Human Immunodeficiency Virus Reverse Transcription Research Articles

An efficient synthesis, structural analysis, and computational studies of benzothiazole derivatives activated by formic acid under solvent-free conditions

In the present work, formic acid was used as a low-cost and eco-friendly catalyst to synthesize 2-(2-hydroxyphenyl)benzothiazole (HB) through a cycloaddition process in a solvent-free reaction medium. Imine molecule was synthesized with the help of acetic acid (B). The ring closure and formation of the thiazole moiety give acetic acid distinctiveness as a catalyst. The cyclization of fused HB failed without an acid catalyst. The chemical structures were characterized using microanalysis techniques such as IR, 1H-NMR, and 13C-NMR. The obtained FT-IR spectra were compared to theoretical data using the B3LYP/6-31G* level of theory. The electronic characteristics of molecules can be predicted utilizing wave function-dependent features, such as Average localized ionization energy (ALIE), Localized orbital locator (LOL), Electron localized function (ELF), Reduced density gradient (RDG), and Noncovalent interactions NCI studies, which have provided a wealth of knowledge. The molecular docking study was investigated to explore binding energy and interaction type. Docking analysis displayed binding energies (kcal/mol) of HB with values of -8.29, -6.80, -6.68, -7.43, and -6.69 of acetylcholinesterase (AChE) and butylcholinesterase (BuChE)), SARS-CoV-2 main protease (Mpro), human immunodeficiency virus reverse transcription (HIV RT)) and cytochrome (P450), respectively, which was the most active compound against all receptors. The dramatic improvement of the proposed approach over the existing methods can be used to derive an efficient and successful route for the synthesis of HB. This process provides a fresh and high-yield method for the synthesis of benzimidazoles.

A proposed new paradigm for an anti-AIDS tolerogenic vaccine

Until now, despite 30 years of intensive work, the RV144 human immunodeficiency virus (HIV) vaccine trial initiated in 2003 remains so far the most protective vaccine prototype of all those tested (32% reduction in the infection rate three years after the vaccination) and the HIV epidemic is still spreading worldwide. In addition, antiretroviral therapy (ART) for people living with HIV is given for life as no other pharmacological intervention has allowed to maintain an undetectable viral load after ART withdrawal. Pr Andrieu and colleagues discovered tolerogenic CD8+T-cells that suppress simian immunodeficiency virus (SIV) specific activation, ensuing SIV reverse transcription suppression and viral replication-defective in Chinese macaques vaccinated by intragastric route with inactivated SIV particles + Lactobacillus rhamnosus. Moreover, in HIV-infected elite controllers with specific genetic features (HLA-1-Bw4-80i and KIR3DL1 genes), Pr Andrieu found out that similar tolerogenic CD8+T-cells suppress in the same manner HIV-specific activation, HIV reverse transcription, and HIV replication. These data justify the development of a tolerogenic vaccine composed of inactivated HIV particles + Lactobacillus rhamnosus that could be used as a preventive or therapeutic vaccine.

Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy

AIDS Research and Human RetrovirusesVol. 30, No. 3 Images in HIV ResearchFree AccessUnraveling the Gymnastics of Reverse Transcription Through Single Molecule SpectroscopyElio A. Abbondanzieri and Stuart F.J. Le GriceElio A. AbbondanzieriDepartment of Bionanoscience, Delft University of Technology, Delft, The Netherlands.Search for more papers by this author and Stuart F.J. Le GriceRT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland.Search for more papers by this authorPublished Online:3 Mar 2014https://doi.org/10.1089/aid.2014.0023AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Synthesizing integration-competent, double-stranded DNA from the single-stranded human immunodeficiency virus (HIV) RNA genome might more aptly be described as molecular gymnastics (Fig. 1). Key elements of this routine include intramolecular and intermolecular transfer of the replication complex between genome termini, strand displacement synthesis, selecting plus-strand RNA primers from within the RNA/DNA replication intermediate, their excision from nascent DNA, and phased A-tract-induced cessation of DNA synthesis near the center of the genome. While site-directed mutagenesis identifies residues of HIV-1 reverse transcriptase (RT) that mediate these functions, and X-ray crystallography gives elegant, but static snapshots of nucleoprotein complexes, they cannot assess conformational dynamics of this remarkably versatile enzyme. To better understand how HIV-1 RT interacts with nucleic acid required designing a single molecule strategy based on Foerster resonance energy transfer (FRET), placing fluorophores into the enzyme and its nucleic acid substrates.FIG. 1. Multistep acrobatics and reverse transcription.Studies with duplex DNA predictably recapitulated an enzyme with its DNA polymerase catalytic center straddled over the primer terminus. However, an unexpected observation of Abbondanzieri et al.1 was that this orientation reversed when the DNA primer was replaced with RNA. Later studies examining polypurine tract recognition not only confirmed that HIV-1 RT could assume different orientations, but also was sufficiently agile to “flip” between these during a single binding event. Such reversal in RT orientation could help explain how its N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) domains can recognize the PPT primer terminus at different steps of plus-strand synthesis.Another concept we accept when studying HIV-1 RT is that it is a distributive polymerase, i.e., it frequently dissociates from its nucleic acid substrate. How then, upon rebinding, does the enzyme locate the primer terminus to reengage in DNA synthesis? Subsequent studies of Liu et al. illustrated the capacity of RT to slide on the nucleic acid duplex, a property that could be enhanced in the presence of nonnucleoside RT inhibitors or restricted by forming a ternary complex. However, the story does not end here. Since there appears to be no orientation (or directional) bias during sliding, the polymerization target site could be accessed by the retroviral polymerase in two different orientations. Single molecule spectroscopy not only confirmed this, but elegantly demonstrated that an enzyme accessing the primer terminus in an “RNase H mode” could spontaneously flip into the polymerization mode, thereby linking the properties of flipping and sliding.2A state-of-the-art technology, high-profile publications, but what is the significance? For many years, we have presumed that nonnucleoside inhibition of HIV-1 RT occurred through a “grinding of the gears” and an inability to support DNA synthesis. However, examining the interaction of HIV-1 RT with its PPT by single molecule spectroscopy revealed that the nonnucleoside reverse transcriptase inhibitor (NNRTI) nevirapine promotes a reversal of enzyme orientation, positioning the RNase H domain over the primer terminus, thereby inhibiting synthesis of plus-strand DNA. These and related studies on tRNA-primed initiation of minus-strand DNA synthesis3 show that, in a different sense, HIV fitness requires nimble and flexible maneuvering of its DNA polymerase!References1 Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang J, Le Grice SFJ, and Zhuang X: Dynamic binding orientations determine the enzymatic activity of HIV reverse transcriptase. Nature 2008;453:184–189. Crossref, Medline, Google Scholar2 Liu S, Abbondanzieri EA, Rausch JW, Le Grice SFJ, and Zhuang X: Slide into action dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 2008;322:1092–1097. Crossref, Medline, Google Scholar3 Liu S, Harada BT, Miller JT, Le Grice SFJ, and Zhuang X: Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat Struct Mol Biol 2010;17:1453–1460. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 30Issue 3Mar 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Elio A. Abbondanzieri and Stuart F.J. Le Grice.Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy.AIDS Research and Human Retroviruses.Mar 2014.209-210.http://doi.org/10.1089/aid.2014.0023Published in Volume: 30 Issue 3: March 3, 2014PDF download

HIV-1 Drug-Resistant Minority Variants: Sweating the Small Stuff

Within each human immunodeficiency virus (HIV)–infected individual, there exists a remarkably heterogeneous collection of viruses. Over the course of a typical HIV infection, a starting population of relatively homogeneous virus may undergo substantial diversification, such that mutations may be detected at >10% of nucleotide positions [1]. This diversity is driven both by the error-prone nature of HIV reverse transcription and the high rate of viral replication. The HIV reverse transcriptase enzyme lacks a proofreading mechanism, and it is estimated that up to 5 mutations may be introduced during each replication cycle [2, 3]. In addition, the virus is thought to undergo 10–100 million rounds of replication daily, leading to the production of more than a billion new virions [4, 5].

