Background:  Analysis of a large clinical genotype-phenotype dataset identified 3 nucleoside reverse transcriptase inhibitor (NRTI) mutations (118I, 208Y, and 215Y) strongly associated with non-NRTI (NNRTI) hypersusceptibility. Site-directed mutagenesis revealed different patterns of NNRTI hypersusceptibility and replication capacity depending on the combination of mutations present. The 208Y/215Y and 118I/208Y/215Y mutant viruses exhibited reduced replication capacity (40 and 35% of wild type). In comparison, the 118I/215Y mutant virus replicated as efficiently as the wild type virus. We investigated a mechanism by which these mutations confer NNRTI hypersusceptibility.

Methods:  Wild type and mutant viruses were produced by transfection of proviral clones into permissive cells. The quantity of HIV-1 RT incorporated into the wild type and mutant viruses was determined by Western blot analysis of viral lysates using monoclonal antibodies that recognized RT and p24. In addition, DNA polymerase activity of RT within viral lysates was measured.

Results:  The amount of HIV-1 RT incorporated into the 208Y/215Y and 118I/208Y/215Y mutant viruses was significantly reduced compared with the wild type virus (47% and 30% of wild type, respectively). The RT content in the 118I/215Y virus was similar to the wild type virus. The polymerase activity of RT from viral lysates correlated with the amount of RT present in virions. Decreases in quantifiable viral RT and polymerase activity are evident for the 208Y/215Y and 118I/208Y/215Y mutants while 118I/215Y viral RT has activity similar to wild type.

Conclusions:  Decreased virion-associated levels of RT, as well as decreased viral enzyme activity, give evidence to a distinct mechanism of NNRTI hypersusceptibility for the 208Y/215Y and 118I/208Y/215Y mutants; whereas the 118I/215Y mutant has levels of RT and polymerase activity similar to wild type providing evidence for a different NNRTI hypersusceptibility mechanism. These data substantiate multiple mechanisms, highlighting the complexity of the NNRTI hypersusceptibility phenotype.