Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs.

Two mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the "primer grip" region and could be influenced by the M230I mutation.IMPORTANCE Although antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.

HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

Post-Catalytic Complexes with Emtricitabine or Stavudine and HIV-1 Reverse Transcriptase Reveal New Mechanistic Insights for Nucleotide Incorporation and Drug Resistance.

Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a "snapshot" for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.

Active-site deformation in the structure of HIV-1 RT with HBV-associated septuple amino acid substitutions rationalizes the differential susceptibility of HIV-1 and HBV against 4'-modified nucleoside RT inhibitors.

Nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs) are major antiviral agents against hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1). However, the notorious insoluble property of HBV RT has prevented atomic-resolution structural studies and rational anti-HBV drug design. Here, we created HIV-1 RT mutants containing HBV-mimicking sextuple or septuple amino acid substitutions at the nucleoside-binding site (N-site) and verified that these mutants retained the RT activity. The most active RT mutant, HIV-1 RT7MC, carrying Q151M/G112S/D113A/Y115F/F116Y/F160L/I159L was successfully crystallized, and its three-dimensional structure was determined in complex with DNA:dGTP/entecavir-triphosphate (ETV-TP), a potent anti-HBV guanosine analogue RT inhibitor, at a resolution of 2.43 Šand 2.60 Å, respectively. The structures reveal significant positional rearrangements of the amino acid side-chains at the N-site, elucidating the mechanism underlying the differential susceptibility of HIV-1 and HBV against recently reported 4'-modified NRTIs.

Risk factors and outcomes for the Q151M and T69 insertion HIV-1 resistance mutations in historic UK data.

BACKGROUND: The prevalence of HIV-1 resistance to antiretroviral therapies (ART) has declined in high-income countries over recent years, but drug resistance remains a substantial concern in many low and middle-income countries. The Q151M and T69 insertion (T69i) resistance mutations in the viral reverse transcriptase gene can reduce susceptibility to all nucleoside/tide analogue reverse transcriptase inhibitors, motivating the present study to investigate the risk factors and outcomes associated with these mutations. METHODS: We considered all data in the UK HIV Drug Resistance Database for blood samples obtained in the period 1997-2014. Where available, treatment history and patient outcomes were obtained through linkage to the UK Collaborative HIV Cohort study. A matched case-control approach was used to assess risk factors associated with the appearance of each of the mutations in ART-experienced patients, and survival analysis was used to investigate factors associated with viral suppression. A further analysis using matched controls was performed to investigate the impact of each mutation on survival. RESULTS: A total of 180 patients with Q151M mutation and 85 with T69i mutation were identified, almost entirely from before 2006. Occurrence of both the Q151M and T69i mutations was strongly associated with cumulative period of virological failure while on ART, and for Q151M there was a particular positive association with use of stavudine and negative association with use of boosted-protease inhibitors. Subsequent viral suppression was negatively associated with viral load at sequencing for both mutations, and for Q151M we found a negative association with didanosine use but a positive association with boosted-protease inhibitor use. The results obtained in these analyses were also consistent with potentially large associations with other drugs. Analyses were inconclusive regarding associations between the mutations and mortality, but mortality was high for patients with low CD4 at detection. CONCLUSIONS: The Q151M and T69i resistance mutations are now very rare in the UK. Our results suggest that good outcomes are possible for people with these mutations. However, in this historic sample, viral load and CD4 at detection were important factors in determining prognosis.

Comparative analysis of drug resistance mutations in the human immunodeficiency virus reverse transcriptase gene in patients who are non-responsive, responsive and naive to antiretroviral therapy.

Drug resistance mutations in the Pol gene of human immunodeficiency virus 1 (HIV-1) are one of the critical factors associated with antiretroviral therapy (ART) failure in HIV-1 patients. The issue of resistance to reverse transcriptase inhibitors (RTIs) in HIV infection has not been adequately addressed in the Indian subcontinent. We compared HIV-1 reverse transcriptase (RT) gene sequences to identify mutations present in HIV-1 patients who were ART non-responders, ART responders and drug naive. Genotypic drug resistance testing was performed by sequencing a 655-bp region of the RT gene from 102 HIV-1 patients, consisting of 30 ART-non-responding, 35 ART-responding and 37 drug-naive patients. The Stanford HIV Resistance Database (HIVDBv 6.2), IAS-USA mutation list, ANRS_09/2012 algorithm, and Rega v8.02 algorithm were used to interpret the pattern of drug resistance. The majority of the sequences (96 %) belonged to subtype C, and a few of them (3.9 %) to subtype A1. The frequency of drug resistance mutations observed in ART-non-responding, ART-responding and drug-naive patients was 40.1 %, 10.7 % and 20.58 %, respectively. It was observed that in non-responders, multiple mutations were present in the same patient, while in responders, a single mutation was found. Some of the drug-naive patients had more than one mutation. Thymidine analogue mutations (TAMs), however, were found in non-responders and naive patients but not in responders. Although drug resistance mutations were widely distributed among ART non-responders, the presence of resistance mutations in the viruses of drug-naive patients poses a big concern in the absence of a genotyping resistance test.

