Kathryn J Howard | Search PubMed

PubMed2006

Effects of efavirenz binding on the subunit equilibria of HIV-1 reverse transcriptase.

Recent studies showed that nonnucleoside reverse transcriptase inhibitors (NNRTIs) have variable effects on dimerization of p66 and p51 subunits of HIV-1 reverse transcriptase (RT). Efavirenz, one of three NNRTIs currently used in highly active anti-retroviral therapy, enhances subunit dimerization. Sedimentation equilibrium experiments on each subunit and equimolar mixtures of both subunits were used to measure dissociation constants for the three coupled dimerization reactions of RT in the absence and presence of saturating concentrations of the drug. The dimerization constants of the p51/p51 homodimer, the p66/p66 homodimer, and the p66/p51 heterodimer increased 600-, 50-, and 25-fold, respectively, upon binding of efavirenz. The effects of NNRTIs on RT dimerization are consistent with a thermodynamic linkage between subunit association/dissociation and inhibitor binding. Analysis of crystal structures of the p66/p51 heterodimer reveals that efavirenz binding induces small structural changes at the dimer interface.

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PubMed2003

Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription.

Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.

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