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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 病毒成熟几乎发生在所有复杂的病毒中。在成熟之后，病毒获得了传染性，通常也获得了细胞外环境所需的稳定性。我们以卡氏新月形体omega病毒(NwV)为模型来了解这一过程，并通过突变来控制它。 NwV Proapsid直径为480A，由240、70 kDa亚基组成。成熟的颗粒直径为410A，由240个残基1-570(β)和240个副本的多肽571-644(伽马)组成。α(1-644)到β+伽马的自身催化是成熟的一个方便的报告。在体外，成熟是通过将pH从7.5降低到4.0来启动的。在pH=5.0时，颗粒大小变化不到一秒，解理的半衰期约为45分钟。根据先前确定的X射线结构，进行了突变，并通过检测亚基蛋白降解的程度进行了表征。一个表现出耐人寻味的表型的突变体是E73Q。在实验误差范围内，50%的亚基被切割，其余的在很长一段时间内没有被切割。这表明我们选择性地扰乱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.