Molecular architecture of the Vaccinia virion by structural proteomics

痘病毒包括医学、生态和农业重要性的主要病毒家族。 作为最臭名昭著的家族成员，天花一直是人类最大的杀手之一。 虽然这种疾病在大约40年前就被根除了，但天花重新流行的可能性仍然存在。 在未来的某个时间出现了不可估量的增加与最近的演示， 一种与天花病毒非常相似的痘病毒可以在实验室里很容易地重新制造出来。 此外，根除和停止接种疫苗的同时， 野生人类痘病毒，包括非洲、美国和英国的人类猴痘病毒。不懂 由于天花病毒是致命因素，这种爆发的全部潜力仍然不确定。的重要性 病毒包膜和衣壳蛋白在介导抗病毒治疗作用中的作用， 疫苗是毋庸置疑的。特别是对于小RNA病毒， 在分子或原子分辨率上的应用指导了治疗剂的开发， 了解感染和疾病的机制。由于其复杂性、不对称性和 然而，由于病毒的异质性，痘病毒粒子一直未能阐明其 分子结构，关闭一个合理设计和干预的潜在途径。私家侦探 假设牛痘病毒粒子的相对复杂性可能是治疗的致命弱点， 脚跟此外，对病毒体形态发生和组织的分子水平理解， 在痘病毒生命周期中最后剩下的黑盒中，至少有五个 病毒复制的七个典型阶段我们对痘病毒体的认识中的一个主要空白 结构存在于分子结构的水平上--一个介于 超微结构特征和病毒体内所含蛋白质分子的清单。的 P.I.已经成功地将蛋白质-蛋白质化学交联方法与 用蛋白质质谱（XLMS）来发现邻近的蛋白质和结构域， 原位未破坏的牛痘病毒体。本提案的目标1旨在深化XLMS数据集， 一个允许蛋白质分子对接的水平。将XLMS与突变病毒颗粒组合 形态发生受阻，没有明显的内部组织， 一项提案提出，病毒体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，没有单一的，占主导地位的途径，从分子 组件组装的病毒体。使用“QconCAT”定量MS，目标3寻求将 通过确定病毒体蛋白质的总体化学计量，将XLMS数据与分子模型相结合。

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.