Molecular architecture of the Vaccinia virion by structural proteomics

痘病毒是一个具有医学、生态和农业重要性的主要病毒家族。 天花是最臭名昭著的家庭成员，一直是人类的最大杀手之一。 虽然这种疾病在大约40年前就被根除了，但天花再次流行的可能性 随着最近的证明，在未来的某个时候出现的次数已经大大增加了 在实验室里，一种与天花非常相似的痘病毒可以很容易地重新创造出来。 此外，根除和停止接种疫苗恰逢出现了 非洲、美国和英国的野生人痘病毒，包括人类猴痘。不知道 尽管天花是致命因素，但这种暴发的全部潜力仍不确定。它的重要性 病毒包膜和衣壳蛋白在介导抗病毒治疗和 疫苗是无可争辩的。特别是对于小RNA病毒，了解病毒粒子的结构 分子或原子分辨率指导了治疗剂和一种 了解感染和疾病的机制。由于它们的复杂性、不对称性和 异质性，然而，痘病毒病毒体一直试图阐明它们的 分子结构，关闭了一条合理设计和干预的潜在途径。私家侦探。 假设痘苗病毒粒子的相对复杂性可能是一种治疗跟腱 跟上。此外，从分子水平上理解病毒粒子的形态发生和组织，一 在痘病毒生命周期中最后剩下的黑匣子中，至少有五个 病毒复制的七个经典阶段。我们对痘病毒粒子认识上的一个主要差距 结构位于分子结构的水平上-介于 病毒粒子中所含蛋白质分子的超微结构特征和清单。这个 P.I.已经成功地将蛋白质-蛋白质化学交联法应用于 使用蛋白质质谱仪(XLMS)来发现邻近的蛋白质和结构域 原位未中断的牛痘病毒粒子。该提案的目标1旨在深化XLMS数据集，以 这一水平将允许蛋白质分子对接。XLMS与突变病毒颗粒的结合 形态发生受阻，没有明显的内部组织，目标2 提案询问病毒粒子的形态发生途径是否遵循经典的程序性线性 等级或自组织过程，没有来自分子的单一的、占主导地位的路径 组装成病毒粒子的组件。使用“QconCAT”定量MS，AIM 3寻求转换 通过确定病毒粒子蛋白质的全局化学计量比，将XLMS数据转换为分子模型。

The poxviruses comprise a major virus family of medical, ecological and agricultural importance. The most notorious family member, smallpox has been one of the great killers of mankind. Although the disease was eradicated some 40 years ago, the possibility of smallpox re- appearance at some future time has increased immeasurably with the recent demonstration that a poxvirus very similar to smallpox could be recreated de novo, in the laboratory, with ease. Moreover, eradication and the cessation of vaccination has coincided with the appearance of feral human poxviruses including human monkeypox in Africa, the US and UK. Not knowing the lethal factor in smallpox, the full potential of such outbreaks remains uncertain. The importance of virus envelope and capsid proteins in mediating the effects of antiviral therapeutics and vaccines is undisputed. For small RNA viruses in particular, an understanding of virion structure at molecular or atomic resolution has instructed the development of therapeutic agents and an understanding of mechanisms of infection and disease. Due to their complexity, asymmetry and heterogeneity, poxvirus virions have, however, persistently eluded attempts to elucidate their molecular structure, closing a potential avenue of rational design and intervention. The P.I. hypothesizes that the relative complexity of the vaccinia virion may be a therapeutic Achilles heel. Moreover, a molecular-level understanding of virion morphogenesis and organization, one of the last remaining black boxes in the lifecycle of the poxviruses, impinges upon at least five of the seven classical stages of virus replication. A major gap in our knowledge of pox virion structure lies at the level of molecular architecture – an intervening organizational level between ultrastructural features and the inventory of protein molecules contained within the virion. The P.I. has successfully applied a protein-protein chemical crosslinking approach in combination with protein mass spectrometry (XLMS) to discover neighboring proteins and domains within the undisrupted vaccinia virion in situ. Aim 1 of this proposal seeks to deepen the XLMS dataset to a level that will allow protein molecular docking. Combining XLMS with mutant virus particles blocked in morphogenesis and displaying no apparent internal organization, Aim 2 of this proposal asks whether the virion morphogenic pathway follows a classical programmed linear hierarchy or a process of self-organization with no single, dominant route from molecular components to assembled virion. Using “QconCAT” quantitative MS, Aim 3 seeks to convert XLMS data to a molecular model by determining the global stoichiometries of virion proteins.