Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

• Published in: Journal of virology 2017-10, Vol.91 (19)
• Main Authors: Ke, Qi, Weaver, Wade, Pore, Adam, Gorgoglione, Bartolomeo, Wildschutte, Julia Halo, Xiao, Peng, Shepherd, Brian S, Spear, Allyn, Malathi, Krishnamurthy, Stepien, Carol A, Vakharia, Vikram N, Leaman, Douglas W
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - antagonists & inhibitors; Animals; Cell Line; Chromatin Immunoprecipitation; Cyprinidae; Fish Diseases - virology; HEK293 Cells; Hemorrhagic Septicemia, Viral - pathology; Hemorrhagic Septicemia, Viral - virology; Humans; Immunity, Innate - immunology; Interferon Type I - immunology; Novirhabdovirus - growth & development; Novirhabdovirus - immunology; Phosphorylation - genetics; Promoter Regions, Genetic - genetics; RNA - genetics; RNA Polymerase II - antagonists & inhibitors; Simian virus 40 - genetics; Transcription, Genetic - physiology; Viral Matrix Proteins - genetics; Viral Matrix Proteins - metabolism; Virus Replication - genetics; Virus-Cell Interactions
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/JVI.00279-17

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression. Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more ro