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.

项目摘要 我们反复观察到，细胞可以在长期内获得从病毒复制/裂解中逃脱的能力 低水平暴露于囊泡口腔炎病毒（VSV）。在研究与 逃脱，我们表明apobec3b是逃避病毒的细胞中突变可塑性的主要效应因子 复制/疗法，包括VSV。逃脱VSV的细胞rnaseq识别〜300个编码突变 使用APOBEC3B签名，我们假设的一些签名将直接影响病毒的能力 在目标细胞内复制。在这些突变中，冷冲击域中的单个C-T点突变 含有E1（CSDE1）基因（CSDE1C-T），该基因产生突变体CSDE1蛋白CSDE15P-S，是 在人类和鼠细胞中以高克隆性表示不同的组织学类型，这成为 对VSV的抗性。据我们所知，这些数据首次表明CSDE1扮演着非常重要的 VSV在复制和吞噬的复制中的作用。我们还观察到VSV可以共同进化以补充细胞 突变，例如CSDE1C-T。那，通过CSDE15P-S细胞强迫野生型VSV的强迫演变使我们得以 跟踪突变病毒VSV-IFNß-MIG P/MC-U的出现。该病毒含有高度特异性的突变 在VSV（IGR P/MC-U）的P和M基因之间的基因间区域（位于唯一的完美 VSV基因组中CSDE1的共识结合位点，并完全恢复了病毒的高水平复制 在表达突变体CSDE15P-S的细胞中。根据这些数据，我们将在这里检验我们的总体假设 CSDE1是VSV复制的关键中介。 CSDE1中的突变，例如在CSDE1C-T， 允许目标细胞从VSV复制中逃脱； VSV可以将补偿性突变发展为 恢复CSDE1（C-T）突出细胞中的复制适应性。为了检验这一假设，我们已经制定了四个 我们将在其中的具体目的：（目标1）定义CSDE1如何介导VSV的复制，以及如何突变中的突变 CSDE1与逃离VSV密切相关； （AIM 2）了解IGR P/MC-U突变如何 VSV完成CSDE15P-S； （AIM 3）随着病毒的经历，跟踪IGR P/MC-U突变的诱导 强烈的选择性压力反对其复制，并确定细胞抗病毒突变途径 将突变特征刻在VSV基因组上； （目标4）利用我们发现CSDE1作为关键共同的发现 VSV复制的因素，以提高效率和疫苗接种，生成表达CSDE1的VSV的因素 溶瘤平台。总体而言，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的途径中 有能力的准物种，并将为新颖，改进VSV的开发提供疫苗接种和肿瘤的发展。