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Previous work on mutations in the thumb of HIV-1 reverse transcriptase (RT) showed that the majority of the mutant RTs were degraded (by the viral protease) to various extents in virions. This degradation was, in most cases, temperature sensitive, and presumably was due to a partial unfolding of the protein at 37°C. We used recombinant proteins to investigate the effects of the mutations on the thermal stability and proteolytic degradation of RT. Both subunits contribute to the stability of RT. In general, the differences in stability between the mutants and WT were greater if the mutation was in p51 rather than p66. Expressing the Pol polyprotein containing the RT mutants in Escherichia coli produced results similar to what was seen in virions; the mutant RTs were misfolded and/or degraded at 37°C, but were better folded and processed at 30°C., (Published by Elsevier Inc.)

As anti-HIV therapy becomes more widely available in developing nations, it is clear that drug resistance will continue to be a major problem. The related viruses HIV-1 and HIV-2 share many of the same resistance pathways to nucleoside reverse transcriptase inhibitors (NRTIs). However, clinical data suggest that while HIV-1 reverse transcriptase (RT) usually uses an ATP-dependent excision pathway to develop resistance to the nucleoside analog zidovudine (AZT), HIV-2 RT does not appear to use this pathway. We previously described data that suggested that wild-type (WT) HIV-2 RT has a much lower ability to excise AZT monophosphate (AZTMP) than does WT HIV-1 RT and suggested that this is the reason that HIV-2 RT more readily adopts an exclusion pathway against AZT triphosphate (AZTTP), while HIV-1 RT is better able to exploit the ATP-dependent pyrophosphorolysis mechanism. However, we have now done additional experiments, which show that while HIV-1 RT can adopt either an exclusion- or excision-based resistance mechanism against AZT, HIV-2 RT can use only the exclusion mechanism. All of our attempts to make HIV-2 RT excision competent did not produce an AZT-resistant RT but instead yielded RTs that were less able to polymerize than the WT. This suggests that the exclusion pathway is the only pathway available to HIV-2.

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 A resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 A away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.