Mechanism of Substrate Recognition by Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variants Revealed by a Novel Structural Intermediate

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual “intermediate” conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.

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High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.11.4546-4554.2005 ◽

2005 ◽

Vol 49 (11) ◽

pp. 4546-4554 ◽

Cited By ~ 16

Author(s):

Reynel Cancio ◽

Romano Silvestri ◽

Rino Ragno ◽

Marino Artico ◽

Gabriella De Martino ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Mechanism Of Action ◽

Drug Resistant ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.

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A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+ T Cells of Viremic Patients

Journal of Virology ◽

10.1128/jvi.79.8.5185-5202.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 5185-5202 ◽

Cited By ~ 25

Author(s):

Daphne Monie ◽

Rachel P. Simmons ◽

Richard E. Nettles ◽

Tara L. Kieffer ◽

Yan Zhou ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Latent Reservoir ◽

Drug Resistant ◽

Plasma Virus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT A latent reservoir for human immunodeficiency virus type 1 (HIV-1) consisting of integrated provirus in resting memory CD4+ T cells prevents viral eradication in patients on highly active antiretroviral therapy (HAART). It is difficult to analyze the nature and dynamics of this reservoir in untreated patients and in patients failing therapy, because it is obscured by an excess of unintegrated viral DNA that constitutes the majority of viral species in resting CD4+ T cells from viremic patients. Therefore, we developed a novel culture assay that stimulates virus production from latent, integrated HIV-1 in resting CD4+ T cells in the presence of antiretroviral drugs that prevent the replication of unintegrated virus. Following activation, resting CD4+ T cells with integrated HIV-1 DNA produced virus particles for several days, with peak production at day 5. Using this assay, HIV-1 pol sequences from the resting CD4+ T cells of viremic patients were found to be genetically distinct from contemporaneous plasma virus. Despite the predominance of a relatively homogeneous population of drug-resistant viruses in the plasma of patients failing HAART, resting CD4+ T cells harbored a diverse array of wild-type and archival drug-resistant viruses that were less fit than plasma virus in the context of current therapy. These results provide the first direct evidence that resting CD4+ T cells serve as a stable reservoir for HIV-1 even in the setting of high levels of viremia. The ability to analyze archival species in viremic patients may have clinical utility in detecting drug-resistant variants not present in the plasma.

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Origin of Human Immunodeficiency Virus Type 1 Quasispecies Emerging after Antiretroviral Treatment Interruption in Patients with Therapeutic Failure

Journal of Virology ◽

10.1128/jvi.76.14.7000-7009.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 7000-7009 ◽

Cited By ~ 47

Author(s):

Gustavo H. Kijak ◽

Viviana Simon ◽

Peter Balfe ◽

Jeroen Vanderhoeven ◽

Sandra E. Pampuro ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Treatment Failure ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Treatment Interruption ◽

Viral Population ◽

Drug Resistant ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.

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X-Ray Crystal Structures of Human Immunodeficiency Virus Type 1 Protease Mutants Complexed with Atazanavir

Journal of Virology ◽

10.1128/jvi.02503-05 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9525-9535 ◽

Cited By ~ 19

Author(s):

Herbert E. Klei ◽

Kevin Kish ◽

Pin-Fang M. Lin ◽

Qi Guo ◽

Jacques Friborg ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Crystal Structures ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Resistant Mutant ◽

X Ray ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Atazanavir, which is marketed as REYATAZ, is the first human immunodeficiency virus type 1 (HIV-1) protease inhibitor approved for once-daily administration. As previously reported, atazanavir offers improved inhibitory profiles against several common variants of HIV-1 protease over those of the other peptidomimetic inhibitors currently on the market. This work describes the X-ray crystal structures of complexes of atazanavir with two HIV-1 protease variants, namely, (i) an enzyme optimized for resistance to autolysis and oxidation, referred to as the cleavage-resistant mutant (CRM); and (ii) the M46I/V82F/I84V/L90M mutant of the CRM enzyme, which is resistant to all approved HIV-1 protease inhibitors, referred to as the inhibitor-resistant mutant. In these two complexes, atazanavir adopts distinct bound conformations in response to the V82F substitution, which may explain why this substitution, at least in isolation, has yet to be selected in vitro or in the clinic. Because of its nearly symmetrical chemical structure, atazanavir is able to make several analogous contacts with each monomer of the biological dimer.