Alkaloids from the Sponge Stylissa carteri Present Prospective Scaffolds for the Inhibition of Human Immunodeficiency Virus 1 (HIV-1).

The sponge Stylissa carteri is known to produce a number of secondary metabolites displaying anti-fouling, anti-inflammatory, and anti-cancer activity. However, the anti-viral potential of metabolites produced by S. carteri has not been extensively explored. In this study, an S. carteri extract was HPLC fractionated and a cell based assay was used to evaluate the effects of HPLC fractions on parameters of Human Immunodeficiency Virus (HIV-1) infection and cell viability. Candidate HIV-1 inhibitory fractions were then analyzed for the presence of potential HIV-1 inhibitory compounds by mass spectrometry, leading to the identification of three previously characterized compounds, i.e., debromohymenialdisine (DBH), hymenialdisine (HD), and oroidin. Commercially available purified versions of these molecules were re-tested to assess their antiviral potential in greater detail. Specifically, DBH and HD exhibit a 30%-40% inhibition of HIV-1 at 3.1 µM and 13 µM, respectively; however, both exhibited cytotoxicity. Conversely, oroidin displayed a 50% inhibition of viral replication at 50 µM with no associated toxicity. Additional experimentation using a biochemical assay revealed that oroidin inhibited the activity of the HIV-1 Reverse Transcriptase up to 90% at 25 µM. Taken together, the chemical search space was narrowed and previously isolated compounds with an unexplored anti-viral potential were found. Our results support exploration of marine natural products for anti-viral drug discovery.

In vitro anti-HIV and antioxidant activity of Hoodia gordonii (Apocynaceae), a commercial plant product.

BACKGROUND: Hoodia gordonii products are widely commercialized for anti-obesity purposes; however, minimal research is available on the other health properties demonstrated by this popular herbal plant. METHODS: H. gordonii crude extracts (ethanol and ethyl acetate) were assayed for in vitro anti-HIV-1 protease (PR), reverse transcriptase (RT) and integrase activity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power assays were used for the antioxidant analysis. In addition, qualitative and quantitative phytochemical analyses of the extracts were determined using standard methods. RESULTS: H. gordonii extract demonstrated good inhibition against HIV RT with IC50 values of 73.55 ± 0.04 and 69.81 ± 9.45 µg/mL for ethanol and ethyl acetate extracts, respectively. Both extracts also demonstrated inhibitory activity against HIV PR with IC50 values of 97.29 ± 0.01 and 63.76 ± 9.01 µg/mL for ethanol and ethyl acetate extracts. In addition, H. gordonii also showed good antioxidant activity with IC50 values of 124.6 ± 11.3 and 126.2 ± 3.15 µg/mL obtained for ethanol and ethyl acetate extracts, respectively. The reducing power of H. gordonii extracts increased as the concentration increased which confirmed the presence of antioxidants (reductants) in the extracts. Phytochemical screening of H. gordonii revealed the presence of phenolics, alkaloids, terpenes, steroids, cardiac glycosides and tannins in the ethanolic extract, while the ethyl acetate extract only showed the presence of phenolics, cardiac glycosides and steroids. The total phenolic content was 420 ± 0.17 and 319.9 ± 0.2 mg GAE/g for the ethanol and ethyl acetate extracts, respectively. The ethanol extract, which revealed the presence of tannins, had a tannin content of 330 ± 0.2 mg TAE/g extract. CONCLUSION: This data suggests that H. gordonii has good in vitro inhibition against selected HIV-1 enzymes as well as antioxidant properties, suggesting new potential uses for this commercial plant.

A cell-based strategy to assess intrinsic inhibition efficiencies of HIV-1 reverse transcriptase inhibitors.

During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a per-stop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability, p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC50s) and this basic probability enabled determination of p by successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8- to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.