Secondary Structure of the HIV Reverse Transcription Initiation Complex by NMR

Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA–RNA complex formed between a host transfer RNA (tRNA Lys 3) and a region at the 5′ end of genomic RNA; the 3′ end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA–human tRNA Lys 3 initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3′ end of tRNA Lys 3 drives large conformational rearrangements of the tRNA at the 5′ end while maintaining the anticodon loop for potential loop–loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.

Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription

Human immunodeficiency virus (HIV) initiates reverse transcription of its viral RNA (vRNA) genome from a cellular tRNALys,3 primer. This process is characterized by a slow initiation phase with specific pauses, followed by a fast elongation phase. We report a single-molecule study that monitors the dynamics of individual initiation complexes, comprised of vRNA, tRNA and HIV reverse transcriptase (RT). RT transitions between two opposite binding orientations on tRNA:vRNA complexes, and the prominent pausing events are caused by RT binding in an flipped orientation opposite to the polymerization-competent configuration. A stem-loop structure within the vRNA is responsible for maintaining the enzyme predominantly in this flipped orientation. Disruption of the stem-loop structure triggers the initiation-to-elongation transition. These results highlight the important role played by the structural dynamics of the initiation complex in directing transitions between early reverse transcription phases.

Genotyping for Guiding Drug Choice in Human Immunodeficiency Virus-Infected Children Failing Multiple Antiretroviral Treatment Regimens

To the Editors: The use of human immunodeficiency virus (HIV) genotyping for identifying the most effective regimens in HIV-infected adults failing highly active antiretroviral therapy led to improved virologic response as compared with regimens selected on the basis of antiretroviral exposure history. In contrast, similar studies conducted in small cohorts of HIV-infected children failed to demonstrate a clear advantage of antiretroviral resistance testing over antiretroviral exposure history. In this study, we have genotyped HIV reverse transcription and protease genes in 17 children (ages 4.5–11.3 years) between 2000 and 2001, at treatment failure and have changed antiretroviral drugs according to HIV genotype profiles. All 17 children who were included showed an elevated viral load (>1000 copies/mL) for at least 9 months during the last year before genotyping and had failed from 1–5 regimens during that time. All the HIV-infected children had received a combination of at least 2 nucleoside reverse transcriptase inhibitors (NRTIs) (17 of 17) and while 8 of 17 had been treated with a PI containing regimen; none of them was previously treated with non-NRTIs (Table 1). Adherence to therapy was based on reports from children and caretakers and on calculation of quantities of drugs that were requested at each visit. At the time of treatment failure, we identified substitutions in both the reverse transcription and the protease genes in samples from 94% of children.TABLE 1: Nucleoside Reverse Transcription Inhibitors Used in HIV TreatmentWild-type sequences could not be identified in any sample from this cohort of HIV-infected children. Drug-specific genotypic resistance, identified on the basis of the IAS mutation list,4 was commonly observed for all the NRTIs as shown in detail in Table 1. Antiretroviral therapy was changed by substitution of at least one antiretroviral medication in all patients, except in patient 17. Since none of the patients had been previously exposed to non-NRTI drugs whereas 8 of 17 had received a drug of the protease inhibitor class, we introduced efavirenz in the therapeutic regimens of 11 children. Virologic and immunologic data were collected at 2- to 3-month intervals for at least 12 months. We observed a significant suppression of viral load (<1000 copies) 3-6 months after treatment change. HIV suppression was detected in 8 of the 17 patients from 6 to 35 months (stably undetectable in 5 cases), whereas in another 5 children viral load remained elevated through 12 months of follow-up. CD4+ percentages showed a significant increase from baseline levels in the same children who showed virologic suppression, except in patient 10 who had already started the treatment with 44% of CD4, suggesting that their treatment regimens were effective to induce immunologic reconstitution. The remaining patients (patients 1, 3, 4 and 13) showed a rapid, but transient, decline of viral load after treatment change. Because of the high risk of false negatives, we have performed an additional HIV genotyping in the remaining 9 patients who showed an unsatisfactory response to medication changes. In patients 1 and 13, we detected additional mutations in reverse transcription gene (Table 1) that conferred resistance to efavirenz, thereby accounting for the inadequate response to the previous change of antiretroviral treatment. HIV genotyping did not reveal any resistance-associated mutation in 7 children who had failed 2 changes of treatment regimens, suggesting that limited adherence may account for this observation. Our study demonstrates that HIV genotyping based treatment choices in 17 infected pretreated children led to persistent viral suppression and immunologic reconstitution in 10 children, suggesting that use of this assay improves clinical outcome of antiretroviral-experienced children. A previous study failed to show benefit of HIV resistance testing in a cohort of 18 children. We believe that the limited availability of novel antiretroviral medications and variable adherence to therapeutic regimens may account the different results that are observed in these studies. Drs Badolato and Schumacher contributed equally to this work. Raffaele Badolato, MD, PhD R. Fabian Schumacher, MD Elisabetta Rodella, MD Franco Gargiulo, MD Carlo Torti, MD Luigi D. Notarangelo, MD Marzia Duse, MD Istituto di Medicina Molecolare “Angelo Nocivelli” Clinica Pediatrica Cattedra di Malattie Infettive Laboratorio di Microbiologia University of Brescia Brescia, Italy

