Control of elF4F complex by Vesicular Stomatitis Virus

在感染多种不同病毒的细胞中，基因表达的最显著特征之一是，当病毒mrna被翻译时，宿主mrna的翻译受到抑制。这种病毒对翻译的控制在完整动物宿主的病毒发病机制中起着关键作用。本研究的重点是了解原型负链RNA病毒水疱性口炎病毒（VSV）与翻译起始因子复合体eIF4F相互作用的机制，eIF4F是一种多蛋白复合体，其功能已知是许多病毒（如脊髓灰质炎病毒、腺病毒和轮状病毒）的靶标。该项目的目的是确定宿主VSV感染如何导致eIF4E （eIF4F复合物的帽结合亚基）的去磷酸化。这将通过直接分析eIF4E和eIF4E结合蛋白（eIF4E- bp1）激酶的活性和感染细胞裂解物中的磷酸酶活性来实现，并通过确定在VSV感染期间eIF4E激酶是否与eIF4F分离来实现。此外，本提案的目的是通过使用标准亲和分离技术，然后在复合物纯化后进行免疫印迹分析和/或质谱测序，确定eIF4F蛋白复合物的蛋白质组成是否有额外的变化。这些实验将有助于理解VSV（一种产生5' cap和3' polyadenylated mRNA的病毒）如何控制宿主翻译装置，排除细胞cap和polyadenylated宿主mRNA并翻译自己的mRNA。

One of the most striking features of gene expression in cells infected with many different viruses is that there is an inhibition of translation of host mRNAs while viral mRNAs are being translated. This viral control of translation plays a critical role in viral pathogenesis in intact animal hosts. This proposal is focused on understanding the mechanism by which the prototype negative strand RNA virus, vesicular stomatitis virus (VSV), interacts with the translation initiation factor complex eIF4F. eIF4F is a multi-protein complex, and its function is know to be the target of many viruses such as poliovirus, adenovirus and rotavirus. The aims of this project are to determine how host VSV infection causes the dephosphorylation of eIF4E, the cap-binding subunit of the eIF4F complex. This will be accomplished by directly analyzing the activity of the eIF4E and eIF4E binding protein (eIF4E-BP1) kinase and phosphatase activity in infected cell lysates, and by determining whether the eIF4E kinases are dissociated from eIF4F during VSV infection. Additionally, the aims of this proposal are to determine whether there are additional changes to the protein makeup of the eIF4F protein complex by using standard affinity isolation techniques followed by immunoblot analysis and/or mass-spec sequencing following complex purification. These experiments will help understand how VSV, a virus that produces 5' capped, and 3' polyadenylated mRNAs manages to dominate the host translation apparatus, excluding cellular capped and polyadenylated host mRNA and translating its own.