HIV-1 Reverse Transcriptase Research Articles

Revealing the Mutation Patterns of Drug-Resistant Reverse Transcriptase Variants of Human Immunodeficiency Virus through Proteochemometric Modeling.

Drug-resistant cases of human immunodeficiency virus (HIV) nucleoside reverse transcriptase inhibitors (NRTI) are constantly accumulating due to the frequent mutations of the reverse transcriptase (RT). Predicting the potential drug resistance of HIV-1 NRTIs could provide instructions for the proper clinical use of available drugs. In this study, a novel proteochemometric (PCM) model was constructed to predict the drug resistance between six NRTIs against different variants of RT. Forty-seven dominant mutation sites were screened using the whole protein of HIV-1 RT. Thereafter, the physicochemical properties of the dominant mutation sites can be derived to generate the protein descriptors of RT. Furthermore, by combining the molecular descriptors of NRTIs, PCM modeling can be constructed to predict the inhibition ability between RT variants and NRTIs. The results indicated that our PCM model could achieve a mean AUC value of 0.946 and a mean accuracy of 0.873 on the external validation set. Finally, based on PCM modeling, the importance of features was calculated to reveal the dominant amino acid distribution and mutation patterns on RT, to reflect the characteristics of drug-resistant sequences.

Using machine learning and big data to explore the drug resistance landscape in HIV.

Drug resistance mutations (DRMs) appear in HIV under treatment pressure. DRMs are commonly transmitted to naive patients. The standard approach to reveal new DRMs is to test for significant frequency differences of mutations between treated and naive patients. However, we then consider each mutation individually and cannot hope to study interactions between several mutations. Here, we aim to leverage the ever-growing quantity of high-quality sequence data and machine learning methods to study such interactions (i.e. epistasis), as well as try to find new DRMs.We trained classifiers to discriminate between Reverse Transcriptase Inhibitor (RTI)-experienced and RTI-naive samples on a large HIV-1 reverse transcriptase (RT) sequence dataset from the UK (n ≈ 55, 000), using all observed mutations as binary representation features. To assess the robustness of our findings, our classifiers were evaluated on independent data sets, both from the UK and Africa. Important representation features for each classifier were then extracted as potential DRMs. To find novel DRMs, we repeated this process by removing either features or samples associated to known DRMs.When keeping all known resistance signal, we detected sufficiently prevalent known DRMs, thus validating the approach. When removing features corresponding to known DRMs, our classifiers retained some prediction accuracy, and six new mutations significantly associated with resistance were identified. These six mutations have a low genetic barrier, are correlated to known DRMs, and are spatially close to either the RT active site or the regulatory binding pocket. When removing both known DRM features and sequences containing at least one known DRM, our classifiers lose all prediction accuracy. These results likely indicate that all mutations directly conferring resistance have been found, and that our newly discovered DRMs are accessory or compensatory mutations. Moreover, apart from the accessory nature of the relationships we found, we did not find any significant signal of further, more subtle epistasis combining several mutations which individually do not seem to confer any resistance.

Chemical Characterization and Anti-HIV-1 Activity Assessment of Iridoids and Flavonols from Scrophularia trifoliata.

Plants are the everlasting source of a wide spectrum of specialized metabolites, characterized by wide variability in term of chemical structures and different biological properties such antiviral activity. In the search for novel antiviral agents against Human Immunodeficiency Virus type 1 (HIV-1) from plants, the phytochemical investigation of Scrophularia trifoliata L. led us to isolate and characterize four flavonols glycosides along with nine iridoid glycosides, two of them, 5 and 13, described for the first time. In the present study, we investigated, for the first time, the contents of a methanol extract of S. trifoliata leaves, in order to explore the potential antiviral activity against HIV-1. The antiviral activity was evaluated in biochemical assays for the inhibition of HIV-1Reverse Transcriptase (RT)-associated Ribonuclease H (RNase H) activity and HIV-1 Integrase (IN). Three isolated flavonoids, rutin, kaempferol-7-O-rhamnosyl-3-O-glucopyranoside, and kaempferol-3-O-glucopyranoside, 8–10, inhibited specifically the HIV-1 IN activity at submicromolar concentration, with the latter being the most potent, showing an IC50 value of 24 nM.

Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT).

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.

