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

Scripps Research Institute, The, La Jolla CA

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Abstract

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.