A pentameric protein ring with novel architecture is required for herpesviral packaging
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Abstract
Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids, which involves essential accessory factors that are poorly understood. Here, we present structures of two such accessory factors from the oncogenic herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV; ORF68) and Epstein–Barr virus (EBV; BFLF1). These homologous proteins form highly similar homopentameric rings with a positively charged central channel that binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection, these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.
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Summary: Didychuk et al. report crystal and cryo-EM structures of the ORF68 protein from KSHV/HHV-8, plus the cryo-EM structure of its homologue BFLF1 from EBV/HHV-4. These structures, along with biochemical data presented in this paper and the group's previous work, demonstrate convincingly that ORF68 is a DNA-binding protein involved in genome packaging. Importantly, the authors show that the conserved cysteine residues in ORF68 mediate zinc ligation, suggesting that they play a structural role rather than a role in intracellular disulfide bond regulation (as had been hypothesised for the HSV-1/HHV-1 homologue pUL32). The work is methodologically sound and provides a structural framework for probing the function of ORF68 and homologues in virus assembly.
Reviewer #1:
The genome packaging machinery of herpesviruses is composed of 6 …
Summary: Didychuk et al. report crystal and cryo-EM structures of the ORF68 protein from KSHV/HHV-8, plus the cryo-EM structure of its homologue BFLF1 from EBV/HHV-4. These structures, along with biochemical data presented in this paper and the group's previous work, demonstrate convincingly that ORF68 is a DNA-binding protein involved in genome packaging. Importantly, the authors show that the conserved cysteine residues in ORF68 mediate zinc ligation, suggesting that they play a structural role rather than a role in intracellular disulfide bond regulation (as had been hypothesised for the HSV-1/HHV-1 homologue pUL32). The work is methodologically sound and provides a structural framework for probing the function of ORF68 and homologues in virus assembly.
Reviewer #1:
The genome packaging machinery of herpesviruses is composed of 6 proteins. The functions of 5 of these have been relatively well characterized, but little is known about the 6th component, the conserved protein termed ORF68 in KSHV. Here, by obtaining a high-resolution structure of ORF68 (and its homolog from a closely related EBV), authors show that it forms a pentameric ring with a positively charged pore that could accommodate dsDNA. Authors further show that the basic residues lining the pore are essential for DNA binding, genome packaging, and viral replication. These data for the first time suggest that ORF68 binds the dsDNA genome and may, in some manner, act as an adaptor bringing the genome and the genome-packaging terminase motor to the capsid portal. Structural analysis suggests that all ORF68 homologs share similar architecture, providing templates for the future mechanistic exploration. The study is well executed, and the manuscript was a pleasure to read. The concerns are minor except for the following.
The functional importance of basic residues lining the pore leaves little doubt that some sort of a quaternary structure with a pore that would accommodate dsDNA is formed in vivo. However, the authors do not formally show that the pentameric assembly observed in vitro is functionally relevant nor consider the possibility that a functionally relevant assembly could be something other than a pentamer. If ORF68 acts as an adaptor that tethers the hexameric terminase motor to the dodecameric capsid portal, it could very well be a hexamer. In principle, it could even form a spiral rather than a ring. Understandably, obtaining additional structures may be beyond the scope of this manuscript whereas mutagenesis of the pentameric interface would not rule out hexamers (pentameric and hexameric interfaces may be quite similar). Nonetheless, the authors could, at least, acknowledge the possibility of alternative oligomeric states.
Reviewer #2:
Didychuk et al. report crystal and cryo-EM structures of the ORF68 protein from KSHV/HHV-8, plus the cryo-EM structure of its homologue BFLF1 from EBV/HHV-4. These structures, along with biochemical data presented in this paper and the group's previous work, demonstrate convincingly that ORF68 is a DNA-binding protein involved in genome packaging. Importantly, the authors show that the conserved cysteine residues in ORF68 mediate zinc ligation, suggesting that they play a structural role rather than a role in intracellular disulfide bond regulation (as had been hypothesised for the HSV-1/HHV-1 homologue pUL32). The work is methodologically sound and provides a structural framework for probing the function of ORF68 and homologues in virus assembly.
