Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection

doi:10.1073/pnas.1932546100

Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection

^*HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702; and ^†Department of Chemistry, Stanford University, Stanford, CA 94305

Edited by John M. Coffin, Tufts University School of Medicine, Boston, MA, and approved July 28, 2003 (received for review April 29, 2003)

Abstract

Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson–Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66/p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA/DNA hybrid was cleaved 3–4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (+) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: slegrice{at}ncifcrf.gov.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: RT, reverse transcriptase; HIV-1, HIV type 1; PPT, polypurine tract; dF, 2,4-difluoro-5-methylbenzene deoxyribonucleoside; dD, 2-fluoro-4-methylbenzene deoxyribonucleoside.