The Pox Virion Molecular Interactome

描述（由申请人提供）：天花在历史上一直是人类的一大杀手。尽管人们认为这种疾病在大约35年前就已被根除，但痘病毒仍然是具有医学、生态和农业重要性的一大病毒家族。对人类来说，除了天花再次引入的可能性之外，在过去40年里，消灭天花的同时，非洲和美国也在过去10年里出现了人类猴痘。由于不知道天花的致命因素，这种暴发的全部潜力仍然不确定。病毒包膜和衣壳蛋白在介导抗病毒治疗和疫苗作用中的重要性是无可争议的。从小核糖核酸病毒到疱疹病毒，在分子或原子分辨率上对病毒粒子外分子结构的理解，指导了治疗剂的合理设计，以及对引起和阻止病毒感染和疾病的机制的理解。然而，由于痘病毒的复杂性、不对称性和异质性，人们一直在试图从分子上理解它们的病毒粒子结构

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.