Synthesis and Formulation of Powerful ZnO Q-Dots/CNTs@Fe3O4 Nanopackage as a Predominant Anti-HIV-1 Infection

Usage of fine and uniform Fe3O4 nanoparticles (NPs) including super-paramagnetic unique properties developed state of the nanobio-formulations in recent years. We have shown a new formulated nanocomposition of super-paramagnetic iron oxide (Fe3O4) NPs (as substrate) with carbon nanotubes (CNTs) (as activator). Besides, ZnO@CNTs was synthesized as a magic assistant/hybrid for ZnO quantum dots nanoparticles (Q-Dots NPs) in this nanopackage. This novel formulated water-based nanofluid product consists of strong stabilizer, suitable dispersant-wetting agent complex and desirable water in oil emulsifier (w/o) package to damage HIV infection (AIDS) type 1. The achieved results demonstrated that smart nanofluid formulation had excellent functions as inhibitor, controller and treatment (Antiretroviral therapy (ART)) for HIV-1 integrase and could act as strong oxidizing agent. The nanofluid product was completely characterized with SEM morphology, TEM images, FTIR spectroscopy, XRD pattern, UV–Vis absorption spectroscopy, EDS and mapping of internal layers for one of the SEM surface morphology. Moreover, HIV-1 replication Assay, RT (reverse transcriptase) Assay, integrase assay, and cytotoxicity tests were performed and compared with Zidovudine (ZDV) and Raltegravir (RAL) as control antiretroviral medications. The specific interaction of this nanopackage with the target RNA and DNA proteins has been very interesting through main redox reactions.

Lentiviral-Driven Discovery of Cancer Drug Resistance Mutations.

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. SIGNIFICANCE: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

CHARACTERISATION AND STUDY OF 1- [2- (2-BENZOYLPHENOXY) ETHYL] -6-METHYLURACIL MECHANISM OF ACTION

The aim of the study is to identify 1-[2-(2-benzoylphenoxy) ethyl]-6-methyluracil using various methods of analysis, as well as to study its action mechanism against wild-type and mutant forms of HIV-1 reverse transcriptase (RT).Materials and methods. To characterize the structure of the test substance, a few kinds of analysis (X-ray diffraction, elemental, thermal) as well as a few kinds of spectroscopy (UV, IR, and NMR) have been used. The study of the action mechanism of the compound as a potential drug was carried out by evaluating the inhibitory activity against HIV-1 RT wild-type and its mutant forms corresponding to drug-resistant viral strains.Results. The studies have been carried out to confirm the structure of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil. The UV spectrum has a pronounced absorption maximum when measuring a solution of the substance in tetrahydrofuran at the concentration of 0.10 mg / ml. In the IR spectrum, there are specific bands in the range of 4000-370 cm–1. These factors make it possible to use UV and IR spectra to identify the test compound in the substance. It has also been established that the number and mutual arrangement of functional groups, the integrated intensity of signals in the 1H-NMR spectrum, as well as the structure of the carbon skeleton, correspond to the structure of 1-[2-(2-benzoylphenoxy) ethyl]-6-methyluracil. The results of studying the action mechanism showed that the test compound is an effective inhibitor of wild-type HIV-1 RT with an inhibition constant of 0.2 µM, as well as an enzyme inhibitor (mutation G190A) with an inhibition constant of 8 µM; enzyme (mutation Y181C) with an inhibition constant of 10 µM, as well as a reverse transcriptase (RT) inhibitor (mutation L100I, K103N, V106A) and a double mutant K103N / Y181C with an inhibition constant of more than 20 µM.Conclusion. As a result of the performed X-ray structural, elemental, 1H-NMR and 13C-NMR analyzes, the structure of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil has been confirmed. The possibility of using UV, IR and NMR spectroscopy, as well as thermal analyzes to confirm the authenticity during the verification of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil, has been shown. The developed methods can be used in the quality control and included in the draft of practice guidelines for the investigated substance. The studies of the action mechanism of the compound of HIV-1 RT reverse transcriptase have shown that this compound belongs to the group of non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1.

Novel pyridinium-type fullerene derivatives as multitargeting inhibitors of HIV-1 reverse transcriptase, HIV-1 protease, and HCV NS5B polymerase

In the present study, we newly synthesized four types of novel fullerene derivatives: pyridinium/ethyl ester-type derivatives 3b−3l, pyridinium/carboxylic acid-type derivatives 4a, 4e, 4f, pyridinium/amide-type derivative 5a, and pyridinium/2-morpholinone-type derivative 6a. Among the assessed compounds, cis-3c, cis-3d, trans-3e, trans-3h, cis-3l, cis-4e, cis-4f, trans-4f, and cis-5a were found to inhibit HIV-1 reverse transcriptase (HIV-RT), HIV-1 protease (HIV-PR), and HCV NS5B polymerase (HCV NS5B), with IC50 values observed in the micromolar range. Cellular uptake of pyridinium/ethyl ester-type derivatives was higher than that of corresponding pyridinium/carboxylic acid-type derivatives and pyridinium/amide-type derivatives. This result might indicate that pyridinium/ethyl ester-type derivatives are expected to be lead compounds for multitargeting drugs to treat HIV/HCV coinfection.

