Structure Of Reverse Transcriptase Research Articles

Synthesis of Novel Indolyl Aryl Sulfone-clubbed Hydrazone Derivatives as Potential HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: Molecular Modeling and QSAR Studies.

Non-Nucleoside Reverse Transcriptases Inhibitors (NNRTIs) are among the most extensively studied enzymes for understanding the biology of Human Immunodeficiency Viruses (HIV) and designing inhibitors for managing HIV infections. Indolyl aryl sulfones (IASs), an underexplored class of potent NNRTIs, require further exploration for the development of newer drugs for HIV. In this context, we synthesized a series of novels by Indolyl Aryl Sulfones with a hydrazone moiety at the carboxylate site of the indole nucleus. A 2D-QSAR model was developed to predict Reverse Transcriptase inhibitory activity against wild-type RT (WT-RT) enzyme. The model was successfully applied to predict the HIV-1 inhibitory activity of known Indolyl Aryl Sulfones. Considering the reliability, robustness, and reproducibility of the 2D-QSAR model, we made an in-silico prediction of the RT inhibition for our synthesized compounds (1-14). Molecular docking and dynamics simulations established our synthesized Indolyl Aryl Sulfones, particularly compounds 23, 24, and 28, as effective NNRTIs by stabilizing HIV reverse transcriptase's structure. Binding energy calculations revealed compound 28 as the strongest inhibitor (-43.21 ± 0.09 kcal/mol), followed by 23 (-40.94 ± 0.10 kcal/mol) and 24 (-39.18±0.08 kcal/mol), emphasizing their binding affinity towards HIV reverse transcriptase. In summary, the synthesized Indolyl Aryl Sulfones, particularly compounds 23, 24, and 28, demonstrate significant potential as Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) against HIV. These results highlight the promising role of these compounds in developing novel NNRTIs for managing HIV infections.

A molecular movie of nucleotide addition by HIV-1 reverse transcriptase.

Historically, interactions between substrates and/or inhibitors and enzymes have been viewed in terms of binding of small molecules to relatively rigid protein targets. However, computational and experimental studies have revealed that many proteins, in particular DNA polymerases, undergo molecular motions over a wide range of timescales. Such conformational flexibility is critical for enzymatic activity, drug action and drug resistance. Moreover, contemporary structural biology approaches, such as X-ray crystallography, only have the ability to resolve the structures of thermodynamically stable species, and cannot inform on kinetic intermediates. To address this issue, we have developed a novel methodology to achieve time-resolved cryo-electron microscopy (TR cryo-EM) that, for the first time, facilitates visualization of short-lived protein conformations, including those transiently occurring during catalysis.Using TR cryo-EM, we have for the first time elucidated four intermediate kinetic states during HIV-1 reverse transcriptase (RT) mediated nucleotide (dATP) incorporation, supporting the feasibility and power of these approaches. Interestingly, we observe the presence of three Mg+2 in the active site during an intermediate state of the reaction, contrasting the kinetically stable structures of RT obtained via X-ray crystallography, where only two Mg+2 can be detected. The information derived from these studies improves our understanding of RT function, which in turn may facilitate development of new drugs.

Cis-Allosteric Regulation of HIV-1 Reverse Transcriptase by Integrase.

Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes of retroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in the context of HIV-1 replication. The mechanism underlying RTIN's effect, however, remains to be elucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations of dNTPs (less than 10 μM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavage of RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, the cis-allosteric regulatory roles of IN in RT structure and enzymatic activity.

Insights into HIV-1 Reverse Transcriptase (RT) Inhibition and Drug Resistance from Thirty Years of Structural Studies.

The enzyme reverse transcriptase (RT) plays a central role in the life cycle of human immunodeficiency virus (HIV), and RT has been an important drug target. Elucidations of the RT structures trapping and detailing the enzyme at various functional and conformational states by X-ray crystallography have been instrumental for understanding RT activities, inhibition, and drug resistance. The structures have contributed to anti-HIV drug development. Currently, two classes of RT inhibitors are in clinical use. These are nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, the error-prone viral replication generates variants that frequently develop resistance to the available drugs, thus warranting a continued effort to seek more effective treatment options. RT also provides multiple additional potential druggable sites. Recently, the use of single-particle cryogenic electron microscopy (cryo-EM) enabled obtaining structures of NNRTI-inhibited HIV-1 RT/dsRNA initiation and RT/dsDNA elongation complexes that were unsuccessful by X-ray crystallography. The cryo-EM platform for the structural study of RT has been established to aid drug design. In this article, we review the roles of structural biology in understanding and targeting HIV RT in the past three decades and the recent structural insights of RT, using cryo-EM.

Structural Studies and Structure Activity Relationships for Novel Computationally Designed Non-nucleoside Inhibitors and Their Interactions With HIV-1 Reverse Transcriptase.

