL52 pentamer.

    In Didychuk et al., 2021, HSV UL32 is shown to form pentameric rings; negative stained 2D class averages were generated from tagged protein (twin strep tag), produced in mammalian cells (HEK293T), and not purified using size exclusion chromatography. In the present study HSV UL32 was not observed to form pentameric complexes "We first attempted to visualize the pentameric species by negative stain electron microscopy but were unable to identify particles of the expected dimensions." However, it is not clear why this was the case. If the pentameric structures were readily produced in previous experiments, why was cross-linking needed in the current study? As such, the tripentamer complexes seem artifactual in nature. Although the data presented in Fig. 4b suggest that interface residues, K532 and C535, might play a role in the formation of the tripentamer and have a minor role in HSV-1 replication, these experiments are incomplete. Single mutations are needed for each residue to assess their individual contribution to tripentamer formation, evidence for a loss of tripentamer formation is needed, and evidence for protein expression is needed. In the previous negative stain electron micrographs reported by Didychuk et al., 2021, were the higher order tripentamer complexes seen?

    Formation of disulphide bonds between cysteine residues in vitro is not indicative of complexes forming in vivo during replication. What evidence is there for disulphide bond formation between packaging accessory factor pentamers for KSHV, EBV, and LCMV? In the present study, the disulphide bond could form due to proximity as a result of the cross-linking and the presence of molecular oxygen rather than a bona fide enzyme catalysed reaction during herpesvirus replication to generate packaging accessory factor tripentamers.

    The DNA densities in Suppl. Fig. 6e to 6g are curious. As noted by the authors, the 30mer dsDNAs do not traverse through the central cavity of the pentamer. They appear to make contact with neighboring pentamers, again suggesting that these complexes are artefacts from cross-linking. This should be discussed more thoroughly.

    Previously proposed functional roles for ORF68 include a scaffold for terminase assembly, association of the terminase with the portal, generation of initial free ends, or coordination with other replication machinery (Didychuk et al., 2021). Presuming that the new structures for HCMV UL52 and HSV-1 UL32 occur naturally, how do they fit with the previously proposed functional roles of the herpesvirus packaging accessory factor? A more in-depth discussion of this would be valuable.

    Minor comments.

    A lack of page numbers and line numbers made reviewing this manuscript more challenging than necessary.

    As noted in the 'General comments' section above, ORF68 (3.37Å) and BFLF1 (3.60Å) both form pentamers (Didychuk et al., 2021) and were produced in mammalian systems HEK293T cells. Protein purification in the present study was performed in insect (SF9 or High Five) cells. Does this affect complex stability. Also, the tag was retained for UL32 in Didychuk et al., 2021; could this provide stability of the pentamer in the original studies?

    Suppl. Fig. 3 is missing.

    "UL52 has two regions remodeled" The use of the word 'remodeled' is not appropriate in this context as it implies a single protein can form two shapes under different conditions rather than distinct structures between two disparate proteins; UL52 compared to ORF68. This should be rephrased.

    What is the density in the central core of UL52 (Fig. 2a; Suppl. Fig. 2e)? Was any form of focused classification performed to establish the identity of the density within the central pseudocavity?

    Does UL52 bind to dsDNA? To support the hypothesis that the herpesvirus packaging accessory factor has conserved functions across the three subfamilies dsDNA binding experiments should be performed. There is no discussion about how these data relate to the previous functional model for ORF68 presented in Didychuk et al., 2021. Do the new data alter the previous functional models?

    There are some interesting grammatical phrases; please address throughout the manuscript. One example - "...a notable shared aspiration..." Proteins do not have aspirations. Please use a more formal scientific statement.

    Fig. 4b - Statistical analyses missing. Please provide.

    Fig. 6c - Statistical analyses are missing. Please provide. Protein folding/expression data missing; see Fig. 5C showing mutations that result in poor protein expression.

    Suppl. Fig. 2 and 5 - FSC curves have oddities, especially in the corrected curves. The cryo-EM resolution estimates calculated by CryoSPARC for the UL52 '3-mer' and 4-mer, and UL32 tripentamer are likely overestimated. In the PDB validation files each of the deposited structures has a warning for the resolution estimate "The value from deposited half-maps intersecting FSC 0.143 CUT-OFF 4.31 differs from the reported value 3.32 by more than 10 %", suggesting that the resolution estimates are inaccurate. The authors should provide a resolution estimate using loose masks and generate FSC curves using another software program such as RELION's postprocess to provide resolution estimates.

    Suppl. Fig. 6f and 6g - Is there any visible density that might resemble the EGS crosslinking reagent?

    Suppl. Fig. 7f - Statistical analyses absent.

    References.

    Didychuk AL, Gates SN, Gardner MR, Strong LM, Martin A, Glaunsinger BA. A pentameric protein ring with novel architecture is required for herpesviral packaging. Elife. 2021 Feb 8;10:e62261. doi: 10.7554/eLife.62261. PMID: 33554858; PMCID: PMC7889075.

