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Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo.

ABSTRACT: Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C(26)S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C(26)S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.

SUBMITTER: Bottcher S

PROVIDER: S-EPMC2395145 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo.

Böttcher Sindy S Maresch Christina C Granzow Harald H Klupp Barbara G BG Teifke Jens P JP Mettenleiter Thomas C TC

Journal of virology 20080409 12

Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotro ...[more]

PMID: 18400848

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Project description:A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.

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.

Dataset Information

Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo.

Publications

Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo.

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