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

描述 CSDE1 作为 VSV 复制关键辅助因子的作用。

• 批准号: 10650485
• 负责人: Richard G. Vile
• 金额: $ 40.35万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-20 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10650485
• 关键词: 2019-nCoV; Affect; Antigens; Binding Sites; Cell Communication; Cells; Clonality; Code; Complement; Consensus; Cytolysis; Data; Development; Dominant-Negative Mutation; Evolution; Exposure to; Genes; Goals; Histologic; Human; Human papillomavirus 16 E1 protein; Immune; In Vitro; Induced Mutation; Intercistronic Region; Mediating; Mediator; Mus; Mutate; Mutation; Oncolytic; Pathway interactions; Phenotype; Play; Point Mutation; Proteins; Resistance; Role; Shock; TYRP1 gene; Testing; Time; Treatment Failure; Tumor Antigens; Vaccination; Vaccines; Vesicular stomatitis Indiana virus; Viral; Viral Genome; Virus; Virus Diseases; Virus Replication; cofactor; fitness; immunogenic; imprint; improved; in vivo; in vivo Model; loss of function; mutant; neoplastic cell; novel; oncolysis; oncolytic Vesicular Stomatitis Virus; oncolytic virotherapy; pressure; prevent; screening; transcriptome sequencing; tumor; viral fitness; virus identification

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.

项目概要 我们反复观察到，细胞在长期的作用下获得了逃避病毒复制/裂解的能力。 在研究与水疱性口炎病毒（VSV）相关的机制时。 逃避，我们发现 APOBEC3B 是逃避病毒的细胞中突变可塑性的主要效应器 逃脱 VSV 的细胞的复制/疗法（包括 VSV）鉴定出约 300 个编码突变。 具有 APOBEC3B 签名，我们帮助的其中一些签名将直接影响病毒的能力 在这些突变中，冷休克域中有一个 C-T 点突变。 含有E1（CSDE1）基因（CSDE1C-T），产生突变CSDE1蛋白CSDE15P-S， 在不同组织学类型的人类和小鼠细胞中以高克隆性表达， 据我们所知，这些数据首次表明 CSDE1 具有显着的抗 VSV 作用。 我们还观察到 VSV 可以共同进化以补充细胞。 因此，野生型 VSV 通过 CSDE15P-S 细胞强制进化使我们能够 追踪突变病毒 VSV-IFNß-IGR P/MC-U 的出现 该病毒含有高度特异性的突变。 位于 VSV 的 P 和 M 基因之间的基因间区域 (IGR P/MC-U)（位于唯一完美的 VSV 基因组中 CSDE1 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 CSDE15P-S 的细胞中，基于这些数据，我们将在这里检验我们的总体假设。 CSDE1 是 VSV 复制的关键介质；CSDE1 中的突变，例如 CSDE1C-T， 允许靶细胞逃避 VSV 的复制；并且 VSV 可以进化出补偿性突变 为了检验这一假设，我们制定了四种方法来恢复 CSDE1(C-T) 突变细胞的复制适应性。 具体目标是：（目标 1）定义 CSDE1 如何介导 VSV 的复制，以及 VSV 的突变如何 CSDE1 与逃离 VSV 密切相关；（目标 2）了解 IGR P/MC-U 突变如何在 VSV 补充 CSDE15P-S；（目标 3）追踪病毒发生过程中 IGR P/MC-U 突变的诱导 对其复制施加强大的选择性压力，并确定细胞抗病毒突变途径 将突变特征印记到 VSV 基因组上；以及（目标 4）利用我们发现的 CSDE1 作为关键的辅助因子 VSV 复制因子，产生表达 CSDE1 的 VSV，其作为疫苗接种和疫苗接种的功效均增强 总体而言，这些研究将对了解 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以进行种子逃逸的途径中- 有能力的准物种，并将为开发用于疫苗接种和溶瘤的新型改进 VSV 提供信息。