MUTAGENICITY AND PAUSING OF HIV REVERSE-TRANSCRIPTASE DURING HIV PLUS-STRAND DNA-SYNTHESIS

The unusually high frequency of misincorporation by HIV-1 reverse tran scriptase (HIV RT) is likely to be the major factor in the rapid accum ulation of viral mutations in AIDS, especially in the env gene. To inv estigate the ability of HIV RT to copy the env gene, we subcloned an H IV env gene fragment into a single-stranded DNA vector and measured th e progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA po lymerase, pauses specifically at polydeoxyadenosine stretches within t he env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hyperva riable region 1 of the env gene (V-l) containing a polydeoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning a nd sequencing the copied fragments. We found that 27 % of the errors i dentified in V-1 sequence were frameshift mutations opposite the polya denosine tract, a site where strong pausing was observed. Pausing of H IV RT at the polyadenosine tract could be enhanced by either distamyci n A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experimen ts in which HIV RT catalyzes gap filling synthesis within the IacZ gen e in double-stranded circular M13mp2 DNA. These combined results sugge st that the enhanced mutation frequency may be due to increased pausin g at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift muta genesis induced by HIV RT and yield predominantly non-viable virus.