Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

Abstract

Two mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation. IMPORTANCE Although antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

APOBEC3 selects V179I in HIV-1 reverse transcriptase to provide selective advantage for non-nucleoside reverse transcriptase inhibitor-resistant mutants

“…Competition experiments were performed comparing Y181C and V179I/Y181C HIV-1 NL4–3 or Y181V and V179I/Y181V HIV-1 NL4–3 in two independent experiments, as previously described ( 50 ). Briefly, HuT-R5 cells were co-infected with each virus at a 1:1 ratio with a total MOI of 0.004 for 2h at 37°C.…”

“…HIV-1 RNA was isolated from cell supernatants at each time point using the RNeasy kit (Qiagen). PrimerID MiSeq was performed to sequence part of the RT coding region as described previously ( 50 ). The average number of paired end reads for each flask in Experiment 1 was 8,927 copies (range from 2,708 to 14,532 copies) and in Experiment 2 was 483 copies (range from 80 to 1054).…”

“…HIV-1 RNA was isolated from supernatant collected at the last time point from each round of selection using the RNeasy kit. PrimerID MiSeq for the RT coding region was performed as previously described ( 50 ) to determine the frequencies of amino acid substitutions at residues 179 and 181. The average number of paired end reads for each flask was 1,514 copies (range from 466 to 4,307 copies) for Experiment 1 and 628 copies (range from 173 to 1,222 copies) for Experiment 2.…”