Two modes of HIV-1 polypurine tract cleavage are affected by introducing locked nucleic acid analogs into the (-) DNA template.

Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (-) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA.rU tract immediately 5' of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT.rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC.rG and neighboring dT.rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3'-end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (+5/+6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 + 5/+6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.