Nanoparticle-Based ARV Drug Combinations for Synergistic Inhibition of Cell-Free and Cell-Cell HIV Transmission.

Nanocarrier-based drug delivery systems are playing an emerging role in human immunodeficiency virus (HIV) chemoprophylaxis and treatment due to their ability to alter the pharmacokinetics and improve the therapeutic index of various antiretroviral (ARV) drug compounds used alone and in combination. Although several nanocarriers have been described for combination delivery of ARV drugs, measurement of drug-drug activities facilitated by the use of these nanotechnology platforms has not been fully investigated for topical prevention. Here, we show that physicochemically diverse ARV drugs can be encapsulated within polymeric nanoparticles to deliver multidrug combinations that provide potent HIV chemoprophylaxis in relevant models of cell-free, cell-cell, and mucosal tissue infection. In contrast to existing approaches that coformulate ARV drug combinations together in a single nanocarrier, we prepared single-drug-loaded nanoparticles that were subsequently combined upon administration. ARV drug-nanoparticles were prepared using emulsion-solvent evaporation techniques to incorporate maraviroc (MVC), etravirine (ETR), and raltegravir (RAL) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. We compared the antiviral potency of the free and formulated drug combinations for all pairwise and triple drug combinations against both cell-free and cell-associated HIV-1 infection in vitro. The efficacy of ARV-drug nanoparticle combinations was also assessed in a macaque cervicovaginal explant model using a chimeric simian-human immunodeficiency virus (SHIV) containing the reverse transcriptase (RT) of HIV-1. We observed that our ARV-NPs maintained potent HIV inhibition and were more effective when used in combinations. In particular, ARV-NP combinations involving ETR-NP exhibited significantly higher antiviral potency and dose-reduction against both cell-free and cell-associated HIV-1 BaL infection in vitro. Furthermore, ARV-NP combinations that showed large dose-reduction were identified to be synergistic, whereas the equivalent free-drug combinations were observed to be strictly additive. Higher intracellular drug concentration was measured for cells dosed with the triple ARV-NP combination compared to the equivalent unformulated drugs. Finally, as a first step toward evaluating challenge studies in animal models, we also show that our ARV-NP combinations inhibit RT-SHIV virus propagation in macaque cervicovaginal tissue and block virus transmission by migratory cells emigrating from the tissue. Our results demonstrate that ARV-NP combinations control HIV-1 transmission more efficiently than free-drug combinations. These studies provide a rationale to better understand the role of nanocarrier systems in facilitating multidrug effects in relevant cells and tissues associated with HIV infection.

Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action.

HIV-1 resistance to zidovudine [AZT (azidothymidine)] is associated with selection of the mutations M41L, D67N, K70R, L210W, T215F/Y and K219Q/E in RT (reverse transcriptase). These mutations decrease HIV-1 susceptibility to AZT by augmenting RT's ability to excise the chain-terminating AZT-MP (AZT-monophosphate) moiety from the chain-terminated DNA primer. Although AZT-MP excision occurs at the enzyme's polymerase active site, it is mechanistically distinct from the DNA polymerase reaction. Consequently, this activity represents a novel target for drug discovery, and inhibitors that target this activity may increase the efficacy of nucleoside/nucleotide analogues, and may help to delay the onset of drug resistance. In the present study, we have developed a FRET (Förster resonance energy transfer)-based high-throughput screening assay for the AZT-MP excision activity of RT. This assay is sensitive and robust, and demonstrates a signal-to-noise ratio of 3.3 and a Z' factor of 0.69. We screened three chemical libraries (7265 compounds) using this assay, and identified APEX57219 {3,3'-[(3-carboxy-4-oxo-2,5-cyclohexadien-1-ylidene)methylene]bis[6-hydroxybenzoic acid]} as the most promising hit. APEX57219 displays a unique activity profile against wild-type and drug-resistant HIV-1 RT, and was found to inhibit virus replication at the level of reverse transcription. Mechanistic analyses revealed that APEX57219 blocked the interaction between RT and the nucleic acid substrate.

L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness.

The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

Increased detection of the HIV-1 reverse transcriptase M184V mutation using mutation-specific minority assays in a UK surveillance study suggests evidence of unrecognized transmitted drug resistance.

