Residues in the alpha H and alpha I helices of the HIV-1 reverse transcriptase thumb subdomain required for the specificity of RNase H-catalyzed removal of the polypurine tract primer

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remo ve the RNA primer for plus-strand DNA synthesis. We have investigated the r ole that human immunodeficiency virus-1 RT residues in the alpha H and alph a 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. Analysis of alanine-scanning mutants revealed that a subgroup exhibit s an unusual phenotype in which the PPT is cleaved up to seven bases from i ts 3'-end. Further analysis of alpha H mutants (G262A, K263A, N265A, and W2 66A) with changes in residues in or near a structural motif known as the mi nor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substra tes. Vertical scan mutants at position 266 were all defective in specific R Nase H cleavage, consistent with conservation of tryptophan at this positio n among lentiviral RTs, Our results indicate that residues in the thumb sub domain and the minor groove binding track in particular, are crucial for un ique interactions between RT and the PPT required for correct positioning a nd precise RNase H cleavage.