The Pox Virion Molecular Interactome

描述(申请人提供)：历史上，天花一直是人类的最大杀手之一。虽然这种疾病被认为在大约35年前就已经根除，但痘病毒仍然构成了一个具有医学、生态和农业重要性的主要病毒家族。对于人类来说，除了天花重新传入的可能性外，根除天花与过去40年人类猴痘在非洲和过去10年在美国出现的情况不谋而合。由于不知道天花的致死因素，这种暴发的全部潜力仍然不确定。病毒包膜和衣壳蛋白在调节抗病毒治疗和疫苗效果方面的重要性是毋庸置疑的。从微小核糖核酸病毒到疱疹病毒，在分子或原子分辨率上了解病毒粒子的外部分子结构，指导合理设计治疗药物，并理解导致和阻止病毒感染和疾病的机制。然而，由于它们的复杂性、不对称性和异质性，痘病毒一直特别坚持不懈地挫败在分子上理解它们的病毒粒子结构的尝试。 因此，避免了一个重要的潜在干预途径。P.I.假设，痘苗病毒的复杂性可能会被证明是一个致命弱点。此外，对病毒粒子结构的全面了解可能被认为是痘病毒生命周期中最后剩下的黑匣子之一-影响痘病毒复制的早期转录、基因组脱壳和病毒粒子组装阶段。我们对痘病毒粒子结构的了解的主要漏洞在于病毒粒子中存在的蛋白质清单之间的水平 (这在很大程度上是已知的)以及完整粒子的基本拓扑和地形特征(也已知)。这一中间区域可能被称为病毒粒子的“分子结构”，或蛋白质“相互作用体”。在这份R21提案中，P.I.选择了结合质谱学的蛋白质-蛋白质交联法。这种相互作用组分析方法在许多方面都是公正的，并且有提供精细细胞组件的分子结构的记录，如核孔复合体、20S蛋白酶体和RNA聚合酶II。这项建议的目标1试图通过共价蛋白质-蛋白质交联/MS鉴定病毒核心内并列的蛋白质，采取“自上而下”(蛋白质水平)和“自下而上”(多肽水平)的方法。P.I.假设，痘病毒核心壁可能与一些被包裹的RNA病毒的基质蛋白层没有根本不同，经典的内部酶与周围有结构蛋白的描述可能不像目前假设的那样清晰。

DESCRIPTION (provided by applicant): Smallpox has, historically, been one of the great killers of mankind. Although this disease is considered to have been eradicated some 35 years ago, the poxviruses, nonetheless, comprise a major family of viruses of medical, ecological and agricultural importance. For humans, aside from the possibility of smallpox re-introduction, eradication has coincided with the appearance of human monkeypox in Africa during the past 40 years and in the US during the past decade. Not knowing the lethal factor in smallpox, the full potential of such outbreaks remains uncertain. The importance of virus envelopes and capsid proteins in mediating the effects of antiviral therapeutics and vaccines is undisputed. From the picornaviruses to the herpesviruses, an understanding of virion outer molecular structures at molecular or atomic resolution, has instructed the rational design of therapeutic agents and an understanding of mechanisms that cause and thwart virus infection and disease. Due to their complexity, asymmetry and heterogeneity, the poxviruses have, however, been particularly persistent in defeating attempts to understand their virion structure at the molecular level, thus evading an important potential avenue for intervention. The P.I. hypothesizes that the complexity of the vaccinia virion may prove to be an Achilles heel. In addition, a full understanding of virion structure may be regarded as one of the last remaining black boxes in the lifecycle of the poxviruses - one which impinges upon the early transcription, genome uncoating and virion assembly stages of poxvirus replication. The major hole in our knowledge of pox virion structure lies at a level between the inventory of proteins present within the virion (which is largely known) and the basic topological and topographical features of the intact particle (also known). This intervening area may be referred to as the virion's "molecular architecture", or protein "interactome". In this R21 proposal, the P.I. has chosen a protein-protein crosslinking approach in combination with mass spectrometry. Such an approach for interactome analysis is unbiased in many respects, and has a track record of informing the molecular architectures of elaborate cellular assemblies such as the nuclear pore complex, 20S proteasome and RNA polymerase II. Aim 1 of this proposal seeks to identify directly juxtaposed proteins within the virion core via covalent protein-protein crosslinking/MS, taking "top-down" (protein-level) and "bottom-up" (peptide-level) approaches. The P.I. hypothesizes that the pox virion core wall may not be fundamentally dissimilar to the matrix protein layers of some enveloped RNA viruses, and that the classical delineation of enzymes in the deep interior with structural proteins surrounding may not be as clear cut as currently supposed.