QSAR Study for Prediction of HIV-1 Protease Inhibitor Using the Gravitational Search Algorithm–Neural Network (GSA-NN) Methods

Human immunodeficiency virus (HIV) is a virus that infects an immune cell and makes the patient more susceptible to infections and other diseases. HIV is also a factor that leads to acquired immune deficiency syndrome (AIDS) disease. The active target that is usually used in the treatment of HIV is HIV-1 protease. Combining HIV-1 protease inhibitors and reverse-transcriptase inhibitors in highly active antiretroviral therapy (HAART) is typically used to treat this virus. However, this treatment can only reduce the viral load, restore some parts of the immune system, and failed to overcome the drug resistance. This study aimed to build a QSAR model for predicting HIV-1 protease inhibitor activity using the gravitational search algorithm-neural network (GSA-NN) method. The GSA method is used to select molecular descriptors, while NN was used to develop the prediction model. The improvement of model performance was found after performing the hyperparameter tuning procedure. The validation results show that model 3, containing seven descriptors, shows the best performance indicated by the coefficient of determination (r2) and cross-validation coefficient of determination (Q2) values. We found that the value of r2 for train and test data are 0.84 and 0.82, respectively, and the value of Q2 is 0.81.

Exploring New Scaffolds for the Dual Inhibition of HIV-1 RT Polymerase and Ribonuclease Associated Functions.

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated functions.

Prevalence of resistance mutations associated with integrase inhibitors in therapy-naive HIV-positive patients in Hungary.

Widespread introduction of HIV integrase inhibitors into clinical care may result in appearance of drug resistance mutations affecting treatment outcome. The aim of our study was to monitor the resistance patterns of integrase inhibitors beside protease and reverse transcriptase inhibitors in newly diagnosed therapy-naive HIV-positive patients in Hungary between 2017 and 2019.Genotype-based resistance testing of HIV integrase, protease and reverse transcriptase was performed by amplification and Sanger population sequencing from plasma samples. Drug resistance mutations were identified by the algorithm of Stanford HIV Drug Resistance Database.Potentially transmitted, non-polymorphic integrase major mutation was detected in 1 out of 249 samples, while accessory mutations were observed in further 31 patients (12.4%). The overall prevalence of transmitted drug resistance (TDR) mutations related to protease and reverse transcriptase inhibitors was 5.8% (10/173) between the end of 2017 and 2019. Nucleoside reverse transcriptase inhibitor associated resistance mutations were the most frequent indicators of TDR (6/173; 3.5%), followed by resistance mutations associated with protease (3/173; 1.7%) and non-nucleoside reverse transcriptase inhibitors (2/173, 1.2%).The first detection of integrase major mutation and the changing patterns of other resistance mutations in Hungarian untreated HIV-positive population indicate the necessity of continuous molecular surveillance of Hungarian HIV epidemic.

Transmitted HIV drug resistance and subtype patterns among blood donors in Poland

Surveillance on the HIV molecular variability, risk of drug resistance transmission and evolution of novel viral variants among blood donors remains an understudied aspect of hemovigilance. This nationwide study analyses patterns of HIV diversity and transmitted resistance mutations. Study included 185 samples from the first time and repeat blood donors with HIV infection identified by molecular assay. HIV protease, reverse transcriptase and integrase were sequenced using population methods. Drug resistance mutation (DRM) patterns were analyzed based on the Stanford Interpretation Algorithm and standardized lists of transmitted mutations. Phylogeny was used to investigate subtyping, clustering and recombination patterns. HIV-1 subtype B (89.2%) followed by subtype A6 (7.6%) were predominant, while in three (1.6%) cases, novel recombinant B/A6 variants were identified. Non-B variants were more common among repeat donors (14.5%) compared to the first time ones (1.8%), p = 0.011, with higher frequency (9.9%) of A6 variant in the repeat donor group, p = 0.04. Major NRTI DRMs were observed in 3.8%, NNRTI and PI in 0.6% and INSTI 1.1% of cases. Additionally, E157Q polymorphism was observed in 9.8% and L74I in 11.5% of integrase sequences. Transmission of drug resistance among blood donors remains infrequent. Subtype patters increase in complexity with emergence of novel intersubtype A6B recombinants.

The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H

RNase H is a prototypical example for two-metal-ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present not only in important drug targets, such as the HIV-1 reverse transcriptase, but also in many other nucleases, such as Homo sapiens (Hs) and Escherichia coli (Ec) RNase H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton-transfer events is not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2′O atom of the sugar. Additionally, we also describe an alternative proton-transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free-energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs, and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol–1, encountered in the phosphate-cleavage step, matches the slow experimental rate of ∼1–100 min–1. Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal-ion catalytic phosphate-cleaving enzymes with a DDE motif.

Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells.

Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity invitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.

Generalizing the Discrete Gibbs Sampler-Based λ-Dynamics Approach for Multisite Sampling of Many Ligands.

