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Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics.

ABSTRACT: Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.

SUBMITTER: Kato A

PROVIDER: S-EPMC7524712 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics.

Kato Akihisa A Adachi Shungo S Kawano Shuichi S Takeshima Kousuke K Watanabe Mizuki M Kitazume Shinobu S Sato Ryota R Kusano Hideo H Koyanagi Naoto N Maruzuru Yuhei Y Arii Jun J Hatta Tomohisa T Natsume Tohru T Kawaguchi Yasushi Y

Nature communications 20200929 1

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increa ...[more]

PMID: 32994400

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Project description:BackgroundIn herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein.ResultsDEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein.ConclusionsDEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and most similar to the genus Mardivirus. The UL15 and/or UL15.5 accumulate(s) in the cytoplasm during early times post-infection and then are translocated to the nucleus at late times.

Project description:Herpes simplex virus replicates in the nucleus, where new capsids are assembled. It produces procapsids devoid of nucleic acid but containing the preVP22a scaffold protein. These thermo-unstable particles then mature into A-, B- or C-nuclear icosahedral capsids, depending on their ability to shed the proteolytically processed scaffold and incorporation of the viral genome. To study how these viral capsids differ, we performed proteomics studies of highly enriched HSV-1 A-, B- and C-nuclear capsids, relying in part on a novel and powerful flow virometry approach to purify C-capsids. We found that the viral particles contained the expected capsid components and identified several tegument proteins in the C-capsid fraction (pUL21, pUL36, pUL46, pUL48, pUL49, pUL50, pUL51 and pUS10). Moreover, numerous ribosomal, hnRNPs and other host proteins, absent from the uninfected controls, were detected on the capsids with some of them seemingly specific to C-capsids (glycogen synthase, four different keratin-related proteins, fibronectin 1 and PCBP1). A subsequent proteomics analysis was performed to rule out the presence of protein complexes that may share similar density as the viral capsids but do not otherwise interact with them. Using pUL25 or VP5 mutant viruses incapable of assembling C-nuclear or all nuclear capsids, respectively, we confirmed the bulk of our initial findings. Naturally, it will next be important to address the functional relevance of these proteins.IMPORTANCE Much is known about the biology of herpesviruses. This includes their unique ability to traverse the two nuclear envelopes by sequential budding and fusion steps. For HSV-1, this implies the pUL31/pUL34 and pUL17/pUL25 complexes that may favor C-capsid egress. However, this selection process is not clear, nor are all the differences that distinguish A-, B- and C-capsids. The present study probes what proteins compose these capsids, including host proteins. This should open up new research avenues to clarify the biology of this most interesting family of viruses. It also reiterates the use of flow virometry as an innovative tool to purify viral particles.

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Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics.

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Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics.

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