scholarly journals Asymmetric conformational maturation of HIV-1 reverse transcriptase

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Xunhai Zheng ◽  
Lalith Perera ◽  
Geoffrey A Mueller ◽  
Eugene F DeRose ◽  
Robert E London
Keyword(s):  
Reverse Transcriptase ◽  
Early Stage ◽  
Active Form ◽  
Hydrophobic Surface ◽  
Nmr Studies ◽  
Inactive Conformation ◽  
Polymerase Domain ◽  
Dynamics Simulations ◽  
Hiv 1 ◽  
Different Time Scales

HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66' homodimer exists as a conformational heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb' interface occurs at an early stage, while maturation of the connection' and unfolding of the RH' domains are linked and occur on a much slower time scale.

scholarly journals Identification of drivers for the metamorphic transition of HIV-1 reverse transcriptase

2017 ◽  
Vol 474 (19) ◽  
pp. 3321-3338 ◽  
Author(s):  
Xunhai Zheng ◽  
Geoffrey A. Mueller ◽  
Kyungmin Kim ◽  
Lalith Perera ◽  
Eugene F. DeRose ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Hydrophobic Surface ◽  
Terminal Segment ◽  
Nmr Studies ◽  
Conformational Preferences ◽  
Starting Point ◽  
Immunodeficiency Virus ◽  
Β Sheet ◽  
Structural Characterizations ◽  
Hiv 1

Recent structural characterizations of the p51 and p66 monomers have established an important starting point for understanding the maturation pathway of the human immunodeficiency virus (HIV)-1 reverse transcriptase p66/p51 heterodimer. This process requires a metamorphic transition of the polymerase domain leading to formation of a p66/p66′ homodimer that exists as a structural heterodimer. To better understand the drivers for this metamorphic transition, we have performed NMR studies of 15N-labeled RT216 — a construct that includes the fingers and most of the palm domains. These studies are consistent with the conclusion that the p66 monomer exists as a spring-loaded complex. Initial dissociation of the fingers/palm : connection complex allows the fingers/palm to adopt an alternate, more stable structure, reducing the rate of reassociation and facilitating subsequent maturation steps. One of the drivers for an initial extension of the fingers/palm domains is identified as a straightening of helix E relative to its conformation in the monomer by eliminating a bend of ∼50° near residue Phe160. NMR and circular dichroism data also are consistent with the conclusion that a hydrophobic surface of palm domain that becomes exposed after the initial dissociation, as well as the intrinsic conformational preferences of the palm domain C-terminal segment, facilitates the formation of the β-sheet structure that is unique to the active polymerase subunit. Spectral comparisons based on 15N-labeled constructs are all consistent with previous structural conclusions based on studies of 13C-methyl-labeled constructs.

scholarly journals Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine

2007 ◽  
Vol 81 (15) ◽  
pp. 7852-7859 ◽  
Author(s):  
Jessica H. Brehm ◽  
Dianna Koontz ◽  
Jeffrey D. Meteer ◽  
Vinay Pathak ◽  
Nicolas Sluis-Cremer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Cross Resistance ◽  
Polymerase Domain ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAI or AZT-resistant HIV-1LAI (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAI were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

scholarly journals Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

2014 ◽  
Vol 89 (1) ◽  
pp. 833-843 ◽  
Author(s):  
Ilona P. Deuzing ◽  
Charlotte Charpentier ◽  
David W. Wright ◽  
Sophie Matheron ◽  
Jack Paton ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Structural Changes ◽  
Novel Mutation ◽  
Rnase H ◽  
Nucleoside Analogues ◽  
Replication Capacity ◽  
Resistance Mutations ◽  
Loop Region ◽  
Polymerase Domain ◽  
Hiv 1

ABSTRACTInfection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity.IMPORTANCEMutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

scholarly journals A small molecule, ACAi-028, with anti-HIV-1 activity targets a novel hydrophobic pocket on HIV-1 capsid

2021 ◽  
Author(s):  
Tomofumi Nakamura ◽  
Travis chia ◽  
Masayuki Amano ◽  
Nobutoki Takamune ◽  
Masao Matsuoka ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Reverse Transcriptase ◽  
Small Molecule ◽  
Early Stage ◽  
Integrase Inhibitor ◽  
Ionization Mass ◽  
Hydrophobic Pocket ◽  
Binding Model ◽  
Anti Hiv ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection, and an attractive therapeutic target for antivirals. We report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is one of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between a reverse transcriptase inhibitor, azidothymidine (AZT), and an integrase inhibitor, raltegravir (RAL), without any effect on RT activity, suggesting that this compound may affect HIV-1 core disassembly (uncoating). Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and non-covalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues.

scholarly journals Computational development of rubromycin-based lead compounds for HIV-1 reverse transcriptase inhibition

2014 ◽  
Author(s):  
Pedro J Silva ◽  
Carlos E. P. Bernardo
Keyword(s):  
Molecular Dynamics ◽  
Molecular Docking ◽  
Reverse Transcriptase ◽  
Molecular Dynamics Simulations ◽  
Complex Interplay ◽  
Lead Compounds ◽  
High Affinity ◽  
Dynamics Simulations ◽  
Hiv 1

The binding of several rubromycin-based ligands to HIV1-reverse transcriptase was analyzed using molecular docking and molecular dynamics simulations. MM-PBSA analysis and examination of the trajectories allowed the identification of several promising compounds with predicted high affinity towards reverse transcriptase mutants which have proven resistant to current drugs. Important insights on the complex interplay of factors determining the ability of ligands to selectively target each mutant have been obtained.

HIV-1 Reverse Transcriptase: Structure Predictions for the Polymerase Domain

1990 ◽  
Vol 6 (9) ◽  
pp. 1061-1072 ◽  
Author(s):  
A.M. BARBER ◽  
A. HIZI ◽  
J.V. MAIZEL ◽  
S.H. HUGHES
Keyword(s):  
Reverse Transcriptase ◽  
Polymerase Domain ◽  
Hiv 1
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