Control of elF4F complex by Vesicular Stomatitis Virus

在感染许多不同病毒的细胞中，基因表达的一个最显著的特征是，当病毒mRNAs被翻译时，宿主mRNAs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致病中起着关键作用。这项建议的重点是了解原型负链RNA病毒水泡性口炎病毒(VSV)与翻译起始因子复合体eIF4F相互作用的机制。EIF4F是一个多蛋白复合体，其功能被认为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去磷酸化，eIF4F复合体的帽结合亚基。这将通过直接分析感染细胞裂解物中eIF4E和eIF4E结合蛋白(eIF4E-BP1)的活性和磷酸酶活性，并通过确定在VSV感染过程中eIF4E激酶是否从eIF4F中解离来实现。此外，这项建议的目的是通过使用标准亲和分离技术，然后在复杂纯化后进行免疫印迹分析和/或质谱仪测序，来确定eIF4F蛋白复合体的蛋白质组成是否有其他变化。这些实验将有助于理解VSV病毒是如何控制宿主翻译机构的，排除细胞帽和多腺化的宿主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.