Reverse transcriptase (RT) from the human immunodeficiency virus continues to be an attractive drug target for antiretroviral therapy. June 2022 will commemorate the 30th anniversary of the first Human Immunodeficiency Virus (HIV) RT crystal structure complex that was solved with non-nucleoside reverse transcriptase inhibitor nevirapine. The release of this structure opened opportunities for designing many families of non-nucleoside reverse transcriptase inhibitors (NNRTIs). In paying tribute to the first RT-nevirapine structure, we have developed several compound classes targeting the non-nucleoside inhibitor binding pocket of HIV RT. Extensive analysis of crystal structures of RT in complex with the compounds informed iterations of structure-based drug design. Structures of seven additional complexes were determined and analyzed to summarize key interactions with residues in the non-nucleoside inhibitor binding pocket (NNIBP) of RT. Additional insights comparing structures with antiviral data and results from molecular dynamics simulations elucidate key interactions and dynamics between the nucleotide and non-nucleoside binding sites.

Retroviral reverse transcriptase: Structure, function and inhibition.

Reverse transcriptase (RT) is a multifunctional enzyme that has RNA- and DNA-dependent DNA polymerase activity and ribonuclease H (RNase H) activity, and is responsible for the reverse transcription of retroviral single-stranded RNA into double-stranded DNA. The essential role that RT plays in the human immunodeficiency virus (HIV) life cycle is highlighted by the fact that multiple antiviral drugs-which can be classified into two distinct therapeutic classes-are routinely used to treat and/or prevent HIV infection. This book chapter provides detailed insights into the three-dimensional structure of HIV RT, the biochemical mechanisms of DNA polymerization and RNase H activity, and the mechanisms by which nucleoside/nucleotide and nonnucleoside RT inhibitors block reverse transcription.

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.

Small Conformational Changes Underlie Evolution of Resistance to NNRTI in HIV Reverse Transcriptase

Despite achieving considerable success in reducing the number of fatalities due to acquired immunodeficiency syndrome, emergence of resistance against the reverse transcriptase (RT) inhibitor drugs remains one of the biggest challenges of the human immunodeficiency virus antiretroviral therapy (ART). Non-nucleoside reverse transcriptase inhibitors (NNRTIs) form a large class of drugs and a crucial component of ART. In NNRTIs, even a single resistance mutation is known to make the drugs completely ineffective. Additionally, several inhibitor-bound RTs with single resistance mutations do not exhibit any significant variations in their three-dimensional structures compared with the inhibitor-bound RT but completely nullify their inhibitory functions. This makes understanding the structural mechanism of these resistance mutations crucial for drug development. Here, we study several single resistance mutations in the allosteric inhibitor (nevirapine)-bound RT to analyze the mechanism of small structural changes leading to these large functional effects. In this study, we have shown that in absence of significant conformational variations in the inhibitor-bound wild-type RT and RT with single resistance mutations, the protein contact network analysis of their static structures, along with molecular dynamics simulations, can be a useful approach to understand the functional effect of small local conformational variations. The simple network analysis exposes the localized contact changes that lead to global rearrangement in the communication pattern within RT. Furthermore, these conformational changes have implications on the overall dynamics of RT. Using various measures, we show that a single resistance mutation can change the network structure and dynamics of RT to behave more like unbound RT, even in the presence of the inhibitor. This combined coarse-grained contact network and molecular dynamics approach promises to be a useful tool to analyze structure-function studies of proteins that show large functional changes with negligible variations in their overall conformation.

Elucidating the molecular interactions of the L‐nucleotide analogs with the Complex of HIV‐1 Reverse Transcriptase and DNA and the mechanism of drug resistance conferred by the M184V mutation

Emtricitabine (FTC) and lamivudine (3TC) are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in antiretroviral therapy for HIV. They contain an oxathiolane ring with unnatural (−)‐stereochemistry as a mimic of the ribose ring found in natural deoxynucleotides (L‐nucleoside). Treatment with FTC or 3TC primarily selects for the HIV‐1 reverse transcriptase (RT) M184V/I resistance mutants that are characterized by a marked decrease of incorporation of these NRTIs. Pre‐steady state kinetic data and crystal structures of both (−) and (+) oxathiolane analog triphosphates were determined in wild‐type and M184V HIV‐1 RT to elucidate the structural features of RT that recognize these substrates and gain insight into how M184V confers resistance. The pre‐steady state kinetic data reveal that (−)‐FTC‐TP and (−)‐3TC‐TP have higher binding affinities (1/Kd) for WT RT than dCTP however they have a slower rate of incorporation (kp). The crystal structures corroborate these results revealing that the oxathiolane sulfur orients toward the DNA primer terminus and two different triphosphate orientations are observed which would reduce the rate of incorporation. The structure of (+)‐FTC‐TP shows a close match to that of the natural substrate dCTP illustrating how the oxathiolane D‐isomers adopt a triphosphate orientation conducive for incorporation. The M184V mutation displays significantly greater (>200‐fold) Kd for the L isomers and moderately higher (>9‐fold) Kd for the D‐isomers compared to dCTP. The structure of M184V RT illustrates how the mutation repositions the oxathiolane ring, inducing a shift of the triphosphates into a conformation that hinders nucleotide incorporation. This combined study provides a comprehensive kinetic and structural basis for inhibiting WT HIV‐1 RT by the active metabolites of 3TC and FTC and the mechanism of M184V resistance.Support or Funding InformationThis work was supported by a grant from the National Science Foundation (grant number MCB‐1716168).

