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

Abstract

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.HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that converts the single-stranded RNA viral genome into double-stranded DNA. It has RNA-and DNA-dependent DNA polymerase activities, which copy RNA or DNA templates, respectively, into double-stranded DNA, as well as RNase H activity that cleaves genomic RNA still hybridized to DNA (40). Although both activities are essential for HIV-1 replication, some studies have shown that polymerization and RNase H activities are not rigorously coupled (11,12,27,36). RT is a heterodimer comprised of two subunits, a 66-kDa (p66) subunit and a 51-kDa (p51) subunit (20). The p66 subunit has both the polymerase and RNase H domains, which make contact simultaneously with the RNA genome (12). The p51 subunit is an N-terminal cleavage product of p66 which lacks the RNase H domain and serves as a structural scaffold for the p66 subunit (2).Because of its essential role in the virus life cycle, RT is an important antiretroviral target for the treatment of HIV-1. Nonnucleoside RT inhibitors (NNRTIs) are one of three classes of antiretroviral RT inhibitors that are commonly used in treatment. They are a group of structurally diverse small hydrophobic compounds, which can specifically bind and inhibit RT (reviewed in reference 34). One limitation of NNRTIs is that they face a low genetic barrier to resistance. Previous studies o...

Interaction of Reverse Transcriptase (RT) Mutations Conferring Resistance to Lamivudine and Etravirine: Effects on Fitness and RT Activity of Human Immunodeficiency Virus Type 1

“…These results provide another example of cross-talk between mutations affecting the nucleoside and nonnucleoside RT inhibitor binding pockets (10,21). Other NNRTI resistance mutations result in a reduction in the amount of virion-associated RT, an effect correlated with observed fitness differences of the mutant viruses (27). Whether the E138K mutation also reduces the amount of virion-associated RT and whether that effect is counteracted by the M184V mutation are important points for future study.…”

“…(These data were presented in part at the following meetings: 18th Conference on Retroviruses and Opportunistic Infections, 27 …”

Nonnucleoside Reverse Transcriptase Inhibitor-Resistant HIV Is Stimulated by Efavirenz during Early Stages of Infection

“…We have previously shown that some NNRTI-resistant mutants have decreased amounts of RT in their virions (37). We wanted to know whether ES, D10, and D10MMTT virions had a reduced amount of RT and whether this reduction could be corrected in the presence of EFV.…”

“…The WT, G190S, K101EϩG190S (ES), and L74VϩK101EϩG190S (VES) RT p51 subunit sequences were subcloned into pET-28a(ϩ) expression vector, and the p66 subunits were cloned into pET-21a(ϩ) vectors (Novagen) (37). The WT and mutant His-tagged p51 (His-p51) subunits and untagged p66 subunits were expressed in E. coli.…”

“…Equal amounts of virus as determined by capsid protein quantitation were pelleted by centrifugation at 150,000 ϫ g for 1 h at 4°C. Detection of virion-associated RT content was performed as previously described (37). Briefly, virus pellets were resuspended in 15 l of NuPAGE 2ϫ sample buffer (Invitrogen), and viral proteins were separated by 4 to 12% Bis-Tris NuPAGE electrophoresis according to the manufacturer's instructions.…”