Mechanisms of nucleoside analog antiviral activity and resistance during human immunodeficiency virus reverse transcription.

Progression to AIDS in human immunodeficiency virus type 1 (HIV-1)-positive individuals is characterized by a slow destruction of the immune system and a depletion of CD4 cells in the peripheral blood. The complex mechanism of CD4 cell disappearance is poorly understood, but this depletion may be attributable in part to a superantigen effect, apoptosis, viral cytopathicity, or combinations of all of these. A rough correlation exists between increases in viral load in lymphoid organs, rate of disease progression, and extent of CD4 cell depletion (118). Anti-HIV chemotherapy with agents such as 39-azido39-deoxythymidine (AZT) has resulted in at least transient decreases in viral load and increases in CD4 cells (66, 166). The anti-HIV drugs currently employed in clinical trials or licensed for AIDS therapy generally fall into three major categories, i.e., nucleoside analogs, nonnucleoside reverse transcriptase (RT) inhibitors, and HIV proteinase inhibitors. This review focuses on the inhibition of HIV-1 RT and reverse transcription by nucleoside analogs and on mechanisms of resistance to nucleoside analogs. AZT possesses activity against a number of retroviruses (83, 115) besides HIV-1 (106). AZT blocks HIV-1 replication at low concentrations; i.e., the effective concentration for 50% percent inhibition (IC50) is approximately 0.01 mM (Table 1). In addition, this drug is largely nontoxic for T lymphocytes; i.e., the cell culture inhibitory dose for 50% inhibition of cell growth (CCID50) is approximately 10 to 50 mM (Table 1) (106, 109). The active form of AZT, i.e., AZT 59-triphosphate (AZT-TP), phosphorylated by cellular nucleoside kinases, is thought to inhibit HIV-1 reverse transcription both as a competitive inhibitor of RT and as a chain terminator of DNA elongation (152). When studied in phase I and phase II clinical trials, AZT treatment resulted in increased numbers of CD4 cells, decreased occurrences of opportunistic infections, and decreased viral loads (20, 34, 42, 173). Many of the initially serious toxic effects of AZT, e.g., bone marrow suppression and anemia, have been alleviated by lowering the dosage and by administering recombinant erythropoietin (41, 134, 163). AZT can cross the blood-brain barrier and may reverse certain neurological abnormalities such as AIDS dementia (52). However, AZT treatment for prolonged periods also resulted in the emergence of drug-resistant viral isolates that displayed normal replication kinetics and up to 100-fold resistance to this drug (89, 138). In addition, a direct correlation between the development of AZT resistance and clinical progression to AIDS and death has been established (33, 108). A number of other dideoxynucleoside triphosphates (ddNTP), analogous to native deoxynucleoside triphosphates (dNTP) but deficient in their 39 hydroxyl group, have also been shown to inhibit HIV replication in CD4 cells. Relatively few of these compounds, e.g., 29,39-dideoxyinosine (ddI or didanosine) and 29,39-dideoxycytidine (ddC or zalcitabine), have IC50s of below 10 mM (Table 1) (32, 104, 