scholarly journals The role of phenylalanine-119 of the reverse transcriptase of mouse mammary tumour virus in DNA synthesis, ribose selection and drug resistance

2002 ◽  
Vol 367 (2) ◽  
pp. 381-391 ◽  
Author(s):  
Michal ENTIN-MEER ◽  
Ziv SEVILYA ◽  
Amnon HIZI
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Three Dimensional ◽  
Mammary Tumour ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Aromatic Side Chain ◽  
Leukaemia Virus ◽  
Mouse Mammary Tumour Virus ◽  
Hiv 1

Phe-119 in the reverse transcriptase (RT) of mouse mammary tumour virus (MMTV) is homologous with Tyr-115 in HIV type 1 (HIV-1) RT and to Phe-155 in murine leukaemia virus (MLV) RT. By mutating these residues in HIV-1 and MLV RTs (which are strict DNA polymerases) the enzymes were shown to function also as RNA polymerases. Owing to the uniqueness of MMTV as a type B retrovirus, we have generated a Phe-119—Val mutant of MMTV RT to study the involvement of this residue in affecting the catalytic features of this RT. The data presented here show that the mutant MMTV RT can incorporate both deoxyribonucleosides and ribonucleosides while copying either RNA or DNA. In addition, this mutant RT shows resistance to nucleoside analogues and an enhanced fidelity of DNA synthesis; all relative to the wild-type enzyme. The Phe-119—Val mutant is also different from the wild-type enzyme in its preference for most template primers tested and in its ability to synthesize DNA under non-processive and processive conditions. Overall, it is likely that the aromatic side chain of Phe-119 is located at the dNTP-binding site of MMTV RT and thus might be part of a putative ‘steric gate’ that prevents the incorporation of nucleoside triphosphates. Since the only three-dimensional structures of RTs published so far are those of HIV-1 and MLV, it is likely that MMTV RT folds quite similarly to these RTs.

scholarly journals Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization

1998 ◽  
Vol 329 (3) ◽  
pp. 579-587 ◽  
Author(s):  
Ran TAUBE ◽  
Shoshana LOYA ◽  
Orna AVIDAN ◽  
Michal PERACH ◽  
Amnon HIZI
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Biochemical Characterization ◽  
Ph Dependence ◽  
Rnase H ◽  
Mammary Tumour ◽  
Nucleoside Triphosphate ◽  
Mouse Mammary Tumour Virus ◽  
Dna Primers ◽  
Hiv 1

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

scholarly journals Thermostable HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase

2011 ◽  
Vol 436 (3) ◽  
pp. 599-607 ◽  
Author(s):  
Verónica Barrioluengo ◽  
Mar Álvarez ◽  
Daniela Barbieri ◽  
Luis Menéndez-Arias
Keyword(s):  
Reverse Transcriptase ◽  
Polymerase Activity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Leukaemia Virus ◽  
Murine Leukaemia Virus ◽  
Subtype B ◽  
Rna Targets ◽  
Forward Mutation ◽  
Hiv 1

Wild-type HIV-1 group O RT (reverse transcriptase) shows increased thermostability in comparison with HIV-1 group M subtype B RT and MLV (murine leukaemia virus) RT. However, its utility in the amplification of RNA targets is limited by the reduced accuracy of lentiviral RTs compared with oncoretroviral RTs (i.e. MLV RT). The effects of the mutations K65R, R78A and K65R/V75I on the fidelity of HIV-1 group O RTs were studied using gel-based and M13mp2 lacZ forward-mutation fidelity assays. Forward-mutation assays demonstrated that mutant RTs K65R, R78A and K65R/V75I showed >9-fold increased accuracy in comparison with the wild-type enzyme and were approximately two times more faithful than the MLV RT. Compared with MLV RT, all of the tested HIV-1 group O RT variants showed decreased frameshift fidelity. However, K65R RT showed a higher tendency to introduce one-nucleotide deletions in comparison with other HIV-1 group O RT variants. R78A had a destabilizing effect on the RT, either in the presence or absence of V75I. At temperatures above 52 °C, K65R and K65R/V75I retained similar levels of DNA polymerase activity to the wild-type HIV-1 group O RT, but were more efficient than HIV-1 group M subtype B and MLV RTs. K65R, K65R/V75I and R78A RTs showed decreased misinsertion and mispair extension fidelity in comparison with the wild-type enzyme for most base pairs studied. These assays revealed that nucleotide selection is mainly governed by kpol (pol is polymerization) in the case of K65R, whereas both kpol and Kd affect nucleotide discrimination in the case of K65R/V75I.

3D-QSAR Studies of S-DABO Derivatives as Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

2019 ◽  
Vol 16 (8) ◽  
pp. 868-881
Author(s):  
Yueping Wang ◽  
Jie Chang ◽  
Jiangyuan Wang ◽  
Peng Zhong ◽  
Yufang Zhang ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Three Dimensional ◽  
Predictive Ability ◽  
3D Qsar ◽  
Binding Mode ◽  
Quantitative Structure Activity Relationship ◽  
Field Analysis ◽  
Reverse Transcriptase Inhibitors ◽  
Qsar Studies ◽  
Hiv 1

Background: S-dihydro-alkyloxy-benzyl-oxopyrimidines (S-DABOs) as non-nucleoside reverse transcriptase inhibitors have received considerable attention during the last decade due to their high potency against HIV-1. Methods: In this study, three-dimensional quantitative structure-activity relationship (3D-QSAR) of a series of 38 S-DABO analogues developed in our lab was studied using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA). The Docking/MMFF94s computational protocol based on the co-crystallized complex (PDB ID: 1RT2) was used to determine the most probable binding mode and to obtain reliable conformations for molecular alignment. Statistically significant CoMFA (q2=0.766 and r2=0.949) and CoMSIA (q2=0.827 and r2=0.974) models were generated using the training set of 30 compounds on the basis of hybrid docking-based and ligand-based alignment. Results: The predictive ability of CoMFA and CoMSIA models was further validated using a test set of eight compounds with predictive r2 pred values of 0.843 and 0.723, respectively. Conclusion: The information obtained from the 3D contour maps can be used in designing new SDABO derivatives with improved HIV-1 inhibitory activity.

