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

Akihisa Kato,Naoto Koyanagi,Jun Arii,Hideo Kusano,Shuichi Kawano,Shungo Adachi,Tomohisa Hatta,Ryota Sato,Yasushi Kawaguchi,Kousuke Takeshima,Shinobu Kitazume,Yuhei Maruzuru,Tohru Natsume,Mizuki Watanabe

doi:10.1038/s41467-020-18718-9

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.

Highlights

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
Among peptides detected in herpes simplex virus 1 (HSV-1)(F)infected cells that were not mapped to host cellular open reading frames (ORFs) in the experiments shown in Fig. 1a and Supplementary Fig. 1a, 97.2% mapped to reported HSV-1 protein coding genes, and 2.8% mapped to previously unidentified HSV-1 ORFs (Fig. 1c–e and Supplementary Data 1)
Based on criteria that (i) at least two distinct peptides detected in HSV-1(F)-infected cells, each of which was identified by more than one peptide spectra match, were mapped to a previously cryptic orphan coding sequences (CDSs), and (ii) the CDS was conserved in another HSV-1 strain {HSV-1 (17)}, we identified 10 previously cryptic orphan CDSs (Fig. 1f, Supplementary Fig. 2, and Supplementary Tables 1 and 2)

Summary

Introduction

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. 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. Viruses need to produce many molecules from a limited set of viral genes to promote their replication, to hijack host cellular mechanisms and to evade host restriction mechanisms To overcome this discrepancy, viruses have evolved multiple strategies: for example, viral genomes harbor a variety of non-canonical translational elements that allow them to densely pack coding information[1,2]. Our results indicate that a complete understanding of viral pathogenesis requires their identification, and their in-depth characterization

Methods

Results

Conclusion