Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type 1 reverse transcriptase that increase resistance to 3'-azido-3'-dideoxythymidine

Abstract

Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3'-azido-3'-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1(LAI) or AZT-resistant HIV-1(LAI) (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1(LAI) were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance ( approximately 16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance approximately 10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

FIG. 1.

Single-cycle and multiple-cycle replication assays of recombinant HIV_LAI containing the A371V and Q509L mutations. (A) Single-cycle replication was measured in P4/R5 cells infected with 10 ng p24 in a 96-well plate. After 48 h, cells were lysed and viral replication was measured using RLU. (B) Multiple-cycle replication was measured in MT-2 cells infected with 10 ng p24. p24 was measured from cell supernatant harvested daily for 7 days. The recombinant viruses analyzed were wild-type HIV_LAI (closed circle), D67N/K70R (open square), D67N/K70R/A371V/Q509L (closed square), D67N/K70R/T215I/A371V/Q509L (open circle), and D67N/K70R/T215F/A371V/Q509L (closed triangle). The data represent the means and standard deviations from three independent experiments. *, P values of <0.05 were considered significant compared to the wild-type control (HIV_LAI).

FIG. 2.

Structural representation of AZT-selected mutations in the p66 subunit of RT. (A) Locations of residues A371 and Q509 in relation to TAMs D67, K70, and T215, the polymerase active site, and the RNase H active site. (B) Both A371 and Q509 are located near the T/P DNA binding tract. A371 is 2.8 Å from K374, whose side chain interacts with the phosphate backbone of the RNA template strand (white line). Q509 is in proximity to I505, a residue that makes up the RNase H primer grip. p66 subunit of RT, cartoon; DNA primer, green ribbon; RNA template, purple ribbon. The structure was drawn using the Molecular Operating Environment, based on coordinates from Sarafianos et al. (30). Protein Data Bank access number 1HYS.