Application of Molecular Docking for the Development of Improved HIV-1 Reverse Transcriptase Inhibitors

2020 ◽  
Vol 16 ◽  
Author(s):  
Arash Soltani ◽  
Seyed Isaac Hashemy ◽  
Farnaz Zahedi Avval ◽  
Houshang Rafatpanah ◽  
Seyed Abdolrahim Rezaee ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Capacity ◽  
Binding Pocket ◽  
Ligand Docking ◽  
Binding Modes ◽  
Wild Type ◽  
Parent Molecule ◽  
Discovery Studio ◽  
Approved Drugs ◽  
Hiv 1

Introoduction: Inhibition of the reverse transcriptase (RT) enzyme of human immunodeficiency virus (HIV) by low molecular weight inhibitors is still an active area of research. Here, protein-ligand interactions and possible binding modes of novel compounds with the HIV-1 RT binding pocket (the wild-type as well as Y181C and K103N mutants) were obtained and discussed. Methods: A molecular fragment-based approach using FDA-approved drugs were followed to design novel chemical derivatives using delavirdine, efavirenz, etravirine and rilpivirine as the scaffolds. The drug-likeliness of the derivatives was evaluated using Swiss-ADME. Then the parent molecule and derivatives were docked into the binding pocket of related crystal structures (PDB ID: 4G1Q, 1IKW, 1KLM and 3MEC). Genetic Optimization for Ligand Docking (GOLD) Suite 5.2.2 software was used for docking and the results analyzed in the Discovery Studio Visualizer 4. A derivative was chosen for further analysis, if it passed drug-likeliness and the docked energy was more favorable than that of its parent molecule. Out of the fifty-seven derivatives, forty-eight failed in druglikeness screening by Swiss-ADME or in docking stage. Results: The final results showed that the selected compounds had higher predicted binding affinities than their parent scaffolds in both wild-type and the mutants. Binding energy improvement was higher for the structures designed based on second-generation NNRTIs (etravirine and rilpivirine) than the first-generation NNRTIs (delavirdine and efavirenz). For example, while the docked energy for rilpivirine was -51 KJ/mol, it was improved for its derivatives RPV01 and RPV15 up to -58.3 and -54.5 KJ/mol, respectively. Conclusion: In this study, we have identified and proposed some novel molecules with improved binding capacity for HIV RT using fragment-based approach.

scholarly journals Structures of Complexes Formed by HIV-1 Reverse Transcriptase at a Termination Site of DNA Synthesis

2001 ◽  
Vol 276 (33) ◽  
pp. 31439-31448 ◽  
Author(s):  
Marc Lavigne ◽  
Lucette Polomack ◽  
Henri Buc
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Hiv 1

scholarly journals Mutating a Region of HIV-1 Reverse Transcriptase Implicated in tRNALys-3Binding and the Consequences for (−)-Strand DNA Synthesis

1998 ◽  
Vol 273 (23) ◽  
pp. 14523-14532 ◽  
Author(s):  
Eric J. Arts ◽  
Jennifer T. Miller ◽  
Bernard Ehresmann ◽  
Stuart F. J. Le Grice
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Hiv 1

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

The double mutation L109M and R448M of HIV-1 reverse transcriptase decreases fidelity of DNA synthesis by promoting mismatch elongation

2015 ◽  
Vol 396 (12) ◽  
pp. 1315-1323
Author(s):  
Bianca Heyn ◽  
Nicole Pogodalla ◽  
Susanne Brakmann
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Error Rate ◽  
Double Mutant ◽  
Dissociation Rate ◽  
Double Mutation ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Simultaneous Substitution

Abstract Changes of Leu109 and Arg448 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have as yet not been associated with altered fitness. However, in a recent study, we described that the simultaneous substitution of L109 and R448 by methionine leads to an error-producing polymerase phenotype that is not observed for the isolated substitutions. The double mutant increased the error rate of DNA-dependent DNA synthesis 3.1-fold as compared to the wildtype enzyme and showed a mutational spectrum with a fraction of 28% frameshift mutations and 48% transitions. We show here that weaker binding of DNA:DNA primer-templates as indicated by an increased dissociation rate constant (koff) could account for the higher frameshift error rate. Furthermore, we were able to explain the prevalence of transition mutations with the finding that HIV-1 RT variant L109M/R448M preferred misincorporation of C opposite A and elongation of C:A mismatches.

scholarly journals Construction of A Preliminary Three-Dimensional Structure Simian betaretrovirus Serotype-2 (SRV-2) Reverse Transcriptase Isolated from Indonesian Cynomolgus Monkey

2020 ◽  
Vol 31 (3) ◽  
pp. 47-61
Author(s):  
Uus Saepuloh ◽  
Diah Iskandriati ◽  
Joko Pamungkas ◽  
Dedy Duryadi Solihin ◽  
Sela Septima Mariya ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Cynomolgus Monkey ◽  
Tertiary Structure ◽  
Vaccine Development ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Nucleotide Position ◽  
Structure Model ◽  
Three Dimensional Structure ◽  
Hiv 1

Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284–4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme.

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