Virulence Mechanisms of Viral Bcl-2 Homologs

病毒 Bcl-2 同源物的毒力机制

• 批准号: 8582057
• 负责人: SHU-ICHI MATSUZAWA
• 金额: $ 48.75万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8582057
• 关键词: Address; Adenoviruses; Apoptosis; Apoptotic; Autophagocytosis; BCL-2 Protein; BCL2 gene; Binding; Biological Models; Caspase-1; Cells; Cultured Cells; DNA Viruses; Disease; Engineering; Family; Family member; Foundations; Genes; Genome; Goals; Herpesviridae; Homologous Gene; Host Defense; Host Defense Mechanism; Immune response; Inflammation; Inflammatory; Investigation; Knock-in Mouse; Laboratories; Learning; Mediating; Mediator of activation protein; Morbidity - disease rate; Mus; Mutation; Natural Immunity; Pathogenicity; Play; Poxviridae; Process; Protein Family; Proteins; Role; Site; Testing; Therapeutic; Vaccinia virus; Viral; Viral Interference; Viral Proteins; Virulence; Virus; Virus Diseases; base; cytokine; defense response; gammaherpesvirus; human disease; in vivo; inhibition of autophagy; inhibitor/antagonist; member; mortality; mutant; novel; novel strategies; pathogen; public health relevance

DESCRIPTION (provided by applicant): DNA viruses are responsible for extensive morbidity and mortality on a worldwide basis. Viral homologs of anti-apoptotic Bcl-2 family proteins are encoded in the genomes of several classes of DNA viruses. In Vaccinia Virus (VV), a poxvirus-family member that has served as a paradigm for investigations of many aspects of host-pathogen interactions, at least two viral Bcl-2 genes have been identified, F1L and N1L. Neither F1L nor N1L is required for VV infection or replication, but both of these genes make strong contributions to virulence in vivo. Thus, these viral Bcl-2 (vBcl-2) homologs are critically important for in vivo viral pathogenicity of VV. While the anti-apoptotic activity of F1L and N1L is an obvious candidate for explaining their contribution to viral virulence, we have discovered that F1L and N1L have additional functions that include binding to and suppressing the pro-inflammatory actions of NLR-family proteins, important mediators of innate immunity. Viral Bcl-2 proteins are also known to bind Beclin and suppress autophagy, recently recognized as a host defense mechanism against pathogens. We hypothesize the vBcl-2 homologs are multifunctional proteins that utilize 3 discrete mechanisms to thwart host defense mechanisms: (a) suppression of apoptosis; (b) inhibition of autophagy; and (c) interference with NLR-mediated innate immune responses. The hypothesis that we will test is that neutralization by vBcl-2 proteins of each of these 3 classes of host cell targets significantly contributes to virulence. Specifically, we will: (1) Produce site-specific mutations in F1L and N1L that selectively abolish their ability to interact with (a) pro-apoptotic Bcl-2 family proteins [apoptosis]; (b) NLRs [inflammation]; and (c) Beclin [autophagy]; (2) Test the effects of the engineered vBcl-2 proteins on apoptosis, inflammation, and autophagy in cultured cells; (3) Produce recombinant vaccinia viruses with knock-in of F1L and N1L mutants; and (4) Compare the virulence of these recombinant vaccinia viruses in mice. By using VV as a model system, our results will lay a foundation for understanding the role of viral Bcl-2 homologs in viral pathogenicity, thus serving as a paradigm for other DNA viruses that contain vBcl-2 genes and that cause debilitating human diseases. Also, by learning how viruses interfere with apoptosis, autophagy, and inflammation, the information generated may reveal novel strategies for mimicking aspects of vBcl-2 function in therapeutically useful ways for addressing disorders in which excessive apoptosis, autophagy, and inflammation play central roles.

描述（由申请人提供）：DNA病毒在世界范围内造成广泛的发病率和死亡率。抗凋亡Bcl-2家族蛋白的病毒同源物被编码在几种DNA病毒的基因组中。牛痘病毒（VV）是痘病毒家族的一员，已成为研究宿主-病原体相互作用许多方面的范例，至少已鉴定出两个病毒Bcl-2基因，F1L和N1L。VV感染或复制都不需要F1L和N1L，但这两个基因都对体内毒力有很强的贡献。因此，这些病毒Bcl-2 （vBcl-2）同源物对VV的体内病毒致病性至关重要。虽然F1L和N1L的抗凋亡活性是解释它们对病毒毒力贡献的一个明显候选，但我们发现F1L和N1L具有其他功能，包括结合和抑制nlr家族蛋白的促炎作用，nlr家族蛋白是先天免疫的重要介质。病毒Bcl-2蛋白也被认为结合Beclin并抑制自噬，最近被认为是宿主对抗病原体的一种防御机制。我们假设vBcl-2同源物是多功能蛋白，利用3种不同的机制来破坏宿主防御机制：(a)抑制细胞凋亡；(b)抑制自噬；(c)干扰nlr介导的先天免疫反应。我们将测试的假设是，这三种宿主细胞靶点的vBcl-2蛋白的中和作用显著地促进了毒力。具体来说，我们将：(1)在F1L和N1L中产生位点特异性突变，选择性地破坏它们与(a)促凋亡的Bcl-2家族蛋白相互作用的能力[凋亡]；(b) NLRs[炎症]；(c) Beclin[自噬]；(2)检测工程化vBcl-2蛋白对体外培养细胞凋亡、炎症和自噬的影响；(3)制备敲入F1L和N1L突变体的重组痘苗病毒；(4)比较这些重组痘苗病毒在小鼠体内的毒力。通过使用VV作为模型系统，我们的研究结果将为理解病毒Bcl-2同源物在病毒致病性中的作用奠定基础，从而为含有vcl -2基因的其他DNA病毒提供范例，并导致使人衰弱的疾病。此外，通过了解病毒如何干扰细胞凋亡、自噬和炎症，所产生的信息可能揭示模仿vBcl-2功能方面的新策略，以治疗有用的方式解决过度细胞凋亡、自噬和炎症起核心作用的疾病。