Mutations in the connection domain of HIV-1 reverse transcriptase increase 3′-azido-3′-deoxythymidine resistance

Abstract

We previously proposed that a balance between nucleotide excision and template RNA degradation plays an important role in nucleoside reverse transcriptase inhibitor (NRTI) resistance. To explore the predictions of this concept, we analyzed the role of patient-derived C-terminal domains of HIV-1 reverse transcriptase (RT) in NRTI resistance. We found that when the polymerase domain contained previously described thymidine analog resistance mutations, mutations in the connection domain increased resistance to 3 -azido-3 -deoxythymidine (AZT) from 11-fold to as much as 536-fold over wild-type RT. Mutational analysis showed that amino acid substitutions E312Q, G335C/D, N348I, A360I/V, V365I, and A376S were associated strongly with the observed increase in AZT resistance; several of these mutations also decreased RT template switching, suggesting that they alter the predicted balance between nucleotide excision and template RNA degradation. These results indicate that mutations in the C-terminal domain of RT significantly enhance clinical NRTI resistance and should be considered in genotypic and phenotypic drug resistance studies.drug resistance ͉ excision ͉ recombination ͉ RNase H ͉ thymidine analog mutations N ucleoside reverse transcriptase inhibitors (NRTIs) constitute a major class of clinically effective antiretroviral drugs (1). HIV-1 populations possess high genetic diversity, which allows them to acquire rapidly resistance to NRTIs and other inhibitors, limiting the effectiveness of antiviral drugs in controlling viral replication and combating AIDS (2). Resistance to the NRTIs 3Ј-azido-3Ј-deoxythymidine (AZT), 2,3-didehydro-2,3-dideoxythymidine (d4T), dideoxyinosine 2Ј,3Ј-dideoxyinosine, 2Ј,3Ј-dideoxycytidine, abacavir, and tenofovir is associated with thymidine analog resistance mutations (TAMs) that are located in the polymerase (pol) domain of HIV-1 reverse transcriptase (RT) (1).We recently observed that AZT treatment increases the frequency of RT template switching in single-cycle assays and that mutations in the RNase H (rh) domain of HIV-1 RT confer high-level resistance to AZT and d4T (3). RT template switching occurs through a proposed mechanism called dynamic copy choice (4), which postulates that a balance between the rates of DNA synthesis and RNA degradation is an important determinant of RT template switching: slowing DNA synthesis increases RT template switching, whereas reducing RNA degradation decreases RT template switching (4, 5). Based on these observations and predictions of the dynamic copy choice model, we proposed a previously undescribed mechanism for NRTI resistance, which states that a balance between degradation of HIV-1 RNA by rh and nucleotide excision from a terminated primer is an important determinant of NRTI resistance. Thus, a reduced rate of RNA degradation is proposed to increase the time period available for excision of incorporated NRTIs, leading to an increase in NRTI resistance.To investigate whether this proposed mechanism contributes to NRTI resistance arising duri...

Connection Domain Mutations N348I and A360V in HIV-1 Reverse Transcriptase Enhance Resistance to 3′-Azido-3′-deoxythymidine through Both RNase H-dependent and -independent Mechanisms

“…In this case, RNase H cleavage appears to force the release of the primer at early time points, and the primer does not rebind to the enzyme under these conditions. The release of the primer is less efficient with the mutant enzyme, which provides experimental evidence for the hypothesis that connection domain mutations can delay the irreversible degradation of the DNA⅐RNA substrate (33).…”

“…Substrates-The combination of A360V and N348I, when present against a background of TAMs was shown to amplify resistance to AZT but not to d4T (33). The crucial role played by TAMs suggests that the combination of TAMs and connection domain mutations further increase efficiency of ATP-mediated AZT-MP excision.…”

“…The efficiency of rescue of DNA synthesis follows the order WT RT Ͻ TAMs Ͻ TAMs/ A360V Ͻ TAMs/N348I Ͻ TAMs/A360V/N348I. A360V and N348I Reduce RNase H Activity and Mediate the Accumulation of Truncated Substrates-N348I-containing mutant enzymes were shown to reduce RNase H activity (26,33). Here, time course experiments were employed to compare RNase H cleavage efficiencies in the context of each of the FIGURE 1.…”

“…The combined mutations were shown to cause marked increases in resistance to AZT, predominantly when present against a background of TAMs (33). N348I was also shown to confer low level resistance to AZT and NNRTIs in the absence of TAMs (26).…”

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

“…Mutations in HIV-1 RT that confer resistance to the NRTI, 3Ј-azidothymidine, increase rates of excision (15)(16)(17)(18)(19)(20)(21). More recently, it has been shown that mutations in the connection and RNase H domains of HIV-1 RT confer resistance to NRTIs and NNRTIs (9,10,(22)(23)(24)(25)(26). These mutations appear to exert their effects on susceptibility to 3Ј-azidothymidine more indirectly.…”