This proposal focuses on the synthesis and the signals that may regulate termination of the (+) strand DNA intermediate of HIV, 18 bases into the tRNA/lys3 primer for (-) strand DNA synthesis. I propose that a methylated adenine at position 58 of tRNA/lys3 may destabilize the polymerase of HIV thereby preventing replication of additional sequence that could impair HIV growth. Experiments are proposed to determine the role of modified bases in generating the HIV (+) strand DNA intermediate, using tRNA/lys3 from rabbit liver with a methylated A58 compared to unmodified tRNA/lys3 produced by T7 RNA polymerase. Because secondary structure in tRNA/lys3 may also cause termination, a synthetic RNA primer with the same 3' sequence but without native secondary structure will be tested to determine whether termination is structurally based. Finally, preliminary observations in our laboratory suggest that the p51 subunit of reverse transcriptase originally thought to be inactive, does generate DNA on a RNA primed DNA template. Thus, mutagenized p51 will be mixed with native p66 to assess its role in generating the (+) strand DNA intermediate. These studies should serve to identify a key control point in the HIV life cycle and perhaps a potential target for therapeutic strategies.
| Burnett, B P; McHenry, C S (1997) Posttranscriptional modification of retroviral primers is required for late stages of DNA replication. Proc Natl Acad Sci U S A 94:7210-5 |
