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The large tegument protein pUL36 is essential for formation of the capsid vertex-specific component at the capsid-tegument interface of herpes simplex virus 1.

ABSTRACT:

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Herpesviruses have a characteristic particle structure comprising an icosahedral capsid, which contains the DNA genome and is, in turn, surrounded by a proteinaceous tegument layer and a lipid envelope. In herpes simplex virus, the interaction between the capsid and tegument is limited to the capsid vertices and involves two minor capsid proteins, pUL17 and pUL25, and the large inner tegument protein pUL36. pUL17 and pUL25 form a heterodimeric structure, the capsid vertex-specific component (CVSC), that lies on top of the peripentonal triplexes, while pUL36 has been reported to connect the CVSC to the penton. In this study, we used virus mutants with deletions in the genes for pUL36 and another inner tegument protein, pUL37, to analyze the contributions of these proteins to CVSC structure. Using electron cryomicroscopy and icosahedral reconstruction of mutants that express pUL17 and pUL25 but not pUL36, we showed that in contrast to accepted models, the CVSC is not formed from pUL17 and pUL25 on their own but requires a contribution from pUL36. In addition, the presence of full-length pUL36 results in weak density that extends the CVSC toward the penton, suggesting either that this extra density is formed directly by pUL36 or that pUL36 stabilizes other components of the vertex-tegument interface.

Importance

Herpesviruses have complex particles that are formed as a result of a carefully controlled sequence of assembly steps. The nature of the interaction between two of the major particle compartments, the icosahedral capsid and the amorphous tegument, has been extensively studied, but the identity of the interacting proteins and their roles in forming the connections are still unclear. In this study, we used electron microscopy and three-dimensional reconstruction to analyze virus particles formed by mutants that do not express particular interacting proteins. We show that the largest viral protein, pUL36, which occupies the layer of tegument closest to the capsid, is essential for formation of structurally normal connections to the capsid. This demonstrates the importance of pUL36 in the initial stages of tegument addition and provides new insights into the process of virus particle assembly.

SUBMITTER: Fan WH

PROVIDER: S-EPMC4300765 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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The large tegument protein pUL36 is essential for formation of the capsid vertex-specific component at the capsid-tegument interface of herpes simplex virus 1.

Fan Wan H WH Roberts Ashley P E AP McElwee Marion M Bhella David D Rixon Frazer J FJ Lauder Rebecca R

Journal of virology 20141119 3

<h4>Unlabelled</h4>Herpesviruses have a characteristic particle structure comprising an icosahedral capsid, which contains the DNA genome and is, in turn, surrounded by a proteinaceous tegument layer and a lipid envelope. In herpes simplex virus, the interaction between the capsid and tegument is limited to the capsid vertices and involves two minor capsid proteins, pUL17 and pUL25, and the large inner tegument protein pUL36. pUL17 and pUL25 form a heterodimeric structure, the capsid vertex-spec ...[more]

PMID: 25410861

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Project description:UnlabelledHerpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs (1766)WD(1767) and (1862)WE(1863) are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17(+))Lox-pUL36-WD/AA-WE/AA and HSV-1(17(+))Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly.ImportanceHerpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread.

Project description:The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28, and pUL33; the capsid vertex-specific component (CVSC) proteins pUL17 and pUL25; and the portal proteins pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging, we isolated a number of recombinant viruses expressing pUL25, pUL17, and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25, (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus, (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the herpes simplex virus 1 (HSV-1) genome, (iv) pUL36 serves no direct role in cleavage/packaging, (v) cleavage and stable packaging of the viral genome involve an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex, and (vi) packaging of the viral genome results in a dramatic displacement of the portal.IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit-length HSV genome. Here, biochemical and structural studies focused on the unique portal vertex of wild-type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal proteins pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.

Project description:UnlabelledpU(L)34 and pU(L)31 of herpes simplex virus (HSV) comprise the nuclear egress complex (NEC) and are required for budding at the inner nuclear membrane. pU(L)31 also associates with capsids, suggesting it bridges the capsid and pU(L)34 in the nuclear membrane to initiate budding. Previous studies showed that capsid association of pU(L)31 was precluded in the absence of the C terminus of pU(L)25, which along with pU(L)17 comprises the capsid vertex-specific complex, or CVSC. The present studies show that the final 20 amino acids of pU(L)25 are required for pU(L)31 capsid association. Unexpectedly, in the complete absence of pU(L)25, or when pU(L)25 capsid binding was precluded by deletion of its first 50 amino acids, pU(L)31 still associated with capsids. Under these conditions, pU(L)31 was shown to coimmunoprecipitate weakly with pU(L)17. Based on these data, we hypothesize that the final 20 amino acids of pU(L)25 are required for pU(L)31 to associate with capsids. In the absence of pU(L)25 from the capsid, regions of capsid-associated pU(L)17 are bound by pU(L)31. Immunogold electron microscopy revealed that pU(L)31 could associate with multiple sites on a single capsid in the nucleus of infected cells. Electron tomography revealed that immunogold particles specific to pU(L)31 protein bind to densities at the vertices of the capsid, a location consistent with that of the CVSC. These data suggest that pU(L)31 loads onto CVSCs in the nucleus to eventually bind pU(L)34 located within the nuclear membrane to initiate capsid budding.ImportanceThis study is important because it localizes pU(L)1, a component previously known to be required for HSV capsids to bud through the inner nuclear membrane, to the vertex-specific complex of HSV capsids, which comprises the unique long region 25 (U(L)25) and U(L)17 gene products. It also shows this interaction is dependent on the C terminus of U(L)25. This information is vital for understanding how capsids bud through the inner nuclear membrane.

Dataset Information

The large tegument protein pUL36 is essential for formation of the capsid vertex-specific component at the capsid-tegument interface of herpes simplex virus 1.

Unlabelled

Importance

Publications

The large tegument protein pUL36 is essential for formation of the capsid vertex-specific component at the capsid-tegument interface of herpes simplex virus 1.

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