Background:  HIV-1 genotypic assays are often limited to the polymerase domain of the reverse transcriptase (RT) and ignore the potential effect of mutations in the RT connection and RNase H domains. Our previous studies showed that N348I, located in the RT connection domain, is significantly more prevalent in ART-experienced (12.1%, n = 368) compared with therapy-naïve (0.8%, n = 1009) individuals (p <<0.01) in our Canadian cohort, was ranked as the ninth most prevalent mutation of all RT drug resistance mutations and appeared relatively early in drug therapy. In an analysis of 74 patients failing drug therapy (viral load >250 copies/mL), N348I was associated with the M184V (77%), K103N (24%), T215Y (18%), and M41L (18%) mutations. Given its prevalence in patients failing therapy, we hypothesise that N348I confers decreased susceptibility to RT inhibitors.

Methods:  Phenotypic susceptibility of HIV-1 engineered with N348I alone or in the context of known resistance mutations was determined in TZM-bl or in MT-2 cells. The significance of differences in the drug 50% inhibitory concentrations was determined using the Wilcoxon Rank Sum test. The biochemical mechanism of resistance was assessed by steady-state kinetic and AZT-monophosphate (AZT-MP) primer unblocking assays.

Results:  N348I conferred 2-fold decreased susceptibility to AZT (p = 0.05) in a wild type backbone. A 2- and 4-fold increase in AZT resistance was also observed when N348I was added to viruses containing M41L+T215Y or M41L+L210W+T215Y, respectively (p = 0.05). N348I alone conferred 6-fold decreased susceptibility to nevirapine (NVP) (p = 0.05) and potentiated NVP resistance in the context of K103N (>>1.25-fold) compared to K103N alone (p = 0.05). N348I had no effect on lamivudine (3TC) susceptibility in the absence or presence of M184V. Molecular dynamics simulations suggest that N348I indirectly affects the thumb and the NNRTI-binding pocket. Biochemical analyses showed that N348I confers NNRTI resistance at the enzyme level. However, N348I did not alter RT ability to discriminate between AZT-TP and TTP or its ability to excise AZT-MP from a DNA/DNA template/primer. Additional studies to define the mechanism by which N348I confers AZT resistance are currently underway.

Conclusions:  N348I confers decreased susceptibility to AZT and NVP. These studies suggest that genotypic assays that do not consider the entire RT coding region might be inadequate for accurate prediction of viral drug susceptibility.