Nucleic Acids Research, 1994, Vol. 22, No. 1 47-52
© 1994
MOLECULAR BIOLOGY |
Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis
The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington Seattle, WA 98195, USA
*To whom correspondence should be addressed
Received October 6, 1993. Accepted November 30, 1993.
The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosls virus, DNA polymerase-
or T7 DNA polymerase, pauses specifically at polydeoxyadenosine stretches within the 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 hypervariable region 1 of the env gene (V-1) containing a polydeoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27 % of the errors Identified in V-1 sequence were frameshift mutations opposite the polyadenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsln-modlfled polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesls induced by HIV RT and yield predominantly non-viable virus.
+Present address: Laboratory of Molecular Genetics, National Institute of Aging, NIH, Baltimore, MD 21224, USA
Present addresses: Laboratoire de Pharmacologie et Toxkologie Fondamentales du CNRS, 205, route de Narbonne, 31077 Toulouse cedex, France
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