Functional Analysis of Amino Acid Residues Constituting the dNTP Binding Pocket of HIV-1 Reverse Transcriptase

ABSTRACT Human foamy virus (HFV), a retrovirus of simian origin which occasionally infects humans, is the basis of retroviral vectors in development for gene therapy. Clinical considerations of how to treat patients developing an uncontrolled infection by either HFV or HFV-based vectors need to be raised. We determined the susceptibility of the HFV to dideoxynucleosides and found that only zidovudine was equally efficient against the replication of human immunodeficiency virus type 1 (HIV-1) and HFV. By contrast, zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), and didanosine (ddI) were 3-, 3-, 30-, and 46-fold less efficient against HFV than against HIV-1, respectively. Some amino acid residues known to be involved in HIV-1 resistance to ddC, 3TC, d4T, and ddI were found at homologous positions of HFV reverse transcriptase (RT). These critical amino acids are located at the same positions in the three-dimensional structure of HIV-1 and HFV RT, suggesting that both enzymes share common patterns of inhibition.

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Characterization of HIV-1 reverse transcriptase encoding mutations at the amino acid residues 161 and 208 involved in phosphonoformate resistance

Antiviral Research ◽

10.1016/0166-3542(95)94777-y ◽

1995 ◽

Vol 26 (3) ◽

pp. A268

Author(s):

E. Tramontano ◽

G. Piras ◽

E. Congeddu ◽

J. Mellors ◽

H. Bazmi ◽

...

Keyword(s):

Amino Acid ◽

Reverse Transcriptase ◽

Amino Acid Residues ◽

Hiv 1

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Analysis of Binding Sites for the New Small-Molecule CCR5 Antagonist TAK-220 on Human CCR5

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.11.4708-4715.2005 ◽

2005 ◽

Vol 49 (11) ◽

pp. 4708-4715 ◽

Cited By ~ 50

Author(s):

Masao Nishikawa ◽

Katsunori Takashima ◽

Toshiya Nishi ◽

Rika A. Furuta ◽

Naoyuki Kanzaki ◽

...

Keyword(s):

Amino Acid ◽

Small Molecule ◽

Conformational Changes ◽

Binding Pocket ◽

Inhibitory Effect ◽

Ccr5 Antagonist ◽

Amino Acid Residues ◽

Ccr5 Antagonists ◽

Human Ccr5 ◽

Hiv 1

ABSTRACT G protein-coupled receptor CCR5 is the main coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1), and various small-molecule CCR5 antagonists are being developed to treat HIV-1 infection. It has been reported that such CCR5 antagonists, including TAK-779, bind to a putative binding pocket formed by transmembrane domains (TMs) 1, 2, 3 and 7 of CCR5, indicating the importance of the conformational changes of the TMs during virus entry. In this report, using a single-round infection assay with human CCR5 and its substitution mutants, we demonstrated that a new CCR5 antagonist, TAK-220, shares the putative interacting amino acid residues Asn252 and Leu255 in TM6 with TAK-779 but also requires the distinct residues Gly163 and Ile198 in TMs 4 and 5, respectively, for its inhibitory effect. We suggested that, together with molecular models of the interactions between the drugs and CCR5, the inhibitory activity of TAK-220 could involve direct interactions with amino acid residues in TMs 4, 5, and 6 in addition to those in the previously postulated binding pocket. The possible interaction of drugs with additional regions of the CCR5 molecule would help to develop a new small-molecule CCR5 antagonist.

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Identification of amino acid residues in HIV-1 reverse transcriptase that are critical for the proteolytic processing of Gag-Pol precursors

FEBS Letters ◽

10.1016/j.febslet.2011.09.034 ◽

2011 ◽

Vol 585 (21) ◽

pp. 3372-3377 ◽

Cited By ~ 4

Author(s):

Hironori Nishitsuji ◽

Masaru Yokoyama ◽

Hironori Sato ◽

Suguru Yamauchi ◽

Hiroshi Takaku

Keyword(s):

Amino Acid ◽

Reverse Transcriptase ◽

Proteolytic Processing ◽

Amino Acid Residues ◽

Hiv 1

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Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53827-8 ◽

1993 ◽

Vol 268 (4) ◽

pp. 2674-2683

Author(s):

S. Volkmann ◽

B.M. Wöhrl ◽

M. Tisdale ◽

K. Moelling

Keyword(s):

Amino Acid ◽

Reverse Transcriptase ◽

Amino Acid Residues ◽

Enzymatic Analysis ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Carboxyl Terminal ◽

Hiv 1 ◽

Ribonucleases H

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Mutational analysis of Lys65 of HIV-1 reverse transcriptase

Biochemical Journal ◽

10.1042/bj3480077 ◽

2000 ◽

Vol 348 (1) ◽

pp. 77-82 ◽

Cited By ~ 29

Author(s):

Nicolas SLUIS-CREMER ◽

Dominique ARION ◽

Neerja KAUSHIK ◽

Henry LIM ◽

Michael A. PARNIAK

Keyword(s):

Amino Acid ◽

Reverse Transcriptase ◽

Mutational Analysis ◽

Catalytic Efficiency ◽

Salt Bridge ◽

Polymerase Activity ◽

Ph Optimum ◽

Modelling Studies ◽

Dntp Binding ◽

Hiv 1

Amino acid Lys65 is part of the highly flexible β3-β4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the ϵ-amino group of Lys65 interacts with the γ-phosphate of the bound deoxynucleoside triphosphate (‘dNTP’) substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys65, we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys65 in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (Km) for all dNTP substrates, in RT catalytic efficiency (kcat/Km) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys65 do not affect the geometry of the loop's α-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys70 and Arg72. In particular, Glu in K65E can form a salt bridge with Arg72, leading to the diminution of the latter residue's interaction with the α-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate (‘ddNTP’) inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.

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Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00224-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 8

Author(s):

Kalyan Das ◽

Sergio E. Martinez ◽

Eddy Arnold

Keyword(s):

Reverse Transcriptase ◽

Binding Pocket ◽

Complex Iii ◽

Structural Basis ◽

Single Mutation ◽

Wild Type ◽

Chain Flexibility ◽

Multiple Drugs ◽

Dntp Binding ◽

Hiv 1

ABSTRACT HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3′-endo and 3′-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

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Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function

FEBS Letters ◽

10.1016/0014-5793(89)81559-5 ◽

1989 ◽

Vol 257 (2) ◽

pp. 311-314 ◽

Cited By ~ 119

Author(s):

Octavian Schatz ◽

Frans V. Cromme ◽

Fiona Grüninger-Leitch ◽

Stuart F.J. Le Grice

Keyword(s):

Amino Acid ◽

Reverse Transcriptase ◽

Point Mutations ◽

Rnase H ◽

Amino Acid Residues ◽

Terminal Domain ◽

Hiv 1

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Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket ofHepatitis B viruspolymerase

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15017896 ◽

2015 ◽

Vol 71 (11) ◽

pp. 1384-1390 ◽

Cited By ~ 7

Author(s):

Akiyoshi Nakamura ◽

Noriko Tamura ◽

Yoshiaki Yasutake

Keyword(s):

Reverse Transcriptase ◽

Binding Pocket ◽

Crystal Form ◽

B Virus ◽

Slight Movement ◽

Inhibitor Resistance ◽

Rt Inhibitors ◽

Dntp Binding ◽

Hiv 1

Hepatitis B viruspolymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stabilityin vitrohas hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol andHuman immunodeficiency virus 1(HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space groupP321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

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

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200628103359 ◽

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.

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