The objective of the proposed work is to solve the mechanism by which poliovirus expresses its genetic information, and uses this information for the replication of its genome. The project can be broadly divided into two parts: First, the production and function of poliovirus RNA replication proteins and second, the mechanism of initiation, elongation, and re-initiation of RNA synthesis. The proposed work will be concentrated particularly on detailed steps of the mechanism by which the terminal protein VPg is uridylylated, and the product is used for elongation, a reaction that has recently been carried out in an aqueous medium. Special emphasis will be given to RNA signals at the 5' and 3' termini that direct the replication proteins to the RNA template. For an elucidation of the function of individual replication proteins, genetic and biochemical procedures as well as the yeast two-hybrid system will be used. With the aid of the procedure of cell-free, de novo synthesis of poliovirus, results of reconstituted biochemical reactions will be tested. Since the cell-free system mimics replication of poliovirus in vivo, this is a useful strategy of bypassing the cell-membrane barrier. Moreover, the cell-free replication system will be applied to study genetic recombination in vitro. Finally, the mechanism by which the poliovirus polyprotein processes itself into functional intermediate and final cleavage produces will be analyzed. The results should be broadly applicable not only to investigations of picornaviruses, but also to RNA viruses with genome-linked proteins as well as RNA viruses that express their ORF in the form of a polyprotein, such as retroviruses and hepatitis C virus.
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