HIV-1 RT Research Articles

Fourier transform infrared spectra and normal mode analysis of drug molecules: Zidovudine

The FTIR spectra of zidovudine molecule have been recorded in the range 4000–400cm−1. The title compound is used as a drug against AIDS or HIV. The molecular structure, fundamental vibrational frequencies and intensities of vibrational bands are evaluated using density functional theory (DFT) using BLYP, B3LYP, B3PW91 and MPW1PW91 methods with 6-31+G(d,p) standard basis set. Comparison of simulated spectra with the experimental spectrum provides important informations and the ability of the computational method to describe the vibrational modes. These calculations have allowed finding most stable conformational structure of AZT. Calculated results of the title compound indicate that the drug molecule has syn orientation. The glycosidic bond in AZT and a minimum-energy structure in which the glycosy torsion angle χ and torsion angle γ values are consistent with those in the conformation of AZT in the AZT5-triphosphate bound to HIV RT is determined.

First biochemical characterization of a novel ribonuclease from wild mushroom Amanita hemibapha

A 45-kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild mushroom Amanita hemibapha. It was adsorbed on DEAE-cellulose, S-sepharose, and finally purified on Superdex 75. The RNase exhibited maximal RNase activity at pH 5 and in a temperature range between 60-70°C. It demonstrated no ribonucleolytic activity toward four polyhomoribonucleotides. The amino acid sequence analysis (GDDETFWEHEWAK) showed this RNase was a ribonuclease T2-like RNase. It exhibited strong inhibitory activity against HIV-1 reverse transcriptase (HIV-1 RT) with an IC50 of 17 μM.

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.

Low recombination activity of R region located at both ends of the HIV-1 genome.

Although two strand transfer events are indispensable for the synthesis of double-stranded DNA and establishing HIV-1 infection, the molecular basis of these phenomena is still unclear. The first obligatory template switching event occurs just at the beginning of the virus replication cycle and involves two copies of the 97-nucleotide long R region, located one each at the both ends of the HIV-1 genome (HIV-1 R). Thus, one can expect that the molecular mechanism of this process is similar to the mechanism of homologous recombination which operates in RNA viruses. To verify the above-mentioned hypothesis, we attempted to assess the recombination activity of HIV-1 R. To this end, we tested in vitro, how effectively it induces template switching by HIV-1 RT in comparison with another well-characterized sequence supporting frequent homologous crossovers in an unrelated virus (R region derived from Brome mosaic virus--BMV R). We also examined if the RNA sequences neighboring HIV-1 R influence its recombination activity. Finally, we tested if HIV-1 R could cause BMV polymerase complex to switch between RNA templates in vivo. Overall, our results have revealed a relatively low recombination activity of HIV-1 R as compared to BMV R. This observation suggests that different factors modulate the efficiency of the first obligatory strand transfer in HIV-1 and the homology-driven recombination in RNA viruses.

Positional Adaptability in the Design of Mutation-Resistant Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors: A Supramolecular Perspective

Drug resistance is a key cause of failed treatment of HIV infection. The efficacy of nonnucleoside reverse transcriptase-inhibiting (NNRTI) drugs is impaired by the rapid emergence of drug-resistant mutations. The literature supports the idea that purposefully designed flexible NNRTIs at an active site may help overcome drug resistance. It is proposed here that the usual "lock and key" model, with respect to NNRTI drug design, be expanded to consider creating "master keys" that would automatically adjust conformations to fit all of the "locks" mutations may make. The present work introduces the novel perspective of designing and creating supramolecular assemblies as potential NNRTIs (instead of the relatively more rigid single-molecule inhibitors). Specifically, flexible self-assembling quinhydrone supramolecular dimers formed from quinonoid monomers (designed to be highly flexible NNRTIs themselves) will be offered as a working example of this new perspective in NNRTI drug design. Quinonoid compounds have demonstrated binding interactions at various sites of the HIV-1 RT enzyme, including the elusive ribonuclease H area. Quinhydrone self-organized dimers have at some point in their molecular architecture a noncovalently interacting donor-acceptor ring pair complex. This complex is at the heart of the increased torsional, rotational, and translational motion this species will experience at a particular active site. Flexible supramolecular assemblies, together with their flexible monomer components, may offer a critical advantage in retaining potency against a wide range of drug-resistant HIV-1 RTs. This new supramolecular perspective may also have broader implications in the general field of antimicrobial drug design.

Differential In Vitro Kinetics of Drug Resistance Mutation Acquisition in HIV-1 RT of Subtypes B and C

BackgroundHIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC).Methods/Principal FindingsMT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F).Conclusions/SignificanceThis study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population.

