The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulated expression of viral genetic information. Studies are being carried out on the functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT). A chimeric RT having the murine leukemia virus (MuLV) polymerase domain fused to E. coli RNase H has been expressed and purified and its RNase H and polymerase activities compared to those of wild-type RT. In the absence of polymerization, the chimeric protein behaves like E. coli RNase H. When there is limited extension of the primer (i.e., addition of 3 dNTPs and 1 ddNTP), we find that with wild-type RT, the RNA is shortened by the same number of bases as the DNA is extended. This result indicates that cleavage by RNase H is normally linked to DNA synthesis. With the chimeric RT, there is little or no effect of dNTPs on RNA cleavage, suggesting that this enzyme is deficient in polymerization. Indeed, direct measurement of polymerase activity shows that unlike wild-type, the chimeric RT makes small products with either an RNA or DNA template. These results indicate that despite the fact that the chimeric RT has a wild-type polymerase region, it is unable to synthesize DNA in a processive manner. Future efforts will include analysis of the RNase H and polymerase activities of other mutant RTs. In other work, translational control of viral gene expression is being investigated by studying readthrough suppression of the UAG codon at the MuLV gag-pol junction. We have now identified the readthrough signal and find that it is entirely contained within the first 57 nucleotides on the 3' side of the UAG codon. The signal is complex and consists of two distinct elements: a highly conserved, eight-nucleotide, purine-rich sequence immediately downstream of the UAG codon, followed by a pseudoknot structure spanning the next 49 nucleotides. Studies to determine how the signal affects translation of a transcript containing CAG instead of UAG at the gag-pol junction will be performed.
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