Virulence Mechanisms of Viral Bcl-2 Homologs

病毒 Bcl-2 同源物的毒力机制

• 批准号: 8197123
• 负责人: JOHN C REED
• 金额: $ 47.75万
• 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/8197123
• 关键词: Address; Adenoviruses; Apoptosis; Apoptotic; Autophagocytosis; 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. PUBLIC HEALTH RELEVANCE: DNA viruses are responsible for extensive morbidity and mortality on a worldwide basis, and yet few therapeutic options are available to counteract these pathogens. Viral homologs of anti-apoptotic Bcl-2 family proteins are encoded in the genomes of several classes of DNA viruses but their roles and mechanism in viral disease are largely unknown. The hypothesis that we will test is that neutralization by viral Bcl-2 proteins of specific classes of host cell proteins significantly contributes to virulence, which will lay a foundation for developing new strategies for viral therapeutics based on disrupting interactions of viral Bcl-2 proteins with their host cell targets.

描述（由申请方提供）：DNA病毒是全球范围内广泛发病率和死亡率的原因。抗凋亡Bcl-2家族蛋白的病毒同源物在几类DNA病毒的基因组中编码。在牛痘病毒（VV）（痘病毒家族成员，其已用作研究宿主-病原体相互作用的许多方面的范例）中，已鉴定出至少两种病毒Bcl-2基因，F1 L和N1 L。VV感染或复制都不需要F1 L或N1 L，但这两个基因对体内毒力都有很大贡献。因此，这些病毒Bcl-2（vBcl-2）同源物对于VV的体内病毒致病性至关重要。虽然F1 L和N1 L的抗凋亡活性是解释其对病毒毒力的贡献的明显候选者，但我们发现F1 L和N1 L具有额外的功能，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蛋白也已知结合Beclin并抑制自噬，自噬最近被认为是针对病原体的宿主防御机制。 我们假设vBcl-2同源物是多功能蛋白质，其利用3种离散机制来阻碍宿主防御机制：（a）抑制细胞凋亡;（B）抑制自噬;和（c）干扰NLR介导的先天免疫应答。我们将检验的假设是，vBcl-2蛋白对这3类宿主细胞靶标中每一类的中和作用显著有助于毒力。具体而言，我们将：（1）在F1 L和N1 L中产生位点特异性突变，其选择性地消除它们与（a）促凋亡Bcl-2家族蛋白[凋亡];（B）NLR [炎症];和（c）Beclin [自噬]相互作用的能力;（2）测试工程化vBcl-2蛋白对培养细胞中的凋亡、炎症和自噬的影响;（3）制备敲入F1 L和N1 L突变体的重组牛痘病毒;（4）比较这些重组牛痘病毒在小鼠中的毒力。通过使用VV作为一个模型系统，我们的研究结果将奠定了基础，了解病毒的Bcl-2同源物在病毒致病性的作用，从而作为一个范例，为其他DNA病毒含有vBcl-2基因，并导致衰弱的人类疾病。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以治疗有用的方式模拟vBcl-2功能方面的新策略，以解决过度细胞凋亡，自噬和炎症发挥核心作用的疾病。 公共卫生相关性：DNA病毒在世界范围内造成广泛的发病率和死亡率，但很少有治疗选择可用于对抗这些病原体。抗凋亡Bcl-2家族蛋白的病毒同源物在几类DNA病毒的基因组中编码，但它们在病毒疾病中的作用和机制在很大程度上是未知的。我们将测试的假设是，病毒Bcl-2蛋白对特定类别的宿主细胞蛋白的中和作用显著有助于毒力，这将为基于破坏病毒Bcl-2蛋白与其宿主细胞靶点的相互作用开发新的病毒治疗策略奠定基础。