Reduced Fitness in Cell Culture of HIV-1 with Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Mutations Correlates with Relative Levels of Reverse Transcriptase Content and RNase H Activity in Virions

摘要

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of multidrug therapy for HIV-1. Understanding the effect of NNRTI-resistant mutants on virus replication and reverse transcriptase (RT) function is valuable for the development of extended-spectrum NNRTIs. We measured the fitness of six NNRTI-resistant mutants, the K103N, V106A, Y181C, G190A, G190S, and P236L viruses, using a flow cytometry-based cell culture assay. K103N and Y181C viruses had fitness similar to that of the wild type while V106A, G190A, G190S, and P236L viruses had reduced fitness. We also determined the biochemical correlates of fitness by measuring the RNase H and polymerization activities of recombinant mutant RTs and virion-associated RTs. The RNase H activities of recombinant and virion-associated RTs correlated with the relative fitness for each mutant. K103N and Y181C mutants had normal RNase H activity; V106A, G190A, and G190S mutants had moderate reductions in activity; and the P236L mutant had substantially reduced activity. With the exception of the P236L mutant, reduced fitness correlates with low virion-associated polymerization efficiency and reduced RT content. Reduced polymerase function in virions derived from low RT content rather than an intrinsic polymerization defect in each RT protein. In conclusion, severe defects in RNase H activity alone, exemplified by the P236L mutant, appear sufficient to cause a substantial reduction in fitness. For the other NNRTI mutants, reductions in RT content decreased both polymerization and RNase H activity in virions. RNase H reduction was compounded by intrinsic RNase H defects in the mutant RTs.