HIV-1 reverse transcriptase (RT) polymorphism 172K suppresses the effect of clinically relevant drug resistance mutations to both nucleoside and non-nucleoside RT inhibitors.

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT(172K)) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the k(cat)/K(m) values for dNTP. Surface plasmon resonance experiments revealed that RT(172K) decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT(172K) results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 Å) crystal structure of RT mutated at 172 and compared crystal structures of RT(172R) and RT(172K) bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between α-helix E (where 172 resides) and the active site ß9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.

The N348I mutation at the connection subdomain of HIV-1 reverse transcriptase decreases binding to nevirapine.

The N348I mutation at the connection subdomain of HIV-1 reverse transcriptase (RT) confers clinically significant resistance to both nucleoside and non-nucleoside RT inhibitors (NNRTIs) by mechanisms that are not well understood. We used transient kinetics to characterize the enzymatic properties of N348I RT and determine the biochemical mechanism of resistance to the NNRTI nevirapine (NVP). We demonstrate that changes distant from the NNRTI binding pocket decrease inhibitor binding (increase K(d)(-NVP)) by primarily decreasing the association rate of the inhibitor (k(on-NVP)). We characterized RTs mutated in either p66 (p66(N348I)/p51(WT)), p51 (p66(WT)/p51(N348I)), or both subunits (p66(N348I)/p51(N348I)). Mutation in either subunit caused NVP resistance during RNA-dependent and DNA-dependent DNA polymerization. Mutation in p66 alone (p66(N348I)/p51(WT)) caused NVP resistance without significantly affecting RNase H activity, whereas mutation in p51 caused NVP resistance and impaired RNase H, demonstrating that NVP resistance may occur independently from defects in RNase H function. Mutation in either subunit improved affinity for nucleic acid and enhanced processivity of DNA synthesis. Surprisingly, mutation in either subunit decreased catalytic rates (k(pol)) of p66(N348I)/p51(N348I), p66(N348I)/p51(WT), and p66(WT)/p51(N348I) without significantly affecting affinity for deoxynucleotide substrate (K(d)(-dNTP)). Hence, in addition to providing structural integrity for the heterodimer, p51 is critical for fine tuning catalytic turnover, RNase H processing, and drug resistance. In conclusion, connection subdomain mutation N348I decreases catalytic efficiency and causes in vitro resistance to NVP by decreasing inhibitor binding.

Amino acid mutation N348I in the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase confers multiclass resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors.

We identified clinical isolates with phenotypic resistance to nevirapine (NVP) in the absence of known nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations. This resistance is caused by N348I, a mutation at the connection subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Virologic analysis showed that N348I conferred multiclass resistance to NNRTIs (NVP and delavirdine) and to nucleoside reverse transcriptase inhibitors (zidovudine [AZT] and didanosine [ddI]). N348I impaired HIV-1 replication in a cell-type-dependent manner. Acquisition of N348I was frequently observed in AZT- and/or ddI-containing therapy (12.5%; n = 48; P < 0.0001) and was accompanied with thymidine analogue-associated mutations, e.g., T215Y (n = 5/6) and the lamivudine resistance mutation M184V (n = 1/6) in a Japanese cohort. Molecular modeling analysis shows that residue 348 is proximal to the NNRTI-binding pocket and to a flexible hinge region at the base of the p66 thumb that may be affected by the N348I mutation. Our results further highlight the role of connection subdomain residues in drug resistance.

K70Q adds high-level tenofovir resistance to "Q151M complex" HIV reverse transcriptase through the enhanced discrimination mechanism.

HIV-1 carrying the "Q151M complex" reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.