    D'Imprima E, Floris D, Joppe M, Sánchez R, Grininger M, Kühlbrandt W. Protein denaturation at the air-water interface and how to prevent it. Elife. 2019 Apr 1;8:e42747. doi: 10.7554/eLife.42747. PMID: 30932812; PMCID: PMC6443348.

    Gardner MR, Glaunsinger BA. Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging. J Virol. 2018 Jul 31;92(16):e00840-18. doi: 10.1128/JVI.00840-18. PMID: 29875246; PMCID: PMC6069193.

    Glaeser RM. PROTEINS, INTERFACES, AND CRYO-EM GRIDS. Curr Opin Colloid Interface Sci. 2018 Mar;34:1-8. doi: 10.1016/j.cocis.2017.12.009. Epub 2017 Dec 22. PMID: 29867291; PMCID: PMC5983355.

    Significance

    General assessment: The strengths of this manuscript are the structural information provide by the cryo-EM maps for the HCMV UL52 and HSV-1 UL32 and the mutagenesis studies that corroborate previous studies for the packaging accessory factor for gammaherpesviruses KSHV and EBV. However, there are limitations. These are centered on whether the structures are representative of UL52 and UL32 complexes produced during replication rather than over expression in insect cells and stabilization using chemical cross-linking.

    There is a lack of novelty in the context of the herpesvirus packaging factor. The pentameric architecture, DNA binding, zinc fingers (4), and charged residues required for DNA binding were conclusively demonstrated in previous studies (Gardner and Glaunsinger, 2018; Didychuk et al., 2021). Thus, the novelty comes from the different pentameric structures; UL52 4-mer and UL32 tripentamer. However, if these are artefactual structures due to the expression system (mammalian versus insect) used, air-liquid interface induced protein instability, or cross-linking, the novelty is lost. That's not to say the data are not informative for the herpesvirus community.

    Advance: The advance in this manuscript is the new structural information for the UL52 and UL32. Even if the higher order complexes are potential artefacts, high resolution structure information for the subunit is especially informative. The mutagenesis data for UL32 are also informative in that the provide important information about a conserved and necessary protein needed for herpesvirus replication and has the potential to be used as a novel druggable target.

    Audience: The manuscript will appeal to specialized and broad audiences and could influence research into antiviral therapies for herpesviruses. My field of expertise is herpesvirology, structural biology, and cryogenic electron microscopy modalities,

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Bailey et al investigate DNA packaging accessory factors of various herpesviruses. The central findings are the cryo-EM structures of the accessory factors from HSV1 and HCMV. Combined with the corresponding author's previous structure of the KSHV accessory factor, these new findings now provide a window into packaging in all three families of human herpesviruses. They reveal that the overall structure of a ring of pentameric symmetry is conserved, the overall oligomeric stabilities are not conserved across all herpesviruses. Moreover, the authors have the important finding that basic residues in the ring pore are required for viral replication. Overall, this study represents a strong extension of the authors' study of packaging accessory proteins, with solid data and very few concerns to be addressed.

    Major issues.

    1. There is a high probability that the tripentamer is an artifact of the cross-linking. Because of this, it'd be great to know more about the cross-linking reaction, ideally mass spec identification and quantification of cross-links. This would also address the authors' speculation of contacts that stabilize the tripentamer. Moreover, the author's claim "However, mutation of K532A/C535A reduced infectious virion production by half (Fig. 4b), suggesting that the tripentamer interface may play a role in the viral life cycle." Seems to be an overreach. Perhaps this is semantics but the data just show that these residues play a role in viral replication (albeit not a huge role based on the modest effect).

    2. The density for the potential DNA does not look very convincing, although it still remains the strongest hypothesis. The authors should try to strengthen their argument. Does this putative DNA would contact residues that they show are necessary for viral replication? Showing seq conservation on the sturcutre could help their argument for the shared function of DNA-binding.

    3. My last major issue is stylistic and concerns the descriptions of cryoEM structures. I found that the paper was a bit of challenge to read when the authors would introduce each structure. It was a bit of a slog to get through. Descriptions of the structures veered off into overly detailed comparisons that required constant comparison with the figure and didn't really advance my understanding past "the outer surfaces of the three orthologs are different." This masked the more interesting aspects of the authors' findings. Perhaps this could be summarized in supplementary figures or a table. Because this is a stylistic suggestion, the authors should feel free to ignore this request.

    Minor comments

    1. The descriptions of structure determination in the text were often unclear. For example, "In the 3-mer map, a poorly-resolved fourth protomer is visible at low contour levels, suggesting that an additional protomer is present but highly flexible in this class (Supplementary Fig. 3a)." Alternatively, it could be that the classification algorithm wasn't able to fully separate particles that were 3-mers from the 4mers. When describing the structure determination of the HSV1 accessory factor, the authors describe no other particles other than the tripentamer. Were there other particles observed? It'd be a bit surprising that all of the protein adopted the tripentamer state. Was symmetry applied, particularly for the tripentamer that appears to have C-3 symmetry? This is in materials and methods but not clear why it isn't mentioned when describing the structure determineation and results.

