Abstract
During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.
Highlights
During retrovirus replication, reverse transcriptase (RT) must interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis
We have investigated the role that residues in the ␣H and ␣I helices in the human immunodeficiency virus type 1 (HIV-1) RT thumb subdomain play in positioning the bound P/T for correct removal of the PPT
Precise recognition and cleavage of the PPT is a crucial event in retrovirus replication and defines one end of proviral DNA
Summary
Reverse transcriptase (RT) must interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the ␣H and ␣I helices in the thumb subdomain play in specific RNase H cleavage at the 3-end of the PPT; an in vitro assay modeling the primer removal step was used. A short, purine-rich sequence known as the polypurine tract (PPT) is almost exclusively used as the primer for plus-strand initiation One possibility is that the PPT sequence is intrinsically resistant to RNase H degradation and survives as the sole RNA primer available for plus-strand initiation. Escherichia coli RNase H catalyzes cleavages within the MuLV PPT [9, 16, 17, 19] as well as within the HIV-1 PPT.
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