105). Derivatives of these analogs, such as the racemic mixture of 29,39-dideoxy-39thiacytidine (BCH-189) and its negative enantiomer (3TC or lamivudine) (25, 26, 142, 150), 29,39-dideoxy-59-fluoro-39thiacytidine (144), 39-fluoro-39-deoxythymidine (FLT), carbocyclic-29,39-didehydro-29,39-dideoxyguanosine (carbovir) (162), 39-azido-29,39-dideoxyuridine, and 29,39-didehydro29,39-dideoxythymidine (d4T) (60), also inhibited HIV infections of CD4 cells and in many cases showed less cytotoxicity and had lower CCID50s than AZT (25, 60, 177). These ddNTPs have all entered all clinical trials, although resistance continues to be a problem (90, 107, 138, 140, 151, 175, 176). The mechanisms of antiviral action by nucleoside analogs in tissue culture are not fully understood. Nucleoside analog triphosphates may block acute infection by HIV and other retroviruses through inhibition of RT and chain termination (112, 178). However, AZT has also been reported to block virus replication in chronically infected cells (139), to interfere with virus maturation (18), and to disrupt syncytium formation (18). However, AZT did not block cell-to-cell HIV transmission (59, 94, 153). In the related feline immunodeficiency virus model, selection of resistance to AZT resulted in mutations outside the RT-coding region, suggesting that viral proteins besides RT may also play a role in resistance (131). Breakthrough HIV replication in CD4 cells was observed despite constant exposure to high AZT concentrations (147). Certain nonnucleoside inhibitors of RT, i.e., phosphonoformate and Nevirapine, and other nucleoside analogs, i.e., carbovir, ddI, FLT, and ddC, may act synergistically with AZT to delay HIV-1 replication in tissue culture (35, 39, 82, 135, 147).

Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription.

Reverse transcription by human immunodeficiency virus (HIV) reverse transcriptase (RT) entails several distinct, early steps in the synthesis of double-stranded DNA from viral RNA templates. These steps include tRNA(lys3) priming of RNA-dependent DNA polymerization and template switching for synthesis of near full-length (-) DNA. However, HIV RT lacks the fluent processivity of other viral reverse transcriptases or cellular polymerases. Previous studies in our laboratory showed that RT reactions primed with tRNA(lys3) had higher efficiencies of template switching than those primed by synthetic oligonucleotides. To further study primer extension, pausing, and template switching, we utilized an in vitro reconstituted reverse transcription/template switching reaction consisting of HIV RNA templates, recombinant HIV RT and primer tRNA(lys3). We observed initial pause sites within the first five nucleotides of the primer terminus; these were extended efficiently to full-length (-) strong-stop DNA with prolonged incubation. Other pause sites occurred in the R/U5/PBS template adjacent to homopolymeric sequences and regions thought to be involved in secondary structure. This assay provides a sensitive means of assessing template switching at times between 10 s and 3 h. Copyright 1995 S. Karger AG, Basel

Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model: kinetics and implications.