    2. Throughout the paper, the authors use the word "remodel" to describe structural differences between orthologs. However, this word usually carries the implication of conformational rearrangement within a protein, and not across orthologs. Please consider a different description.

    3. Figure 2F is confusing and difficult to interpret. It seems that the main point is that these interfaces are conserved, which might be more easily displayed as a standard sequence conservation score mapped onto the structure. I'm also not sure that this figure is necessary as a main figure and could be supplemental.

    4. The authors write "UL32 bound to the shortest probe tested (10 bp, Supplementary Fig. 4a)." This implies that ONLY the shortest probe is bound and that others are not bound. Consider rephrasing.

    5. Frustum is misspellt. ;)

    6. In the discussion, the authors speculate that the variability of the outer surface is due to "virus- or host-specific interactions". I'm confused by "host-specific interactions", because the host is the same for all three viruses. Perhaps the authors mean that the different accessory factors could interact with different host factors? If so, are the authors making a Red Queen argument? If so, it'd be pretty cool to do dN/dS analysis to test that hypothesis.

    Significance

    This paper represents an advance in the field of genome packaging. The herpesvirus packaging mechanism is still mysterious, and the role of this accessory factor is one of the biggest gaps in knowledge. Although this study doesn't uncover the role, this provides new details into the evolution of this factor across the herpesvirus lineages. To me, this window into evolution of this factor is the biggest advance of the work, and tbh I felt that the authors could lean into this a bit more in the discussion section. Are there any differences in the packaging mechanisms of the different herpes families that can be related to their different behavior? Any other molecular evolution analyses (e.g. dN/dS ratio analysis) that could inform their study?

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Bailey et al. present the results of their structural analysis of packaging factors encoded by the β-herpesvirus human cytomegalovirus (UL52) and the ⍺-herpesvirus herpes simplex virus type 1 (UL32). The authors have previously published structures for orthologous proteins in the γ-herpesviruses Kaposi sarcoma associated herpes virus (ORF68) and Epstein-Barr virus (BFLF-1), showing both to form pentameric rings having a positively charged central channel. Here HCMV UL52 is found to form 3-mer and 4-mer assemblies that resemble incomplete pentameric rings. The complexes are formed by a screw displacement however, having both rotation about- and translation along the central axis. The structure shows fold conservation with the previously described structures, including preservation of the positively charged central channel.

    Attempts to image HSV-1 UL32 were initially unsuccessful, despite light-scattering analysis indicating the presence of pentamers. DNA binding is shown by EMSA, but these complexes were also not stable for cryo-EM analysis. Chemical crosslinking of the DNA bound complex was therefore employed, resulting in production of higher-order assemblies including one comprising three pentamers, that was successfully resolved by cryo-EM. Interestingly focussed classification analysis highlighted the presence of rod-shaped density passing through the central channels of two pentameric rings in this complex. Mutation of the interface that gives rise to the formation of tripentamers reduced progeny virion production by half, leading the authors to suggest that this complex may be a biologically important assembly.

    The importance of zinc-fingers identified in these structures was probed showing that mutation abolishes protein production. Similarly, mutation of the positively charged residues lining the central channel of HSV-1 UL32 greatly reduced or completely ablated progeny virion production in an assay where either WT or mutant UL32 was transfected into cells to complement UL32 knockout virus.

    Overall, I found the manuscript very easy to read and the analysis appears to be expertly performed. I have no substantive criticisms of the work and think it would be suitable for publication in its current form, or subject to some small edits.

    Minor comments

    The authors suggest that the weak 4th protomer in the HCMV UL52 3-mer map is a consequence of flexibility. This may be the case, but it may also be the case that the class is polluted with 4-mer particles leading to reduced occupancy. Erasing the weak density and running a multi-model 3D classification providing the erased 3-mer and a 4-mer starting map may separate these.

    I found the supplemental figure to show the DNA in the tripentamer map too small, this is an interesting finding and should be shown more clearly.

    Significance

    Herpesviruses are important pathogens of humans and are biologically complex systems. The structural analysis of this essential packaging co-factor is an important contribution to the field. It builds on the previous work by this group concerning the packaging factors of the gamma-herpesviruses KSHV and EBV. I consider this paper to be high-quality and worthy of publication in a very good journal with a microbiology/virology, biochemistry or molecular biology focussed readership. The process of genome packaging in herpesviruses is not as well characterised as in bacteriophages (and even in that case it is not well understood). This work provides important knowledge that will support future studies on this critical process in herpesvirus replication.

  5. Version published to 10.64898/2026.01.22.701024 on bioRxiv