OBJECTIVES: The aim of the study was to estimate the levels of transmitted drug resistance (TDR) in HIV-1 using very sensitive assays to detect minority drug-resistant populations. METHODS: We tested unlinked anonymous serum specimens from sexual health clinic attendees, who had not received an HIV diagnosis at the time of sampling, by both standard genotyping and using minority detection assays. RESULTS: By standard genotyping, 21 of 165 specimens (12.7%) showed evidence of drug resistance, while, using a combination of standard genotyping and minority mutation assays targeting three commonly observed drug resistance mutations which cause high-level resistance to commonly prescribed first-line antiretroviral therapy (ART), this rose to 32 of 165 (19.4%). This increase of 45% in drug resistance levels [95% confidence interval (CI) 15.2-83.7%; P=0.002] was statistically significant. Almost all of this increase was accounted for by additional detections of the M184V mutation. CONCLUSIONS: Future surveillance studies of TDR in the United Kingdom should consider combining standard genotyping and minority-specific assays to provide more accurate estimates, particularly when using specimens collected from chronic HIV infections in which TDR variants may have declined to low levels.

Retroviral reverse transcriptases.

Reverse transcription is a critical step in the life cycle of all retroviruses and related retrotransposons. This complex process is performed exclusively by the retroviral reverse transcriptase (RT) enzyme that converts the viral single-stranded RNA into integration-competent double-stranded DNA. Although all RTs have similar catalytic activities, they significantly differ in several aspects of their catalytic properties, their structures and subunit composition. The RT of human immunodeficiency virus type-1 (HIV-1), the virus causing acquired immunodeficiency syndrome (AIDS), is a prime target for the development of antiretroviral drug therapy of HIV-1/AIDS carriers. Therefore, despite the fundamental contributions of other RTs to the understanding of RTs and retrovirology, most recent RT studies are related to HIV-1 RT. In this review we summarize the basic properties of different RTs. These include, among other topics, their structures, enzymatic activities, interactions with both viral and host proteins, RT inhibition and resistance to antiretroviral drugs.

Design, synthesis, and evaluation of "dual-site"-binding diarylpyrimidines targeting both NNIBP and the NNRTI adjacent site of the HIV-1 reverse transcriptase.

Inspired by our previous efforts to improve the drug-resistance profiles of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), a novel series of "dual-site" binding diarylpyrimidine (DAPY) derivatives targeting both the NNRTI adjacent site and NNRTIs binding pocket (NNIBP) were designed, synthesized, and evaluated for their anti-HIV potency in TZM-bl and MT-4 cells. Eight compounds exhibited moderate to excellent potencies in inhibiting wild-type (WT) HIV-1 replication with EC50 values ranging from 2.45 nM to 5.36 nM, and 14c (EC50 = 2.45 nM) proved to be the most promising inhibitor. Of note, 14c exhibited potent activity against the single mutant strain E138K (EC50 = 10.6 nM), being comparable with ETR (EC50 = 9.80 nM) and 3.5-fold more potent than that of compound 7 (EC50 = 37.3 nM). Moreover, 14c acted as a classical NNRTI with high affinity for WT HIV-1 RT (IC50 = 0.0589 µM). The detailed structure-activity relationships (SARs) of the representative compounds were also determined, and further supported by molecular dynamics simulation. Overall, we envision that the "dual-site"-binding NNRTIs have significant prospects and pave the way for the next round of rational design of potent anti-HIV-1 agents.

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening.

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.

Homodimerization of the p51 subunit of HIV-1 reverse transcriptase.

The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-(13)C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The (1)H-(13)C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a "p66-like" conformation. SAXS data obtained for p51 samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, "p51C" conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, "p51E" conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51(L289K) has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of the p51E(L289K) to p51C(L289K) monomers.

Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance.

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg(72) were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg(72) were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.

Subtype-specific differences in the human immunodeficiency virus type 1 reverse transcriptase connection subdomain of CRF01_AE are associated with higher levels of resistance to 3'-azido-3'-deoxythymidine.

We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3'-azido-3'-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance.

The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) plays a major role in treatment resistance, from the development of vaccines to therapeutic drugs. In addressing the crux of the issue, various attempts to estimate the mutation rate of HIV-1 resulted in a large range of 10-5-10-3 errors/bp/cycle due to the use of different types of investigation methods. In this review, we discuss the different assay methods, their findings on the mutation rates of HIV-1 and how the locations of mutations can be further analyzed for their allosteric effects to allow for new inhibitor designs. Given that HIV is one of the fastest mutating viruses, it serves as a good model for the comprehensive study of viral mutations that can give rise to a more horizontal understanding towards overall viral drug resistance as well as emerging viral diseases.