Virulence Mechanisms of Viral Bcl-2 Homologs

描述(申请人提供)：DNA病毒在全球范围内造成广泛的发病率和死亡率。抗细胞凋亡的Bcl2家族蛋白的病毒同源物在几类DNA病毒的基因组中被编码。痘苗病毒(Vaccinia Virus，VV)是研究宿主-病原体相互作用多方面的一个典型的痘病毒家族成员，目前已鉴定出至少两个病毒Bcl2基因：F1L和N1L。VV感染或复制不需要F1L或N1L，但这两个基因对体内毒力都有很大贡献。因此，这些病毒的Bcl2(VBcl2)同源物对于VV体内的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因素，但我们发现F1L和N1L还有额外的功能，包括结合和抑制NLR家族蛋白的促炎作用，NLR家族蛋白是天然免疫的重要介质。病毒的Bcl2蛋白还可以结合Beclin并抑制自噬，最近被认为是一种抵抗病原体的宿主防御机制。我们假设vBcl2同源物是一种多功能蛋白，它利用三种不同的机制来阻止宿主防御机制：(A)抑制细胞凋亡；(B)抑制自噬；(C)干扰NLR介导的天然免疫反应。我们将测试的假设是，这三类宿主细胞靶标中的每一种都被vBcl2蛋白中和，这对毒力有重要贡献。具体地说，我们将：(1)产生F1L和N1L的位点特异性突变，从而选择性地消除它们与(A)促凋亡Bcl-2家族蛋白[细胞凋亡]；(B)NLRs[炎症]；以及(C)Beclin[自噬]；(2)检测vBcl-2基因工程蛋白对培养细胞的凋亡、炎症和自噬的影响；(3)生产具有敲入F1L和N1L突变体的重组痘苗病毒；以及(4)比较这些重组痘苗病毒在小鼠中的毒力。通过使用VV作为模型系统，我们的结果将为了解病毒Bcl2同源基因在病毒致病性中的作用奠定基础，从而为其他含有vBcl2基因并导致人类衰弱疾病的DNA病毒提供范例。此外，通过了解病毒如何干扰细胞凋亡、自噬和炎症，产生的信息可能揭示新的策略，以模拟vBcl2功能的某些方面，以治疗有用的方式来解决过度凋亡、自噬和炎症发挥核心作用的疾病。 与公共卫生相关：DNA病毒在全球范围内造成广泛的发病率和死亡率，但几乎没有可用的治疗方案来对抗这些病原体。抗细胞凋亡的Bcl2家族蛋白的病毒同源物在几类DNA病毒的基因组中都有编码，但它们在病毒疾病中的作用和机制尚不清楚。我们将测试的假设是，病毒Bcl2蛋白对特定类型的宿主细胞蛋白的中和显著有助于毒力，这将为开发基于破坏病毒Bcl2蛋白与宿主细胞靶点相互作用的病毒治疗新策略奠定基础。

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.