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.

项目总结