Integrative structural biology studies of HIV-1 reverse transcriptase binding to a high-affinity DNA aptamer.

The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2′-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2′-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2′-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.

Structural and Functional Aspects of Foamy Virus Protease-Reverse Transcriptase.

Reverse transcription describes the process of the transformation of single-stranded RNA into double-stranded DNA via an RNA/DNA duplex intermediate, and is catalyzed by the viral enzyme reverse transcriptase (RT). This event is a pivotal step in the life cycle of all retroviruses. In contrast to orthoretroviruses, the domain structure of the mature RT of foamy viruses is different, i.e., it harbors the protease (PR) domain at its N-terminus, thus being a PR-RT. This structural feature has consequences on PR activation, since the enzyme is monomeric in solution and retroviral PRs are only active as dimers. This review focuses on the structural and functional aspects of simian and prototype foamy virus reverse transcription and reverse transcriptase, as well as special features of reverse transcription that deviate from orthoretroviral processes, e.g., PR activation.

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 4ʹ-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 Å and 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 4ʹ-modified NRTIs.

Molecular Docking Studies of HIV-1 Resistance to Reverse Transcriptase Inhibitors: Mini-Review.

Currently, millions of people are living with human immunodeficiency virus type 1 (HIV-1), which causes acquired immunodeficiency syndrome. However, the spread of the HIV-1 resistance to antiviral agents is the major problem in the antiretroviral therapy and medical management of HIV-infected patients. HIV-1 reverse transcriptase (RT) is one of the key viral targets for HIV-1 inhibition. Therefore, the studies on the combatting the HIV resistance that occurs due to the structural changes in RT, are in great demand. This work aims to provide an overview of the state-of-the-art molecular docking approaches applied to the studies of the HIV-1 resistance, associated with RT structure changes. We have reviewed recent studies using molecular docking with mutant forms of RT. The work discusses the modifications of molecular docking, which have been developed to find the novel molecules active against resistance mutants of RT and/or recombinant strains of HIV-1. The perspectives of the existing algorithms of molecular docking to the studies on molecular mechanisms of resistance and selection of the correct binding poses for the reverse transcriptase inhibitors are discussed.

A computational study for rational HIV-1 non-nucleoside reverse transcriptase inhibitor selection and the discovery of novel allosteric pockets for inhibitor design.

HIV drug resistant mutations that render the current Highly Active Anti-Retroviral Therapy (HAART) cocktail drugs ineffective are increasingly reported. To study the mechanisms of these mutations in conferring drug resistance, we computationally analyzed 14 reverse transcriptase (RT) structures of HIV-1 on the following parameters: drug-binding pocket volume, allosteric effects caused by the mutations, and structural thermal stability. We constructed structural correlation-based networks of the mutant RT–drug complexes and the analyses support the use of efavirenz (EFZ) as the first-line drug, given that cross-resistance is least likely to develop from EFZ-resistant mutations. On the other hand, rilpivirine (RPV)-resistant mutations showed the highest cross-resistance to the other non-nucleoside RT inhibitors. With significant drug cross-resistance associated with the known allosteric drug-binding site, there is a need to identify new allosteric druggable sites in the structure of RT. Through computational analyses, we found such a novel druggable pocket on the HIV-1 RT structure that is comparable with the original allosteric drug site, opening the possibility to the design of new inhibitors.

The structure of FIV reverse transcriptase and its implications for non-nucleoside inhibitor resistance.

Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the “closed” pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more “closed” conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.

Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid

HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.

Molecular Modeling of Potential Dual Inhibitors of HIV Reverse Transcriptase and Integrase

With the goal of suggesting dual inhibitors of HIV reverse transcriptase (RT) and integrase (IN), herein we report the molecular docking of an initial set of 556 compounds related to the pyridinone class. Docking with multiple crystallographic structures of HIV-1 RT led to 160 potential binders of RT interacting with key amino acid residues at the enzyme’s allosteric site. Compounds selected from the docking with RT were further docked with a crystallographic structure of HIV-1 IN. A total of 31 structures had the potential to make contacts with Mg2+ ions located in a small space between DNA and IN. Interactions with Mg2+ ions are relevant because they participate in the stabilization of the IN-DNA complex. In conclusion, 31 compounds synthetically accessible are proposed as dual inhibitors of RT and IN. It is hypothesized that the suggested compounds will inhibit RT by occupying the allosteric site for NNRTIs and will inhibit the catalytic activity of IN by destabilizing the IN-DNA complex. The main perspective of this work is the synthesis and biological testing of the candidate molecules.

