Background:  Previously, our group showed that subtype C HIV-1 selects more rapidly for K65R than subtype B HIV-1 in cell culture. However, our biochemical studies that compared subtype B and C reverse transcriptase (RT) enzymes did not reveal any major differences that would explain these observations. Recently, we presented subtype-specific differences in fidelity of RT in (+) strand DNA synthesis at the site responsible for the K65R mutation. This novel nucleotide template-based mechanism may therefore be decisive in the selection of the K65R resistance mutation pathway in subtype C, as compared to subtype B. In this study, we confirm the suggested mutational pathway outcome in cell culture.

Methods:  NL4-3 (C 64/65) plasmid (subtype B with subtype C nucleotide sequence at residues 64/65) was generated from NL4-3 (wt) plasmid (subtype B) by site-directed mutagenesis. MT2 cells were infected with viruses harvested from 293T cells (multiplicity of infection, 0.01) over 2 hours and subsequently washed. Infected MT2 cells were seeded and drugs (tenofovir [TFV]/lamivudine [3TC]/abacavir [ABC]) were added. Drug pressure was increased when the culture consistently peaked at the same interval as the control well in repeated passages without any drugs. Collected culture supernatants were sequenced for genotypic analysis.

Results:  NL4-3 (wt)acquired K65R after 15 passages with TFV, whereas it developed M184I after 7 to 13 passages with TFV+3TC or ABC. Strikingly, NL4-3 (C 64/65) developed K65R after 7 to 11 passages with any of TFV, TFV+3TC or ABC. These observations indicate that template subtype C sequences increase the propensity for K65R acquisition. Furthermore, the selective pressure for M184I/V by 3TC is outcompeted by TFV under these circumstances, although, as expected, treatment with 3TC led to the development of M184I in both cases after 7 passages. Single mutations in NL4-3 (wt) that yielded either NL4-3 (C 64) or NL4-3 (C 65) did not increase the probability for K65R acquisition.

Conclusions:  These results, together with our previous mechanistic data, demonstrate that nucleotide sequence changes at both positions 64 and 65 within subtype C, distinct from subtype B, redirect the selection pattern with multiple N(t)RTI toward the K65R resistance mutation pathway, which is not the case for single changes at either of these positions. Hence, the 64/65 changes in subtype C are required in tandem and represent signature polymorphisms for the development of K65R in this subtype in regard to N(t)RTI selective pressure.