In this study, we showed that AZT selects novel mutations in RT, specifically A371V in the connection domain and Q509L in the RNase H domain, that increase AZT resistance up to 50-fold when combined with the TAMs in the polymerase domain. This provides the first clear virologic evidence that mutations in both the connection and RNase H domains of RT can be selected by AZT. In addition, we show that these mutations, when combined with TAMs, confer greater cross-resistance to 3TC and ABC, with a trend toward greater TNV resistance.
The only mutations that arose during the selection that started with AZT-resistant virus encoding the TAMs M41L, L210W, and T215Y were D67N and L214F in the polymerase domain (Table ), which increased AZT resistance to >1,000-fold at passage 35. No mutations were detected in the connection or RNase H domains. This indicates that very high-level AZT resistance is possible with mutations restricted to the polymerase domain. Nevertheless, we determined the effects of A371V and Q509L mutations on the AZT susceptibility of virus with M41L/L210W/T215Y. AZT resistance was increased 10-fold by the addition of A371V and Q509L to the M41L/L210W/T215Y background, indicating that the phenotypic effects of A371V/Q509L are not specific for a single TAM pattern.
Other evidence suggests that mutations outside of the polymerase domain of HIV-1 RT are involved in resistance to NRTI. For example, Nikolenko et al. recently demonstrated that mutations that reduce RNase H activity, such as D549N and H539N, increase AZT resistance (22
), but these mutations have not been identified in viruses from antiretroviral-experienced patients, nor have they been selected for by AZT in vitro. Initial analyses of clinical samples, however, have identified mutations in the connection and RNase H domains of RT that can increase AZT resistance (7
). For example, mutations G335C, N348I, and A360I reduce AZT susceptibility 30-, 35-, and 30-fold, respectively, when present with TAMs (9
). In addition, a polymorphism at RT amino acid 333 (G to E) has been observed in samples from patients on combination therapy with AZT and 3TC (15
). The G333E polymorphism counteracts the increase in AZT sensitivity of virus with the 3TC resistance mutation, M184V (33
Several retrospective statistical analyses of clinical-genotype databases have identified other mutations in the connection and RNase H domains of RT that appear more frequently in samples from antiretroviral-experienced patients than antiretroviral-naïve patients (5
). However, the roles of these mutations in NRTI resistance have not been proven. The A371V mutation has been identified in patient genotypes in the Stanford HIV Drug Resistance Database (26
), and our preliminary analysis of this database revealed that patients treated with AZT showed an increase in the frequencies of several mutations in the C terminus of RT (amino acids 350 to 560). For example, A371V was detected in 5.6% of 160 samples from treatment-naïve individuals and in 10.9% of 91 samples from patients treated with AZT monotherapy. Another mutation at codon 371 (A to T) was also seen at 2.1% frequency in AZT monotherapy samples. In addition, A371V was associated with mutations at T215 (Y/F/I/S) in 77% of the AZT monotherapy samples and with 46%, 23%, 31%, 23%, and 15% of the samples with M41L, D67N, K70R, L210W, and K219Q, respectively. Only 16 full-length sequences (to codon 560) from AZT monotherapy samples are available in the Stanford database, and none of these have mutations at codon 509. Additional full-length RT sequences from patients who have received AZT therapy are being generated to examine the RNase H domain, including codon 509.
Two phenotypic mechanisms of NRTI resistance have been proposed. The first is NRTI discrimination, and it involves mutations in RT (such as K65R, K70E, L74V, Q151M, and M184V) that enable RT to preferentially incorporate the natural dNTP substrate versus the NRTI-triphosphate (2
). The second mechanism has been termed NRTI excision associated with TAMs. The available biochemical evidence suggests that TAMs increase the ability of HIV-1 RT to phosphorolytically excise AZT-MP from the chain-terminated T/P (3
). Because A371V and Q509L were selected in combination with TAMs and do not confer resistance to AZT alone, we hypothesize that these mutations enhance the RT-mediated excision reaction.
Analysis of the crystal structure of RT bound to an RNA/DNA T/P showed that A371V and Q509L reside close to the DNA binding tract in RT (Fig. ). This suggests that the mutations may affect either T/P interactions (in the case of A371V and Q509L) or RNase H activity (in the case of Q509L). With regard to the latter, several studies have clearly demonstrated that mutations in the RNase primer grip can significantly impact the rates and efficiency of RNase H cleavage (14
). Mechanistic studies are currently under way to define the biochemical mechanisms by which A371V and Q509L increase AZT resistance.
Because there was only a small difference between the IC50 values of viruses with D67N/K70R/A371V/Q509L and D67N/K70R/T215I/A371V/Q509L (Table ), replication capacity and kinetics assays were performed to determine whether the T215I mutation affected viral replication capacity/kinetics. Single-cycle and multiple-cycle replication assays clearly showed that the T215I mutation restored the replication capacity and kinetics of the D67N/K70R/T215I/A371V/Q509L mutant to wild-type levels (Fig. ). This likely explains why the T215I mutant emerged without having a significant impact on AZT resistance. The T215I mutation was subsequently replaced by T215F at higher AZT selective concentrations. This replacement is likely explained by the fact that the T215F mutation conferred ~25-fold-greater AZT resistance than T215I, but at the cost of reduced replication capacity and kinetics in the absence of AZT.
In summary, we have selected mutations in vitro in the 3′ region of RT that increase AZT resistance and cross-resistance to other nucleoside analog RT inhibitors. Biochemical analyses are in progress to define the mechanisms involved, and additional studies of clinical isolates are planned to define the occurrence and clinical significance of the mutations.