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.

IMPORTANCE 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 robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

病毒性败血败血病病毒（VHSV）是一种致病性鱼弹状病毒，在北半球各地分布。鱼细胞的VHSV感染导致宿主病毒检测反应的上调，但该病毒迅速抑制了干扰素（IFN）的产生和抗病毒基因的表达。通过系统地筛选六个VHSV结构和非结构基因，我们确定了基质蛋白（M）是病毒最有效的抗宿主蛋白。VHSV基因型IV亚系b（VHSV-IVb）中只有M剂量依赖性地抑制线粒体抗病毒信号蛋白（MAVS）和I型干扰素诱导的基因表达。M还抑制了组成型活性猿猴病毒40（SV40）启动子，并整体降低了细胞RNA水平。染色质免疫沉淀（ChIP）研究表明，在VHSV感染期间，M抑制了RNA聚合酶II（RNAP II）募集到基因启动子，并降低了RNAP II C末端域（CTD）Ser2磷酸化。但是，M抑制了RNAP I到III的转录。为鉴定具有功能重要性的区域，在基于细胞的转录抑制试验中测试了来自多种VHSV菌株的M蛋白。特定VHSV-1a菌株F1的M在抑制SV40 /萤光素酶（Luc）表达上的效力明显低于IVb M，但仅相差4个氨基酸。单独将D62突变为丙氨酸，或与E181突变为丙氨酸（D62A E181A）结合，会大大降低IVb M抑制宿主转录的能力。

重要性病毒必须抑制宿主抗病毒反应，以便在宿主之间复制和传播。在这些研究中，我们确定了致命的鱼无病毒家禽病毒VHSV的基质蛋白是感染期间宿主抑制的关键介质。我们的研究表明，单独的M可以以非常低的表达水平阻断细胞基因的表达。我们发现了M中的一些细微突变，这些突变在抑制宿主转录方面的效力较弱。当将这些突变改造回重组病毒后，所得病毒能够很好地复制，但在被感染的细胞中毒性较低，并且可以更有效地激活宿主固有的免疫应答。这些数据表明，VHSV M在介导病毒诱导的细胞毒性和病毒复制中起着重要作用。