CARDIOVIRAL PROTEASES AND COMPARATIVE GENOME STRUCTURE

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The medical and agricultural importance of picornavirus-caused disease has long targeted these agents for intensive laboratory study. The family encompasses a diverse variety of pathogenic agents, such as polio, rhino, foot-and mouth-disease, hepatitis A, coxsackie, and the murine cadioviruses (among others). The small size of the RNA genome (about 8,000 bases) and the relatively simple nature of the icosametric capsid shell have allowed facile manipulation of genomes as infectious CDNA copies, and determination of several virion structures to atomic resolution. These powerful tools not withstanding, there still remain many unresolved questions about how or why a specific infection does indeed bring about a particular course of disease within its natural host. Central to these processes is the molecular examination of all facets of a virus life cycle, especially those ensuing within the infected cell. Such studies not only bring to light the (exploitable) individualities of particular viral phenotypes, but also serve as critical probes to the inner workings of elementary biological phenomena whose disruption through infection can mediate the expression of disease. This program focuses on the murine cardioviruses, encephalomyocarditis virus (EMCV) and Mengovirus. The special propensity of cardioviral RNAs facilitate efficient translation in cell-free extracts, is a hallmark of these genomes, and makes these isolates exceptionally useful experimental subjects for molecular dissection of the pico mavirus life cycle. The goals of this investigation are to explore and define the relationship of the cardiovirus genus to other members of the piconavirus family, and to exploit the unique features of cardioviruses to examine fundamental molecular questions about piconaviral translation, proteolytic processing and morphogenesis. The specific aims are: (1) To define the sequence and structural contexts required for the novel NPGP "suicide" cleavage that is responsible for primary proteolytic processing of cardioviral polyprotein; (2) To evaluate the in vivo effects of engineered mutations introduced into the viral 3C protease, its processing sites, or other relevant genomic locations, and to correlate these results with specific phases or defects in the cardioviral life cycle; (3) To probe distinctive sequence and structural elements within the 5' noncoding region of cardioviral RNAs, and assess their responsibilities in cap-independent translation initiation and in viral-induced host protein shut-off during natural infection; and (4) To compare and contrast all available piconavirus sequence data by computer analysis, with special emphasis of correlative support for a revised taxonomic ordering of the family. Innovative, computer-generated graphic displays of internal and external virion capsid surfaces will also be advanced, for detailed molecular surface maps and topographical feature comparisons.