Synthesis, and in vitro Enzymatic and Antiviral Evaluation of d4T Polyphosphate Derivatives as Chain Terminators

A series of d4T di- or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV-1 RT, and also tested for their in vitro anti-HIV activity. The steady-state kinetic study of compounds 1-4 in an enzymatic incorporation assay by HIV-1 RT follows Michaelis-Menten profile. In addition, compounds 2-4 are able to inhibit HIV-1 replication to the same extent as d4T and d4TMP in MT-4 cells, as well as in CEM/0 cells and CEM/TK(-) cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.

Molecular Mechanism of Antagonism between the Y181C and E138K Mutations in HIV-1 Reverse Transcriptase

Etravirine (ETR) is an expanded-spectrum nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use as an antiretroviral agent in treatment-experienced patients. Y181C and E138K in HIV-1 RT are among 20 different drug resistance mutations associated with ETR. However, E138K can be consistently selected by ETR when wild-type viruses but not viruses containing Y181C are grown in tissue culture. This study was carried out to evaluate any possible mechanisms that might explain antagonism between the Y181C and E138K mutations. Accordingly, we performed tissue culture studies to investigate the evolutionary dynamics of E138K in both a wild-type (WT) and a Y181C background. We also generated recombinant enzymes containing Y181C and E138K alone or in combination in order to study enzyme processivity, rates of processive DNA synthesis, enzyme kinetics, and susceptibility to ETR. We now show that the presence of the Y181C mutation prevented the emergence of E138K in cell culture and that the simultaneous presence of E138K and Y181C impaired each of enzyme activity, processivity, rate of processive DNA synthesis, and deoxynucleoside triphosphate (dNTP) affinity. The addition of E138K to Y181C also decreased the level of resistance to ETR compared to that obtained with Y181C alone.

The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding

BackgroundThe ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity.ResultsThe solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding.ConclusionsThe structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

A prime-boost immunization with rBCG expressing HIV-1 Gag, RT and gp120 and SAAVI MVA-C elicits immune responses in blood and MALT of rhesus macaques

Methods Chinese rhesus macaques (n=8) were primed twice with a mixture of rBCG, 12 weeks apart. A control group (n=4) was mock-primed with a control BCG. Both groups were boosted with MVA. Two weeks after the MVA vaccination, two macaques from the rBCG-primed group were euthanased and jejunum, spleen and inguinal, mesenteric, iliac and bronchial lymph nodes were harvested for isolation of mononuclear cells. HIV-1-specific IFN-gamma ELISPOT responses were measured in the blood and these tissues using pools of overlapping HIV-1 peptides.

E138K and M184I mutations in HIV-1 reverse transcriptase coemerge as a result of APOBEC3 editing in the absence of drug exposure

Recent clinical trials with rilpivirine combined with emtricitabine and tenofovir revealed that patients failing treatment, frequently, harbored viruses encoding resistance-associated mutations in the HIV-1 reverse transcriptase at position E138K and M184I. We show here that APOBEC3 proteins play a role in the emergence of these drug resistance mutations. We used a Vif mutant that has suboptimal activity against APOBEC3 to assess the in-vitro frequency of APOBEC3-induced resistance mutations in reverse transcriptase. To assess the degree of in-vivo G-to-A viral hypermutation, a large amount of data of HIV-1 RT proviral sequences from peripheral blood mononuclear cells (PBMCs) recovered from infected patients under HAART was analyzed. In-vitro replication experiments in cell lines with and without APOBEC3 expression suggest that APOBEC3-driven mutagenesis contributes to the generation of both M184I and E138K within HIV proviral repository in the absence of drug exposure. Additionally, analysis of 601 patients PBMCs sequences revealed that the copresence of mutations E138K and M184I were never detected in nonhypermutated sequences, whereas these mutations were found at a high frequency (24%) in the context of APOBEC3 editing and in the absence of exposure to etravirine-rilpivirine. We demonstrate using in-vitro experiments and analyzing patients PBMCs sequences that M184I and E138K resistance-associated mutations may pre-exist in proviral reservoir at a high frequency prior to drug exposure, as a result of APOBEC3 editing. Thus, incomplete neutralization of one or more APOBEC3 proteins may favor viral escape to rilpivirine-emtricitabine.

