We have a long-standing interest in HIV-1 reverse transcriptase (RT) and, more recently, have begun work on another essential viral enzyme, integrase (IN). Although much of our work is focused on understanding the mechanisms that underlie drug resistance, and using that information to develop more effective anti-HIV drugs, we are, in both the RT and IN projects, doing experiments that are intended to better understand the roles that these enzymes play in viral replication. _____BACKGROUND: HIV-1 is the causative agent of AIDS. The three viral enzymes -- RT, IN, and protease (PR) -- have essential roles in the replication of HIV-1 and are the targets for all of the most potent anti-HIV drugs. Although considerable progress has been made in treating HIV-infected patients with three- and four-drug regimens, there is an immediate need for the development of effective ways to prevent new infections. A potent preventive vaccine would be ideal; however, despite a huge effort, the goal of developing an effective vaccine remains elusive. In the absence of an effective vaccine, reducing the transmission of HIV-1 must rely on barrier methods and/or drug treatments. There are two ways that anti-HIV drugs can be used to reduce viral transmission: (1) effective therapy in infected patients can reduce the viral load, making it less likely that an infected individual will transmit the virus to a partner; and (2) treating the uninfected partner with an anti-HIV drug can block transmission. Because most new infections are caused by a single virus, blocking transmission is an attractive option and there is now good evidence that giving an anti-HIV drug to the uninfected partner can significantly reduce viral transmission if the uninfected partner is compliant. Because of the problem of drug resistance, it would be better to use drugs with nonoverlapping resistance profiles for treatment and prophylaxis. Treatment would have to be long term and, for this reason, drug toxicity is an important consideration, which argues against the use of nucleoside RT inhibitors (NRTIs). It would also be better to block infection before the viral DNA is integrated, which argues against the use of PR inhibitors. Therefore, the two remaining options among the major classes of anti-HIV drugs are nonnucleoside RT inhibitors (NNRTIs) and IN strand-transfer inhibitors (INSTIs). We are using a combined approach that involves structural analysis, biochemistry, virology, modeling, toxicity testing, and chemistry to design, synthesize, and evaluate new NNRTIs and INSTIs. We have made good progress in developing new compounds that are effective against the wild-type (WT) and common drug-resistant viruses and that have good therapeutic indexes in tests done in cultured cells. Our progress with the HIV-1 RT research is reported below; progress with the IN research is reported separately for Project ZIA BC 011426. _____HIV-1 RT: There are two clinically important classes of inhibitors of HIV-1 RT: NRTIs and NNRTIs. In the past, a major focus of our work has been on the mechanism(s) of RT inhibitor resistance. 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 some important issues remain. We are continuing to investigate the mechanisms of resistance to nucleoside analogs (NRTIs), and we are also interested in elucidating the mechanism of action of compounds that do not cause an immediate or complete block to DNA synthesis. Ongoing structural analysis in Dr. Eddy Arnold's group should shed light on the underlying mechanism(s). However, in terms of developing new anti-HIV compounds, we have shifted our focus to NNRTIs. Our chemistry collaborators, Drs. Joel Schneider, Gary Pauly, and Craig Thomas, have developed several promising compounds that are able to inhibit, in a one-round replication assay, WT and several common NNRTI-resistant viruses with IC50s below 5 nM. In cultured cells, these compounds have CC50s that are more than 4 logs higher than their IC50s. Additional compounds are being designed, synthesized, and tested. Recently, our collaborator, Dr. Zandrea Ambrose, selected a novel resistance mutation with one of our new NNRTIs. This mutation has some interesting properties (for example, it appears to make the RT of some, but not all, strains of RT temperature sensitive), and we are attempting to understand the mechanisms that underlie the effects of the new mutation. _____In addition to the experiments designed to understand resistance to anti-RT drugs and to develop new RT inhibitors, we are studying the effects of RT mutations on the stability of RT in virions and on the fidelity of HIV-1 replication. We showed that a large percentage of the mutations we tested in the thumb subdomain make RT susceptible to PR cleavage; in some cases, this susceptibility creates a temperature-sensitive phenotype. We proposed that the mutations that lead to PR susceptibility partially unfold RT, exposing sites where PR can cleave. The data show that mutations that affect the stability of RT can have a significant impact on the ability of the virus to replicate, and by extension, on viral fitness. We recently showed that mutations that affect the stability of RT are found only very rarely in the Stanford database; this shows that these mutations not only affect the fitness of the virus in cultured cells, but also affect the fitness of the virus in patients. _____We are using a LacZalpha complementation assay similar to the assays used by the Pathak and Mansky labs to measure the fidelity of HIV-1 replication in cultured cells. We improved the efficiency of the assay, which allows us not only to measure the mutation rate, but also to determine the positions in LacZalpha where mutations frequently arise (hotspots). The data show that (1) all of the published in vitro assays using purified RT overestimate the in vivo error rate and fail to correctly identify the mutational hotspots; (2) HIV-1 replication is not more error prone than the replication of other retroviruses; (3) which strand of LacZalpha is in the RNA genome does not affect the overall error rate, or the types of errors made, but it does affect the hotspots; (4) based on preliminary data, mutations in RT (including mutations that confer resistance to NRTIs) affect which sites are mutational hotspots. _____We showed that some of the compounds that Dr. Terrence Burke prepared that were intended to inhibit HIV-1 IN can inhibit the polymerase and/or the RNase H activities of HIV-1 RT. We are studying the effects of the compounds using purified HIV-1 RT, and HIV-1-based viral vectors, and our collaborator, Dr. Arnold, is preparing co-crystals of RT and three of the compounds. _____PATENTS LINKED TO THIS PROJECT: (1) Vu BC, Siddiqui MA, Marquez VE, Hughes SH, Boyer PL: C4'-substituted-2-deoxyadenosine Analogs and Methods of Treating HIV. U.S. Patent #8,513,214, issued August 20, 2013. (2) Michejda CJ, Hariprakasha HK, Hughes SH, Szekely Z: Benzidole Derivatives and Method of Treating HIV/AIDS. Patent pending: PCT/US2007/080957 (PC application), submitted in 2006. (3) Hughes SH, Boyer PL, Linial M, Stenbak C, Clark P: Foamy virus mutant reverse transcriptase. Patent issued: 7,560,117 (US in 2009). _____[Corresponds to Hughes Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]