The long-term objective of this project is to determine how virus infection alters host cell metabolism, with particular emphasis on host protein synthesis. During the next five years, the genome of the encephalomyocarditis virus will be mapped to determine the location of regions whose products inhibit host translation, or cause other toxic effects in the host cells. Of particular interest will be genomic regions whose expression results in cell death (lysis). Subsequently, the genome of poliovirus, and perhaps other viruses, will be mapped in the same way. The mechanism by which individual virus gene products cause cytotoxic will also be investigated. Concurrently, the molecular features which enable some host and viral mRNAs to resist viral cytotoxic effects, and to maintain their translation rates in infected cells, will be investigated. Of particular interest will be 5' proximal secondary and tertiary structures, which are expected to influence the interaction of the mRNA with host initiation factors in important ways. The primary experimental tool to be employed is an inducible expression vector, that consists of a polylinker region immediately downstream from the transcription start site of the mouse metallothionein promoter, both carried on a bovine papilloma virus-based shuttle vector. Virus genes inserted into the polylinker can be introduced into mouse C127 cells, which are stably transformed by this vector. Expression of these virus genes can then be induced by more than 50-fold (employing recently developed techniques), and the effects of their products on the host cell can be monitored. This same expression vector will be used to study the translation of mRNAs bearing a variety of 5' proximal secondary and tertiary structures. The inducibility of the expression vector will be further increased by introducing regions of the ferritin L chain genome that respond to iron. The presence of these iron responsive elements in a transcription product should render it iron-inducible at the translational level, thereby increasing its range of induction to between 100-and 500-fold, depending upon cell growth conditions. The implications of this work for human health are difficult to predict, since there is little understanding of how any mammalian virus kills its host cell. Thus many of the answers sought in this study will break entirely new ground. If novel cytotoxicity genes can be identified, then novel antiviral agents may suggest themselves.
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