A laccase with inhibitory activity against HIV-1 reverse transcriptase from the mycorrhizal fungus Lepiota ventriosospora

► A laccase from mycorrhizal not white-rot fungus is reported. ► We evaluate its physical, chemical, and bioactive properties. ► It manifests unique amino acid sequence comparing with other laccases. ► It is stable at pH 4.0 after an incubation of 1 h. ► It possesses a very high inhibitory activity toward HIV-1 RT. An isolation procedure that comprised three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and one gel-filtration step by fast protein liquid chromatography on Superdex 75 was utilized to isolate a laccase with a molecular mass of 65 kDa from fresh fruiting bodies of the mycorrhizal fungus Lepiota ventriosospora. Laccase activity was adsorbed on both DEAE-cellulose and Q-Sepharose but unadsorbed on CM-cellulose. An overall 26.3-fold of purification was obtained. The enzyme demonstrated an optimum temperature at 60 °C and an optimum pH 4.0. The purified laccase was quite stable at pH range of 3.6–4.4, but only 17.8% of total activity left after 1 h incubating at 60 °C. The ranking of its degradative activity toward aromatic substrates was catechol > hydroquinone > ABTS > 2,6-dimethoxy-phenol. It demonstrated the highest inhibitory activity toward HIV-1 reverse transcriptase with an IC 50 value of 0.60 μM among fungal laccaes reported up to now.

HIV-1 Reverse Transcriptase: a potential target for marine products

HIV-1 reverse transcriptase (HIV-1 RT) is a therapeutic target for the treatment of HIV-positive individuals or those already showing AIDS symptoms. In this perspective, the identification of new inhibitors for this enzyme is of great importance in view of the growing viral resistance to the existing treatments. This resistance has compromised the quality of life of those infected with multidrug-resistant strains, whose treatment options are already limited, putting at risk these individuals lives. The literature has recognized marine organisms and their products as natural sources for the identification of new therapeutic options for different pathologies. In this brief review, we consider the structure of HIV-1 RT and its most common inhibitors, as well as some marine diterpenes originally reported as HIV-1 RT inhibitors to encourage the identification and development of new marine antiviral prototypes.

Synthesis of betulinic acid derivatives as entry inhibitors against HIV-1 and bevirimat-resistant HIV-1 variants

Betulinic acid derivatives modified at the C28 position are HIV-1entry inhibitors such as compound A43D; however, modified at the C3 position instead of C28 give HIV-1 maturation inhibitor such as bevirimat. Bevirimat exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative drug binding site. In an effort to determine whether the viruses with bevirimat resistant polymorphism also altered their sensitivities to the betulinic acid derivatives that inhibit HIV-1 entry, a series of new betulinic acid entry inhibitors were synthesized and tested for their activities against HIV-1 NL4-3 and NL4-3 variants resistant to bevirimat. The results show that the bevirimat resistant viruses were approximately 5- to10-fold more sensitive to three new glutamine ester derivatives (13, 15 and 38) and A43D in an HIV-1 multi-cycle replication assay. In contrast, the wild type NL4-3 and the bevirimat resistant variants were equally sensitive to the HIV-1 RT inhibitor AZT. In addition, these three new compounds markedly improved microsomal stability compared to A43D.

Anti-HIV, antimycobacterial and antimicrobial studies of newly synthesized 1,2,4-triazole clubbed benzothiazoles

In an attempt to synthesize pharmacologically active molecules, we report here the synthesis and in vitro anti-HIV, antimycobacterial and antimicrobial activity of various series of 3-(3-pyridyl)-5-(4-substitutedphenyl)-4-(N-substituted-1,3-benzothiazol-2-amino)-4H-1,2,4-triazole. The anti-HIV activity of the title compounds was evaluated as selective human immunodeficiency virus type-1 and -2 (HIV-1, HIV-2 RT) inhibitors, antitubercular activity against H37Rv using Lowenstein-Jensen agar method and antimicrobial activity against certain bacterial and fungal strains using the broth microdilution method. The synthesized compounds were characterized by FTIR,1H-NMR,13C-NMR and mass spectral data together with elemental analysis.

Biochemical mechanism of clinical resistance to rilpivirine

Background The introduction of HAART has significantly prolonged the life span of HIV-infected patients. However, the error-prone nature of HIV-1 reverse transcriptase (HIV-1 RT) results in the emergence of drug-resistant viruses and threatens the effectiveness of HAART. HIV-1 RT is a primary target of two classes of drugs: nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Recent phase III clinical trials have shown that two HIV-1 RT mutations, E138K and M184I, were the most frequent mutations found in patients that experienced virological failure during therapy that included rilpivirine (RPV), emtricitabine (FTC), and tenofovir (TDF).

Pre-steady state kinetic analysis of cyclobutyl derivatives of 2′-deoxyadenosine 5′-triphosphate as inhibitors of HIV-1 reverse transcriptase

Pre-steady state kinetic analysis was utilized for biochemical evaluation of a series of cyclobutyl adenosine nucleotide analogs with HIV-1 RTWT. The phosphonyl-diphosphate form of the cyclobutyl nucleotide, 5, was the most efficiently incorporated of the series. Nucleotide 5 was fourfold more efficiently incorporated than the FDA approved TFV-DP by RTWT. The kinetics of incorporation for 5 using the drug resistant mutant enzyme K65R was also determined. Compound 5 was threefold more efficiently incorporated compared to TFV-DP with RTK65R. These results demonstrate cyclobutyl adenosine analogs can act as substrates for incorporation by HIV-1 RT and be a potential scaffold for HIV inhibitors.

