Efavirenz stimulates HIV-1 reverse transcriptase RNase H activity by a mechanism involving increased substrate binding and secondary cleavage activity.

Treatment and prevention of HIV/AIDS remains a challenging endeavor and a current focus of basic research. A major target of antiretroviral therapies against HIV-1 is the viral reverse transcriptase (RT) 1. HIV-RT is a low fidelity DNA polymerase and highly error prone2,3. Because of this characteristic, mutations are common during reverse transcription. Furthermore, HIV packages two similar RNA genomes, allowing for facile production of recombinant progeny 4,5. Currently two main classes of approved drugs antagonize the RT, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) 6. NRTIs inhibit RT through competitive binding to the active site, while NNRTIs bind to a separate pocket away from the active site 7–9. Both classes of drugs target RT polymerase activity. Development of resistance to existing NRTIs and NNRTIs is common during treatment as a result of the high rates of both mutagenesis and recombination during reverse transcription. Therefore, there is a continuing search for new drugs. RT also possesses RNase H, strand transfer and strand displacement activities 10,11. There are currently no approved drugs directed toward these additional activities, so they remain available targets for drug development. HIV-1 converts its single stranded RNA genome into double stranded DNA that is incorporated into the host cell genome, serving to transcribe more viral RNA 12. The viral RNA genome is first converted into an RNA/DNA hybrid, and then the RNA is degraded to make way for synthesis of the second strand of DNA13. The RT, having both polymerization and RNase H functions, is able to make cuts in the RNA of the RNA/DNA hybrid as it is being made. This is called DNA 3′-end-directed, polymerization-dependent cleavage 14. The cleavage mechanism involves making primary cuts about 18 nt from the DNA 3′ end. At frequent synthesis pause sites, the RT shifts to make secondary cuts about 9 nt from the end. To complete RNA degradation, additional RTs position at the 5′ ends of the RNA fragments to carry out RNA 5′-end-directed cleavage15. Again, primary cuts are made about 18 nt from the RNA 5′ and then secondary cuts are made about 9 nt from the end. All of these classes of cuts are thought to be necessary for efficient genomic RNA removal. RNase H activity is a particularly attractive drug target because of this central role in reverse transcription13,16. Efavirenz (Figure 1a) is one of the most frequently prescribed NNRTIs, used as a first-line treatment for HIV in highly active antiretroviral therapy, HAART 17,18. As with other NNRTIs, rapid development of resistance is a major problem5,19. A more recent disturbing observation was the ability of efavirenz to stimulate virus growth after the virus acquired certain NNRTI resistant mutations 20,21. Although the mechanism of stimulation is largely unknown, it was shown that the effect was caused by changes in the early stages of the viral life cycle 22. Efavirenz has also been shown to stimulate viral RT RNase H activity 23–25, a characteristic that may serve as a basis of virus growth stimulation. Another NNRTI nevirapine was also reported to stimulate HIV-1 RNase H activity, and the mechanism was probed in detail 26, with results suggesting that nevirapine specifically alters DNA 3′-directed RNase H cleavage among other functions. Figure 1 The E478Q RNase H negative mutant RT is more sensitive to efavirenz Since, RNase H activity is an important component of the reverse transcription pathway, a potential drug target, and may connect NNRTI activity to more efficient viral replication; we examined the relationships between efavirenz and HIV-1 RNase H in more detail. We report that a RNase H negative mutant of RT is much more sensitive to efavirenz than the wild type RT, and that efavirenz specifically stimulates certain RNase H functions in both the wild type and a patient-derived drug resistant mutant RT. We discuss how the drug and the RNase H activity influence each other and how these influences might explain promotion of mutant virus growth by efavirenz.