Evolutionary Analysis of HIV-1 Pol Proteins Reveals Representative Residues for Viral Subtype Differentiation.

RNA viruses have been used as model systems to understand the patterns and processes of molecular evolution because they have high mutation rates and are genetically diverse. Human immunodeficiency virus 1 (HIV-1), the etiological agent of acquired immune deficiency syndrome, is highly genetically diverse, and is classified into several groups and subtypes. However, it has been difficult to use its diverse sequences to establish the overall phylogenetic relationships of different strains or the trends in sequence conservation with the construction of phylogenetic trees. Our aims were to systematically characterize HIV-1 subtype evolution and to identify the regions responsible for HIV-1 subtype differentiation at the amino acid level in the Pol protein, which is often used to classify the HIV-1 subtypes. In this study, we systematically characterized the mutation sites in 2,052 Pol proteins from HIV-1 group M (144 subtype A; 1,528 subtype B; 380 subtype C), using sequence similarity networks. We also used spectral clustering to group the sequences based on the network graph structures. A stepwise analysis of the cluster hierarchies allowed us to estimate a possible evolutionary pathway for the Pol proteins. The subtype A sequences also clustered according to when and where the viruses were isolated, whereas both the subtype B and C sequences remained as single clusters. Because the Pol protein has several functional domains, we identified the regions that are discriminative by comparing the structures of the domain-based networks. Our results suggest that sequence changes in the RNase H domain and the reverse transcriptase (RT) connection domain are responsible for the subtype classification. By analyzing the different amino acid compositions at each site in both domain sequences, we found that a few specific amino acid residues (i.e., M357 in the RT connection domain and Q480, Y483, and L491 in the RNase H domain) represent the differences among the subtypes. These residues were located on the surface of the RT structure and in the vicinity of the amino acid sites responsible for RT enzymatic activity or function.

Reverse Transcription in the Saccharomyces cerevisiae Long-Terminal Repeat Retrotransposon Ty3.

Converting the single-stranded retroviral RNA into integration-competent double-stranded DNA is achieved through a multi-step process mediated by the virus-coded reverse transcriptase (RT). With the exception that it is restricted to an intracellular life cycle, replication of the Saccharomyces cerevisiae long terminal repeat (LTR)-retrotransposon Ty3 genome is guided by equivalent events that, while generally similar, show many unique and subtle differences relative to the retroviral counterparts. Until only recently, our knowledge of RT structure and function was guided by a vast body of literature on the human immunodeficiency virus (HIV) enzyme. Although the recently-solved structure of Ty3 RT in the presence of an RNA/DNA hybrid adds little in terms of novelty to the mechanistic basis underlying DNA polymerase and ribonuclease H activity, it highlights quite remarkable topological differences between retroviral and LTR-retrotransposon RTs. The theme of overall similarity but distinct differences extends to the priming mechanisms used by Ty3 RT to initiate (−) and (+) strand DNA synthesis. The unique structural organization of the retrotransposon enzyme and interaction with its nucleic acid substrates, with emphasis on polypurine tract (PPT)-primed initiation of (+) strand synthesis, is the subject of this review.

Computational prediction of human immunodeficiency resistance to reverse transcriptase inhibitors

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) and leads to over one million of deaths annually. Highly active antiretroviral treatment (HAART) is a gold standard in the HIV/AIDS therapy. Nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT) are important component of HAART, but their effect depends on the HIV susceptibility/resistance. HIV resistance mainly occurs due to mutations leading to conformational changes in the three-dimensional structure of HIV RT. The aim of our work was to develop and test a computational method for prediction of HIV resistance associated with the mutations in HIV RT. Earlier we have developed a method for prediction of HIV type 1 (HIV-1) resistance; it is based on the usage of position-specific descriptors. These descriptors are generated using the particular amino acid residue and its position; the position of certain residue is determined in a multiple alignment. The training set consisted of more than 1900 sequences of HIV RT from the Stanford HIV Drug Resistance database; for these HIV RT variants experimental data on their resistance to ten inhibitors are presented. Balanced accuracy of prediction varies from 80% to 99% depending on the method of classification (support vector machine, Naive Bayes, random forest, convolutional neural networks) and the drug, resistance to which is obtained. Maximal balanced accuracy was obtained for prediction of resistance to zidovudine, stavudine, didanosine and efavirenz by the random forest classifier. Average accuracy of prediction is 89%.