Background:  Mutations introduced into the RNase H domain of HIV-1 reverse transcriptase (RT) have been reported to retard RNase H activity and also cause resistance to 3’-azidothymidine (AZT). Hypothetically, these mutations in the RNase H domain reduce RNA template degradation, thereby prolonging the window of opportunity for AZT-monophosphate to be excised from the terminated primer. It is not known, however, whether mutations in the RNase H domain are selected by AZT. We therefore performed in vitro selections of HIV-1 with AZT and examined the entire coding sequence of RT for the emergence of resistance-related mutations.

Methods:  Separate selections with AZT were carried out starting with wild type or mutant virus encoding 3 AZT-resistance mutations (41L, 210W, 215Y). The viruses were passaged in MT-2 cells in increasing concentrations of AZT, or no drug as a control. AZT susceptibility (IC₅₀) was determined in P4/R5 cells that express β-galactosidase under the control of the HIV-1 LTR. The entire coding region of RT from passaged viruses was real-time polymerase chain reaction (RT-PCR) amplified from HIV-1 RNA and DNA sequenced. 

Results:  After 60 passages of wild type HIV-1 in AZT (32 mM maximum concentration), the AZT IC₅₀ increased from 0.362 mM to 29.22 mM (242- fold resistance). Sequencing of passage-60 virus identified 5 mutations in RT:  D67N, K70R, T215I/F, A371V, and Q509L. Q509 is a highly conserved residue located near the RNase H primer grip region of RT. A371 is a polymorphic residue (A/V) located in the connection domain of RT. Residues in the vicinity of A371 contact the RNA or DNA template strands of template/primer complexes. After 35 passages of mutant virus (41L, 210W, 215Y) in AZT (150 mM maximum concentration), the AZT IC₅₀ increased from 6.1 mM to >810 mM (>1630-fold resistance). Sequencing of passage 35 virus identified two new mutations in the polymerase domain of RT (67N and 214F) but no mutations in the RNase H domain.

Conclusions:  Starting with wild type HIV-1, AZT selected the Q509L mutation in the RNase H domain of RT in addition to known polymerase mutations (67N, 70R, T215I/F). Q509 is highly conserved among wild type HIV-1 isolates suggesting a role in AZT resistance. Site-directed mutagenesis is in progress to assess this possibility.