HIV-1 Reverse Transcriptase

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The life cycle of retroviruses is characterized by two steps that are carried out by two essential virally encoded enzymes, reverse transcriptase (RT) and integrase (IN). In the first of these steps, RT converts the single-stranded genomic viral RNA into a linear double-stranded DNA that is longer than the genomic RNA from which it is derived. In the second step, IN inserts this linear viral DNA into the host genome. Both steps are essential for the retroviral life cycle; both RT and IN are key anti-HIV drug targets. The development of new, broadly effective, low-toxicity anti-HIV drugs is one of the high-priority research goals of the NIH, in part because of problems with emerging drug resistance in both the developed and the developing world. The conversion, by RT, of retroviral genomic RNA into DNA involves the two enzymatic activities: a polymerase that can copy either RNA or DNA, and a ribonuclease H (RNase H) that cleaves RNA if it is part of an RNA/DNA hybrid. There are two clinically important classes of anti-RT drugs: nucleoside analogs (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), both of which target the polymerase. Although the RNase H of RT is essential for viral replication, there are no anti-HIV drugs that target RNase H. The NRTIs that are currently used to treat HIV-1 infections lack the 3'-OH found on normal deoxynucleosides. If an NRTI is incorporated into viral DNA by RT, polymerization is blocked. NNRTIs bind to a hydrophobic pocket that is near the polymerase active site. Because NRTIs can also be incorporated into the mitochondrial and nuclear DNA of host cells, these drugs can be toxic to patients, particularly because HIV drug therapies are usually lifelong. A bound NNRTI distorts the structure around the active site, blocking the chemical step of DNA synthesis. However, NNRTIs do not affect host DNA polymerases and, as a consequence, are relatively nontoxic. A major focus of our work has been to understand the mechanism(s) of RT inhibitor resistance to NRTIs and NNRTIs. There is, at this point, a reasonably good understanding of the mechanism(s) of NNRTI resistance, and considerable progress has been made in understanding NRTI resistance. Although NNRTIs are, as a group, relatively nontoxic, they are more susceptible to the development of resistance than NRTIs. We have worked to develop NNRTIs that are more broadly effective against the known drug-resistant mutants of HIV-1 and on understanding NRTI resistance. Most of the analysis that has been done on the susceptibility of HIV to various drugs has been done with subgroup B viruses. Subgroup B viruses are common in the United States, but B is not the major subgroup worldwide. Subgroup C is much more prevalent worldwide and it is important to know if therapies developed based on subgroup B viruses will be equally effective against subgroup C. In particular, we wanted to know if complex group of resistance mutations behaved similarly in subgroups B and C RTs. We identified a complex drug resistant RT mutant in a subgroup C RT and asked if various combinations of these mutations had similar effects in subgroup B and subgroup C RTs. The subgroup C virus was obtained from a donor who was on combination therapy with abacavir, AZT, and 3TC. An RT was sequenced that had mutations at positions 67, 70, 184, 219, and a threonine insertion after position 69. We found that these mutations affected the ability of NRTIs to inhibit DNA synthesis by both the subgroup B and C RTs. Although there were minor differences in the behavior of the RTs, it appears that these mutations behave similarly in both B and C subgroup RTs, which suggests that similar therapies can be used successfully with both subgroups. However, the presence of the inserted threonine reduced the susceptibility of both the subgroup B and subgroup C mutant RTs to inhibition by tenofovir, which is a potential problem, given the importance of that drug in anti-viral therapies.