HIGH RESOLUTION EM OF NUDAURELIA CAPENSIS OMEGA VIRUS (NWV) MUTANTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 病毒成熟发生在几乎所有复杂的病毒中。成熟后，病毒获得感染性，通常获得细胞外环境所需的稳定性。我们使用Nudaurelia Capensis omega病毒（NwV）作为模型来理解这一过程并通过突变来控制它。 NwV原衣壳直径为480埃，由240，70 kDa的亚基组成。成熟颗粒直径为410 A，由240个拷贝的残基1-570（β）和240个拷贝的多肽571-644（γ）形成。α（1-644）到β + γ的自催化作用是成熟的方便报道者。在体外，通过将pH从7.5降低到4.0来启动成熟。颗粒尺寸在不到一秒的时间内发生变化，并且在pH=5.0下裂解的半衰期约为45分钟。基于先前确定的X射线结构，进行突变，并通过检查亚基蛋白水解的程度来表征。一个表现出有趣表型的突变体是E73 Q。在实验误差范围内，50%的亚基裂解，其余的亚基在很长一段时间内未裂解。这表明，我们选择性地扰动了一半亚基中的自催化位点，这可能是由于完成活性位点所需的亚基接触的扰动。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Virus maturation occurs in virtually all complex viruses. Following maturation the virus acquires infectivity and usually the stability required for the extra-cellular environment. We used Nudaurelia Capensis omega virus (NwV) as a model to understand this process and to control it by mutation. The NwV procapsid is 480 A diameter and formed by240, 70kDa subunits. The mature particle is 410 A in diameters and is formed by 240 copies of residues 1-570 (beta) and 240 copies of the polypeptide 571-644 (gamma). The autocatalysis of alpha (1-644) to beta + gamma is a convenient reporter of maturation. In vitro, maturation is initiated by lowering the pH from 7.5 to 4.0. The particle size changes in less than one second and the cleavage has a half life of approximately 45 min at pH=5.0. Based on the previously determined X-ray structure mutations were made and characterized by examining the extent of subunit proteolysis. One mutant displaying an intriguing phenotype was E73Q. Within experimental error 50% of the subunits cleaved, leaving the rest uncleaved for an extended period of time. This suggested that we selectively perturbed the autocatalytic site in half the subunits and that this was probably due to a perturbation of subunit contacts that are required for completing the active site.