In this work, the discrete λ variant of the Gibbs sampler-based λ-dynamics (d-GSλD) method is developed to enable multiple functional group perturbations to be investigated at one or more sites of substitution off a common ligand core. The theoretical framework and special considerations for constructing discrete λ states for multisite d-GSλD are presented. The precision and accuracy of the d-GSλD method is evaluated with three test cases of increasing complexity. Specifically, methyl → methyl symmetric perturbations in water, 1,4-benzene hydration free energies and protein-ligand binding affinities for an example HIV-1 reverse transcriptase inhibitor series are computed with d-GSλD. Complementary MSλD calculations were also performed to compare with d-GSλD's performance. Excellent agreement between d-GSλD and MSλD is observed, with mean unsigned errors of 0.12 and 0.22 kcal/mol for computed hydration and binding free energy test cases, respectively. Good agreement with experiment is also observed, with errors of 0.5-0.7 kcal/mol. These findings support the applicability of the d-GSλD free energy method for a variety of molecular design problems, including structure-based drug design. Finally, a discussion of d-GSλD versus MSλD approaches is presented to compare and contrast features of both methods.

Effect of N'-Benzyl Substituted Uracil and the Analogues on HIV-1 Inhibition

The aim of the research is to study the effect of the synthesis of uracil derivatives on the HIV-1 activity. To achieve the goal, the following tasks were determined: to study the specificity of possible compounds for HIV-1 treatment; to synthesize uracil derivatives; to study the effect of the compounds on HIV-1 replication in vitro and select the most optimal concentrations, considering the cytotoxic effect; to determine the most effective anti-HIV-1 compounds for further research. Thus, nine new uracil analogues have been synthesized and proved to be inhibitors of HIV-1. Key structural modifications included replacement of the 6-chloro group of 1-benzyl-6-chloro-3-(3,5-dimethylbenzyl) uracil by other functional groups or N (1)-alkylation of 3-(3,5-dimethylbenzyl)-5-fluorouracil. These compounds showed only micromolar potency against HIV-1 in MT-4, though two of them; 6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were highly potent (half maximal effective concentration =0.081 and 0.069μM) and selective (selectivity index =679 and 658), respectively. Structure-activity relationships among the newly synthesized uracil analogues suggest the importance of the H-bond formed between 6-amino group of 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil and amide group of HIV-1 reverse transcriptase. Two 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils, (6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil) were discovered as novel anti-HIV agents. Compound’s activity against HIV-1 was determined based on inhibition of virus-induced cytopathogenicity in MT-4 cells. The compounds were tested for efficacy in infected cells and cytotoxicity. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against non-nucleoside reverse transcriptase inhibitor-resistant strains. Thus, it will contribute to the development of a new generation of compounds effective against different viruses, considering their quickly mutation and increased resistance.

HIV Reverse Transcriptase Pre-Steady-State Kinetic Analysis of Chain Terminators and Translocation Inhibitors Reveals Interactions between Magnesium and Nucleotide 3'-OH.

Deoxythymidine triphosphate analogues with various 3′ substituents in the sugar ring (−OH (dTTP)), −H, −N3, −NH2, −F, −O–CH3, no group (2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3′-OH but with 4′ additions (4′-C-methyl, 4′-C-ethyl) or sugar ring modifications (d-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg2+ with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants (kobs) compared to dTTP ranging from about 2-fold (3′-H, −N3, and d4TTP with high Mg2+) to >10-fold (3′-NH2 and 3′-F with low Mg2+), while 3′-O-CH3 dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg2+ and the 3′-OH, kobs using 5 μM dTTP and 0.5 mM Mg2+ was only modestly slower (1.6-fold) than with 6 mM Mg2+, while analogues with 3′ alterations incorporated 2.8–5.1-fold slower in 0.5 mM Mg2+. In contrast, 4′-C-methyl and d-carba dTTP, which retain the 3′-OH, were not significantly affected by Mg2+. Consistent with these results, analogues with 3′ modifications were better inhibitors in 6 versus 0.5 mM Mg2+. Equilibrium dissociation constant (KD) and maximum incorporation rate (kpol) determinations for dTTP and analogues lacking a 3′-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogues but did not significantly affect KD, results consistent with a role for 3′-OH/Mg2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg2+ and the 3′-OH of the incoming nucleotide and suggest that inhibitors with 3′-OH groups may have advantages in low free Mg2+ in physiological settings.

HIV reverse transcriptases defective in strand displacement activity

HIV reverse transcriptases defective in strand displacement activity

Reciprocal Inhibition of Immunogenic Performance in Mice of Two Potent DNA Immunogens Targeting HCV-Related Liver Cancer.

Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. A possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, but also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor-associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Additionally, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescence signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.

Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana.

Background: HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs). Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods: A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results: Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants. There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion: The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.