We have investigated the kinetics of human immunodeficiency virus (HIV) reverse transcription in infected T cells, using a synchronized, one-step, cell-to-cell infection model and quantitative PCR assays for the different DNA intermediate structures that are found sequentially during reverse transcription. Different efficiencies that might arise from the use of different primers and other PCR conditions were normalized by conversion of each PCR product signal to copy numbers by comparing with standards. After an initial lag period, the minus-strand strong-stop viral DNA was detected first. This was followed by the post-transfer newly extended minus-strand viral DNA and then by the plus-strand strong-stop DNA and fully extended minus-strand DNA. Kinetic data indicated that, once reverse transcription was initiated, the HIV reverse transcriptase synthesized minus-strand DNA at a rate of 150-180 bases/min, and that the first template transfer and the initiation of the plus-strand DNA synthesis imposed specific time delays. In contrast, minus-strand viral DNA synthesized after the second template transfer appeared at a time point very close to the time of the appearance of the last piece of DNA synthesized just before the second template switch, suggesting that the second switch occurred very rapidly. Taken together, our results define more accurately than was previously possible the rates of several of the steps in HIV reverse transcription in infected T cell lines and indicate different mechanisms for the two distinct template switches during retrovirus reverse transcription.

Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription

Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription

Preferential incorporation of nucleoside analogs after template switching during human immunodeficiency virus reverse transcription

We assessed the effects of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC) on reverse transcription in CD4-positive cells by isolating truncated human immunodeficiency virus (HIV) DNA fragments. Jurkat cells were treated with AZT (2 microM), ddI (200 microM), or 3TC (50 microM) prior to infection with HIV. Low-molecular-weight DNA was isolated and amplified by PCR with primer pairs which identify different segments of HIV proviral DNA. We found that the HIV DNA fragments generated from drug-treated, HIV-exposed Jurkat cells were truncated at a ratio of 15:1 [i.e., (-) strong-stop DNA to HIV DNA generated after the first template switch]. Full-length DNA was observed in the case of untreated, HIV-infected cultures. Following nucleoside analog treatment of HIV-exposed Jurkat cells, reverse transcription was terminated only after the synthesis of (-) strong-stop DNA. The nucleoside analogs tested, i.e., AZT, ddI, and 3TC, preferentially chain terminated viral DNA synthesis immediately following the first template switch. The (-) strong-stop HIV DNA was present in AZT-treated and untreated cultures for at least 6 days. We also carried out cell-free reverse transcription/template-switching reactions involving tRNA(Lys3) or a deoxyoligonucleotide as a primer, as a means of studying the selective incorporation of AZT triphosphate into proviral DNA. When reactions were primed with tRNA(Lys3), we found that AZT triphosphate was preferentially incorporated after template switching.

The Kinetics of Human Immunodeficiency Virus Reverse Transcription Are Slower in Primary Human Macrophages Than in a Lymphoid Cell Line

Reverse transcription is a critical event in the life of a retrovirus and a potential determinant of viral infectivity. The kinetics of "endogenous" reactions are relatively well defined but little is known about HIV reverse transcription during infection. In this report, we have estimated the rate and efficiency of HIV-1 reverse transcription in primary macrophages and H9 lymphoid cells using quantitative PCR to detect intermediate cDNA structures. DNA synthesis is completed 12-16 hr after infection of H9 cells, but requires more than 36 hr in Mφ, owing to slower rates of extension and template switching. Reverse transcription was inefficient in both cell types and in H9 cells the kinetics were sufficiently well resolved to estimate that only one in three transcripts initiated were extended to full-length viral DNA. Slower DNA synthesis largely accounts for the longer replicative cycle of HIV in macrophages. The rate of reverse transcription, which may depend upon levels of deoxynucleotide triphosphate substrates, is a potential factor in modulating the permissiveness of macrophage populations in vivo.