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A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

ABSTRACT: Inside the virions of ?-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a three-dimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the ?-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9?Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsid-associated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the ?-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsid-tegument interface.

SUBMITTER: Liu YT

PROVIDER: S-EPMC5718256 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

Liu Yun-Tao YT Jiang Jiansen J Bohannon Kevin Patrick KP Dai Xinghong X Gant Luxton G W GW Hui Wong Hoi WH Bi Guo-Qiang GQ Smith Gregory Allan GA Zhou Z Hong ZH

The Journal of general virology 20171016 11

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of exist ...[more]

PMID: 29035172

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Project description:UnlabelledHerpesviruses 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.ImportanceHerpesviruses 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.

Project description:The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-DeltaUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-DeltaUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.

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A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

Publications

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

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