Limitations of the study are that it does not identify any specific interactions with other members of the terminase/packaging complex, so the exact role of ORF68 and homologues remains enigmatic. However, several compelling hypotheses are presented in Figure 6 and this work will undoubtedly stimulate further investigations to unravel the precise function of ORF68.
Substantive issues:
The authors assert that ORF68, BFLF1 and UL32 all form pentamers, and that this is the active form of these proteins. While this is supported by the EM analysis of ORF68 and UL32, the assertion that BFLF1 is also most likely active as a pentamer (lines 166-7) is not supported by data. Ideally the authors would use analytical ultracentrifugation or MALS to define the oligomeric state of the particles in solution, but analytical size exclusion chromatography would be sufficient to confirm that ORF68, BFLF1 and UL32 all form similarly sized particles in solution.
The structural work presented in this manuscript show compellingly that ORF68 and BFLF1 share the same fold, and sequence conservation suggests that this fold will be conserved across alpha- and beta-herpesvirus homologues, UL32 and UL52 (respectively). However, building a homology model of UL32 and UL52 using ORF68 as a template structure does not provide additional support to this hypothesis - by definition a homology model will always look similar to its template structure. Figures 3(c,d) and discussion of the homology models should be removed in favour of a discussion of sequence conservation (Figure S4).
The authors use EMSAs to probe the affinity ORF68 for 'cognate' (GC-rich) or scrambled DNA. While the similar binding affinity can be easily seen, the estimated dissociation constant (Kd) is likely significantly wrong because the Langmuir-Hill equation used by the authors does not take into account ligand depletion and the assumption that the [ORF68]total equals [ORF68]free is not valid when using nM concentrations of both fluorescent DNA probe and ORF68. The authors should either quote the effective binding affinity in their assay (EC50) or fit their data to a model that takes into account ligand depletion.
Reviewer #3:
This paper by Didychuk et al. focused on determining the structure and possible functions of the proteins encoded by the KSHV (orf68) and EBV (BFLF1) that are required for genome packaging. The cleavage and packaging of herpesvirus genomes involves a number of viral proteins. These homologous proteins form pentameric rings with channels that bind dsDNA. The authors present a number of structural and biochemical studies focused on determining the role of these proteins in the cleavage and packaging of the herpesvirus genomes. The work answers questions of significance regarding the novel biochemical activities of ORF68 protein and several models are proposed on how these proteins may function in the packaging of the herpesvirus genomes. The paper is well written, very concisely presented considering the large amount of data, and will be important to those studying DNA packaging of herpesviruses as well as other DNA viruses. Although there are a large number of experiments they all contribute to a very extensive analysis of this very interesting protein whose role in DNA packaging has been unknown.
Specific Points:
p. 18. Lines 335-339. The authors might want to point out that HSV-1 DNA replication produces branched, head-to-tail concatemers of viral genomes that must be cleaved and packaged into capsids as individual, unit-length monomers. PFGE studies have shown that in HSV infected cells the replicated viral genome produces concatemers that are cleaved only at the UL-end of the viral genome (PMID: 9222355). A number of studies with HSV mutants indicated that all of the cleavage packaging proteins (except UL25) along with capsid proteins are required for this initial cleavage reaction. Also the portal protein has been shown to interact with replicating HSV genomes and the role of UL32 and its homologs may facilitate the first cleavage as part complex (PMID: 28095497). Also of interest, these studies (iPOND/aniPOND) did not detect a DNA interaction of UL32.
Discussion: In contrast to the KSHV and HSV proteins the EBV BFLF1 protein forms a decameric ring. What might be the significance of this and why would this not be the case for the other two proteins?
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