FUNCTIONS AND INHIBITION OF HIV POLYMERASE

Retrovirus reverse transcriptases (RT) catalyze a reaction called transfer synthesis, whereby a DNA primer is moved form one viral template to another. The reaction is essential for viral replication, and likely an important step in recombination. It is probable that this type of reaction, when it occurs between internal regions of the viral chromosome, contributes to diversity in the viral genome, improving viral survival. We intent to understand and eventually prevent internal strand transfer from occurring in vivo, since it may be one of the features that improves virulence. A strand transfer assay in vitro will be used to measure the capacity of the reverse transcriptase to transfer DNA synthesis from a natural sequence donor template to an acceptor template. The role of template sequence and secondary structure, and of RNA cleavage, in the strand transfer reaction will be tested. Effects of specific alterations in gap size produced in the RNA template, the size of specifically introduced hairpin loops, and degree of homology between donor and acceptor templates on the efficiency of strand transfer will be examined. The effect of covalent modifications to the donor RNA forming cisplatin adducts will also be determined. Clinically relevant experiments will be done determining the effects of BI-RG-587 and its analogues on processive primer elongation necessary for strand transfer. In summary, the experiments are designed to determine the characteristic of the templates, RT and reaction conditions that favor the internal strand transfer reaction.