RNA-Binding Proteins and Host Shut-Off by VSV

RNA 结合蛋白和 VSV 关闭宿主

• 批准号: 9020822
• 负责人: DOUGLAS S. LYLES
• 金额: $ 43.56万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9020822
• 关键词: Address; Affect; Antiviral Response; Attenuated; Binding; Cell Nucleus; Cells; Chemicals; Chromatin; Complex; Cytoplasm; Data; Defective Viruses; Development; Disease; Employee Strikes; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic Translation; Goals; Health; High-Throughput Nucleotide Sequencing; Homebound Persons; Human; Immunoblotting; Immunoprecipitation; Integration Host Factors; Laboratories; Malignant Neoplasms; Messenger RNA; Molecular; Nuclear; Nuclear Envelope; Nuclear Pore Complex Proteins; Nuclear RNA; Pathogenicity; Process; Proteins; Proteomics; RNA Interference; RNA Transport; RNA-Binding Proteins; Rhabdoviridae; Ribonucleoproteins; Role; Testing; Translating; Translations; Vaccines; Vesicular stomatitis Indiana virus; Viral; Viral Genes; Viral Matrix Proteins; Viral Proteins; Virus; Virus Assembly; Virus Diseases; Work; base; cellular targeting; chromatin immunoprecipitation; crosslink; inhibitor/antagonist; messenger ribonucleoprotein; multiple myeloma M Protein; mutant; novel; novel virus; particle; prototype; research study

DESCRIPTION (provided by applicant): Many viruses suppress host antiviral responses through the global inhibition of host gene expression, often referred to as "host shut-off". Vesicular stomatitis virus (VSV), the prototype rhabdovirus, is a well-established example of a virus that suppresses antiviral responses through the global inhibition of host gene expression. The host shut-off in VSV-infected cells is particularly extensive, resulting in the inhibition of hst transcription, nuclear-cytoplasmic transport of host mRNA, and translation of host proteins. The work of our laboratory and others has shown that the VSV matrix (M) protein is the major inhibitor of host gene expression in VSV-infected cells, and that the ability of M protein to inhibt host gene expression is genetically separable from its function in virus assembly. The goal of this project is to determine how a relatively small (229 a.a.) protein can have such a profound effect on host gene expression. M protein appears to inhibit host gene expression by binding to host components and interfering with their function. Thus far the only host component identified that binds to M protein and not to mutant M proteins is the RNA-binding protein Rae1. Rae1 is a multifunctional protein that was originally thought to be involved in mRNA transport. However, our recent experiments have shown that the M protein-Rae1 complex in VSV-infected cells primarily inhibits host transcription. Furthermore, silencing Rae1 expression has no effect on nuclear accumulation of host mRNAs induced by VSV infection, demonstrating that the block in their transport is independent of Rae1 expression. Finally, our data indicate that the inhibition o translation by VSV is a downstream effect of the inhibition of transcription, thus raising the question of how the virus counters this inhibition in order for viral mRNAs to be translated. The stimulation of translation of viral mRNAs appears to be due to an activity of M protein that is independent of its host shut-off function and involves novel assembly of ribonucleoprotein particles (RNP) that promote translation. The mechanisms by which M protein affects these three levels of gene expression will be determined according to the following aims: 1) Determine the mechanism by which M protein-Rae1 complexes inhibit host transcription. 2) Determine the role of host RNA-binding proteins in mRNA transport in VSV-infected cells. 3) Determine the role of M protein and host RNA-binding proteins in promoting the selective translation of viral mRNAs in VSV-infected cells. These experiments will use state-of-the-art chromatin and RNP immunoprecipitation approaches combined with high throughput sequencing to analyze host and viral gene expression in cells infected with well-characterized M protein mutants or cells in which expression of key host factors is suppressed by RNA silencing. These experiments should reveal novel mechanisms by which VSV inhibits host gene expression. In addition, they should provide novel information about how cellular RNA-binding proteins like Rae1 can regulate host transcription, and how networks of RNA-binding proteins can control translation that is applicable to other viruses that suppress antiviral responses by the global inhibition of host transcription and RNA transport.

描述（由申请方提供）：许多病毒通过全面抑制宿主基因表达（通常称为“宿主关闭”）来抑制宿主抗病毒反应。水泡性口炎病毒（VSV），原型弹状病毒，是一个很好的例子，病毒抑制抗病毒反应，通过全面抑制宿主基因的表达。在VSV感染的细胞中，宿主关闭是特别广泛的，导致hst转录、宿主mRNA的核质转运和宿主蛋白的翻译的抑制。我们实验室和其他实验室的工作表明，VSV基质（M）蛋白是VSV感染细胞中宿主基因表达的主要抑制剂，并且M蛋白抑制宿主基因表达的能力与其在病毒装配中的功能在遗传上是分离的。该项目的目标是确定一个相对较小的（229 a.a.）蛋白质可以对宿主基因表达产生如此深远的影响。M蛋白似乎通过结合宿主组分并干扰其功能来抑制宿主基因表达。到目前为止，唯一鉴定的与M蛋白结合而不与突变M蛋白结合的宿主组分是RNA结合蛋白Rae 1。Rae 1是一种多功能蛋白，最初被认为参与mRNA转运。然而，我们最近的实验表明，在VSV感染的细胞中的M蛋白-Rae 1复合物主要抑制宿主转录。此外，沉默Rae 1表达对VSV感染诱导的宿主mRNA的核积累没有影响，表明其转运的阻断与Rae 1表达无关。最后，我们的数据表明VSV对翻译的抑制是转录抑制的下游效应，因此提出了病毒如何对抗这种抑制以使病毒mRNA被翻译的问题。病毒mRNA翻译的刺激似乎是由于M蛋白的活性，该活性独立于其宿主关闭功能，并且涉及促进翻译的核糖核蛋白颗粒（RNP）的新组装。M蛋白影响这三个水平的基因表达的机制将根据以下目的确定：1）确定M蛋白-Rae 1复合物抑制宿主转录的机制。2)确定宿主RNA结合蛋白在VSV感染细胞中mRNA转运中的作用。3)确定M蛋白和宿主RNA结合蛋白在VSV感染细胞中促进病毒mRNA选择性翻译的作用。这些实验将使用最先进的染色质和RNP免疫沉淀方法结合高通量测序，以分析感染了充分表征的M蛋白突变体的细胞或关键宿主因子表达被RNA沉默抑制的细胞中的宿主和病毒基因表达。这些实验将揭示VSV抑制宿主基因表达的新机制。此外，他们应该提供有关细胞RNA结合蛋白如Rae 1如何调节宿主转录，以及RNA结合蛋白网络如何控制翻译的新信息，这些信息适用于通过全面抑制宿主转录和RNA转运来抑制抗病毒反应的其他病毒。