Structural determinants of polymerase fidelity in human immunodeficiency virus type 1 reverse transcriptase

Abstract

Genetic variation in human immunodeficiency virus type 1 (HIV-1) poses a major problem in the eradication of HIV infection, and the low fidelity of the viral polymerase, reverse transcriptase (RT), makes a key contribution to this variation. Major structural determinants of RT fidelity include elements that contact incoming dNTP or those that contact the bound nucleic acid. Alterations in RT residues which confer resistance to nucleoside RT inhibitors (NRTIs) have previously been shown to confer decreased rates of several types of polymerase error, and the present work serves to characterize the roles of four NRTI-resistance mutations in RT fidelity. Two of these mutations occur at residues which contact incoming dNTP in the wild type RT (K65R and M184V), while the other two alterations occur at residues which interact with the bound nucleic acid in the wild type enzyme (L74V and E89G). While the NRTI-resistance mutations E89G and M184V had previously been shown to confer higher fidelity of dNTP insertion versus wild type, I found that purified RT harboring either or both of these two mutations did not result in a higher fidelity of extension past mispaired primer termini. This may contribute to the lack of change in overall fidelity demonstrated by these mutant RTs. I also showed that the NRTI-resistance mutation K65R resulted in an increase in base substitution fidelity while the L74V mutation did not alter the base substitution fidelity versus wild type RT. Interestingly, the K65R mutation was also shown by others in the same study to confer an increase in overall RT fidelity while the L74V mutation did not. Finally, I examined the influence of residue 89 on several aspects of RT fidelity via vertical scanning mutagenesis of this residue. I found that alteration of E89 to any of eight other amino acid residues resulted in an increase in (-1) frameshift fidelity and an increase in base substitution fidelity but a decrease in (+1) frameshift fidelity. The dramatic influence of altering E89 on these aspects of RT fidelity suggests that residue 89 plays a key role in the global fidelity of HIV-1 RT.