Chiral resolution, absolute configuration assignment and biological activity of racemic diarylpyrimidine CH(OH)-DAPY as potent nonnucleoside HIV-1 reverse transcriptase inhibitors

(+)-3a and (−)-3a were successfully separated from racemate (±)-3a by the chiral technique of supercritical fluid chromatography (SCF) with enantiomeric excess (ee%) >99% and purity >99%, and assigned for their absolute configuration as R and S, respectively, by the experimental electronic circular dichroism (ECD) spectrum and simulated ECD spectra calculated by time-dependent density functional theory (TDDFT) calculations. (+)-(R)-3a displayed excellent activity with an EC50 of 5.3nM against wild-type HIV-1, which was 12-fold more potent than (−)-(S)-3a. However, (−)-(S)-3a showed higher potency than (+)-(R)-3a against the double HIV-1 RT mutant (K103N+Y181C) as well as HIV-2 strain ROD. The possible reason for the difference of (R)- and (S)-3a in anti-HIV-1 activity was interpreted by molecular docking.

Pingry School SMART Team Project: Modeling Current Strategies in HIV‐1 Reverse Transcriptase Inhibitors

Pingry School's 2012 S.M.A.R.T (Students Modeling A Research Topic) Team with Eddy Arnold from The Center for Advanced Biotechnology and Medicine (CABM) at Rutgers University who utilized X‐ray crystallography to determine the structure of HIV‐1 reverse transcriptase (HIV‐1 RT) in multiple functional states which led to detailed understanding of multiple nucleoside RT inhibitors (NRTIs) and and the design of non‐nucleoside RT inhibitors (NNRTIs), including two anti‐AIDS drugs. NRTIs inhibit RT by attaching to the dNTP substrate‐binding pocket, while NNRTIs are allosteric inhibitors. Our project models the NRTI and NNRTI inhibition sites on HIV‐1 RT and demonstrates the efficacy of NRTIs in comparison to NNRTIs on various mutations of HIV‐1 RT. Our physical models contrast the binding of NRTIs and NNRTIs to RT. Specifically, we examine the NRTI drug, AZT, and the NNRTI drugs, etravirine and rilpivirine. The latter two have strategic flexibility and their structures “wiggle and jiggle” inside the binding pocket, consequently making it more difficult for a mutated RT to evade resistance. The Pingry S.M.A.R.T. Team used the findings as a basis for the design of a set of three 3‐dimensional models using Jmol and a 3‐dimensional printer to demonstrate the drug‐RT interactions. These models show interactions that reflect the current research of HIV‐1 RT inhibition. Supported by a grant from the NIH‐SEPA.

AF4 HIV Drug Resistance in Nigeria, a Cost-Effective New Methodology

Since 2004, we have provided PEPFAR support to HIV prevention, care and treatment programs at 35 hospitals in 10 states in Nigeria, providing ART to over 85,000 adult and 4,300 pediatric patients. A diverse array of non-subtype B HIV-1 viruses circulate in Africa and dominate the global pandemic. HIV-1 RT and PR sequences from 338 patients with treatment failure to first-line ART regimens were evaluated. Multivariate logistic regression examined the effect of subtype on each mutation controlling for regimen, time on therapy, and total mutations. CRF02_AG was less likely to have the M41L mutation compared to other subtypes (AOR. 0.35, P = 0.022). Subtype A patients showed a 42.5-fold increased risk (AOR. 42.5, P = 0.001) for the L210W mutation. Among NNRTI mutations, subtype G patients had an increased risk for A98G (AOR. 2.40, P = 0.036) and V106I (AOR. 6.15, P = 0.010). Given the shortcomings of genotyping systems that limit their utility in the developing world, we have created a less expensive, yet more sensitive, method using the ligation-amplification technique. We have designed sets of Nigerian subtype specific oligonucleotides that are used as probes in establishing the presence of the specific HIV drug resistance mutations K103N, M184V, and Y181C/I. Preliminary data have already shown high concordance with sequence data, and ability to amplify samples with low levels of the K103N, M184V and Y181C/I mutations. The lig-amp assay has the potential to provide high sensitivity at a 10 fold reduction in cost. Further study to understand the clinical impact of subtype-specific diversity on drug resistance will be critically important to the continued success of ART scale-up in resource-limited settings.