Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription
Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription
复制标题
DOI：
10.1128/jvi.00279-17
复制
发表时间：
2017-10-01
影响因子：
5.4
通讯作者：
Leaman, Douglas W.
中科院分区：
文献类型：
--
作者：
Ke, Qi;Weaver, Wade;Leaman, Douglas W.
病毒性出血性败血症病毒（VHSV）是一种致病性鱼类弹状病毒，在整个北方半球的离散地区发现。VHSV感染鱼细胞导致宿主的病毒检测反应上调，但病毒迅速抑制干扰素（IFN）的产生和抗病毒基因的表达。通过系统地筛选VHSV的6个结构和非结构基因，我们确定了基质蛋白（M）是病毒最有效的抗宿主蛋白。只有M的VHSV基因型IV亚系B（VHSV-IV B）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的剂量依赖性方式。M还抑制了组成型活性猿猴病毒40（SV 40）启动子，并全面降低细胞RNA水平。染色质免疫沉淀（ChIP）的研究表明，M抑制RNA聚合酶II（RNAP II）招募基因启动子和降低RNAP II C-末端结构域（CTD）Ser 2磷酸化在VHSV感染。然而，RNAP I至III指导的转录被M抑制。为了鉴定功能重要性区域，在基于细胞的转录抑制测定中测试来自多种VHSV株的M蛋白。M的一个特定的VHSV-Ia株，F1，是显着低于IVb M在抑制SV 40/荧光素酶（Luc）的表达，但只有4个氨基酸的差异。单独将D 62突变为丙氨酸，或与E181突变为丙氨酸（D 62 A E181 A）组合，显著降低了IVb M抑制宿主转录的能力。通过反向遗传学将M D 62 A或D 62 A E181 A突变引入VHSV-IVb导致病毒有效复制，但表现出较小的细胞毒性和降低的抗转录活性，这暗示M是致细胞病变和宿主转录抑制的主要调节因子。在这些研究中，我们确定了致命的鱼类novirhabdovirus VHSV的基质蛋白作为感染过程中宿主抑制的关键介质。我们的研究表明，M单独可以在非常低的表达水平上阻断细胞基因表达。我们在M中发现了几个细微的突变，这些突变在抑制宿主转录方面不那么有效。当这些突变被重新设计到重组病毒中时，产生的病毒复制良好，但在感染细胞中引起的毒性较小，并且更强烈地激活宿主先天免疫应答。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重要作用。我们的数据表明，它在这两个过程中的作用可以分开，以设计有效的减毒病毒的候选疫苗。
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 anti-transcriptional 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 virusinduced 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.