Characterizing the role of CSDE1 as a critical co-factor for VSV replication.

项目摘要 我们已经反复观察到，细胞在长期感染后获得逃避病毒复制/裂解的能力。 低水平暴露于水泡性口炎病毒（VSV）。在研究与 逃避，我们表明APOBEC 3B是逃避病毒的细胞中突变可塑性的主要效应子。 包括VSV在内的复制/治疗。逃避VSV的细胞的RNAseq鉴定了约300个编码突变 与APOBEC 3B签名，其中一些我们假设将直接影响病毒的能力， 在靶细胞内复制。在这些突变中，冷休克结构域中的单个C-T点突变 将含有E1（CSDE 1）基因（CSDE 1C-T）的重组质粒转化到大肠杆菌中，得到CSDE 1突变蛋白CSDE 15 P-S， 在不同组织学类型的人和鼠细胞中以高克隆性表达， 抗VSV。这些数据表明，就我们所知，CSDE 1第一次发挥了非常重要的作用。 在VSV复制和溶瘤中的作用。我们还观察到VSV可以协同进化，以补充细胞内 突变，如CSDE 1C-T。因此，野生型VSV通过CSDE 15 P-S细胞的强制进化使我们能够 跟踪病毒VSV-IFN-γ-IGRP/MC-U突变体的出现。这种病毒含有一种高度特异性的突变 在VSV的P和M基因之间的基因间隔区（IGRP/MC-U）（位于VSV的唯一完美的 VSV基因组中CSDE 1的共有结合位点），并完全挽救了病毒的高水平复制 在表达突变体CSDE 15 P-S的细胞中。在这些数据的基础上，我们将在这里测试我们的总体假设 CSDE 1是VSV复制的关键介质; CSDE 1中的突变，如CSDE 1C-T， 允许靶细胞逃避VSV的复制;并且VSV可以进化补偿突变， 在CSDE 1（C-T）突变细胞中恢复复制适应性。为了验证这一假设，我们制定了四个 具体目的，我们将：（目的1）定义CSDE 1如何介导VSV的复制，以及VSV中的突变如何介导VSV的复制。 CSDE 1与VSV逃逸密切相关;（目的2）了解IGRP/MC-U突变在VSV中的作用。 VSV补充CSDE 15 P-S;（目的3）追踪IGRP/MC-U突变的诱导，因为病毒经历了 强大的选择性压力，对它的复制，并确定细胞抗病毒突变途径， 将突变标记印记到VSV基因组上;以及（目标4）利用我们对CSDE 1的发现作为一种关键的协同作用， VSV复制因子，以产生表达CSDE 1的VSV，其作为疫苗接种和 溶瘤平台。总之，这些研究将对理解VSV/宿主细胞产生重大影响。 相互作用，在定义病毒可以篡夺抗病毒诱变途径种子逃逸的途径- 有能力的准种，并将为开发用于疫苗接种和溶瘤的新型改良VSV提供信息。

PROJECT SUMMARY We have repeatedly observed that cells acquire the ability to escape from viral replication/lysis upon long-term low-level exposure to Vesicular Stomatitis Virus (VSV). Whilst investigating the mechanisms associated with escape, we showed that APOBEC3B is a major effector of mutational plasticity in cells which evade viral replication/therapies including VSV. RNAseq of cells which escaped VSV identified ~300 coding mutations with APOBEC3B signatures, some of which we hypothesized would directly affect the ability of the virus to replicate within target cells. Of these mutations, a single C-T point mutation in the Cold Shock Domain Containing E1 (CSDE1) gene (CSDE1C-T), which generates a mutant CSDE1 protein CSDE15P-S, was expressed at high clonality in both human and murine cells, of different histological types, which became resistant to VSV. These data show, for the first time to our knowledge, that CSDE1 plays a highly significant role in replication of, and oncolysis by, VSV. We also observed that VSV can co-evolve to complement cellular mutations such as CSDE1C-T. Thus, forced evolution of wild type VSV through CSDE15P-S cells allowed us to track the emergence of a mutant virus VSV-IFNß-IGR P/MC-U. This virus contained a highly specific mutation in the Intergenic region between the P and M genes of VSV (IGR P/MC-U) (located in the only perfect consensus binding site for CSDE1 in the VSV genome) and completely rescued high level replication of virus in cells expressing mutant CSDE15P-S. On the basis of these data, here we will test our overarching hypothesis that CSDE1 is a critical mediator of the replication of VSV; that mutation in CSDE1, such as at CSDE1C-T, allows for target cell escape from replication of VSV; and that VSV can evolve compensatory mutations to recover replication fitness in CSDE1(C-T)-mutated cells. To test this hypothesis, we have formulated four Specific Aims in which we will: (Aim 1) define how CSDE1 mediates replication of VSV, and how mutations in CSDE1 are critically associated with escape from VSV; (Aim 2) understand how the IGR P/MC-U mutation in VSV complements CSDE15P-S; (Aim 3) track the induction of the IGR P/MC-U mutation as the virus undergoes strong selective pressures against its replication and identify the cellular anti-viral mutational pathways which imprint mutational signatures onto VSV genomes; and (Aim 4) exploit our discovery of CSDE1 as a critical co- factor for VSV replication to generate CSDE1-expressing VSV with enhanced efficacy as both vaccination and oncolytic platforms. Overall, these studies will have significant impact in understanding VSV/host cell interactions, in defining pathways by which viruses can usurp anti-viral mutagenic pathways to seed escape- competent quasi species and will inform development of novel, improved VSV for vaccination and oncolysis.