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Design and Expression of a Dimeric Form of Human Immunodeficiency Virus Type 1 Antibody 2G12 with Increased Neutralization Potency

Journal of Virology ◽

10.1128/jvi.01564-08 ◽

2008 ◽

Vol 83 (1) ◽

pp. 98-104 ◽

Cited By ~ 39

Author(s):

Anthony P. West ◽

Rachel P. Galimidi ◽

Christopher P. Foglesong ◽

Priyanthi N. P. Gnanapragasam ◽

Kathryn E. Huey-Tubman ◽

...

Keyword(s):

Molecular Weight ◽

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Structural Model ◽

Passive Immunization ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Antibody 2G12

ABSTRACT The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.

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Efficient Identification of Human Immunodeficiency Virus Type 1 Mutants Resistant to a Protease Inhibitor by Using a Random Mutant Library

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00687-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5090-5098 ◽

Cited By ~ 1

Author(s):

Sanggu Kim ◽

Yun-Cheol Kim ◽

Hangfei Qi ◽

Kunkai Su ◽

Sherie L. Morrison ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Drug Resistance ◽

Protease Inhibitor ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Drug Resistant ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTEmergence of drug-resistant mutant viruses during the course of antiretroviral therapy is a major hurdle that limits the success of chemotherapeutic treatment to suppress human immunodeficiency virus type 1 (HIV-1) replication and AIDS progression. Development of new drugs and careful patient management based on resistance genotyping data are important for enhancing therapeutic efficacy. However, identifying changes leading to drug resistance can take years of clinical studies, and conventionalin vitroassays are limited in generating reliable drug resistance data. Here we present an efficientin vitroscreening assay for selecting drug-resistant variants from a library of randomly mutated HIV-1 strains generated by transposon-directed base-exchange mutagenesis. As a test of principle, we screened a library of mutant HIV-1 strains containing random mutations in the protease gene by using a reporter T-cell line in the presence of the protease inhibitor (PI) nelfinavir (NFV). Analysis of replicating viruses from a single round of infection identified 50 amino acid substitutions at 35 HIV-1 protease residue positions. The selected mutant viruses showed specific resistance to NFV and included most of the known NFV resistance mutations. Therefore, the new assay is efficient for identifying changes leading to drug resistance. The data also provide insights into the molecular mechanisms underlying the development of drug resistance.

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Influence of Reverse Transcriptase Variants, Drugs, and Vpr on Human Immunodeficiency Virus Type 1 Mutant Frequencies

Journal of Virology ◽

10.1128/jvi.77.3.2071-2080.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2071-2080 ◽

Cited By ~ 52

Author(s):

Louis M. Mansky ◽

Erwann Le Rouzic ◽

Serge Benichou ◽

Lisa C. Gajary

Keyword(s):

Human Immunodeficiency Virus ◽

Drug Resistance ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Drug Resistant ◽

Immunodeficiency Virus ◽

Virus Mutant ◽

Hiv 1

ABSTRACT The evolution of drug resistance is a major complication of human immunodeficiency virus type 1 (HIV-1) chemotherapy. HIV-1 reverse transcriptase (RT) is a major target of antiretroviral therapy and ultimately the target of drug resistance mutations. Previous studies have indicated that drug-resistant HIV-1 RTs can alter HIV-1 mutant frequencies. In this study, we have tested a panel of HIV-1 RT variants for their ability to influence virus mutant frequencies. The RT variants tested included drug-resistant RT variants as well as other variants analyzed in enzyme fidelity studies with the lacZα gene as a mutation target and/or implicated as being important for enzyme fidelity by structural studies. Combinations of mutations that alone had a statistically significant influence on virus mutant frequencies resulted in different mutant frequency phenotypes. Furthermore, when virus replication occurred in the presence of drugs [e.g., 3′-azido-3′-deoxythymidine, (−)2/,3′-dideoxy-3′-thiacytidine, hydroxyurea, thymidine, or thioguanine] with selected RT variants, virus mutant frequencies increased. Similarly, Vpr variants deficient for binding to the uracil DNA glycosylase repair enzyme were observed to influence HIV-1 virus mutant frequencies when tested alone or in combination with RT variants. In summary, these observations indicate that HIV-1 mutant frequencies can significantly change by single amino acid substitutions in RT and that these effects can be altered by additional mutations in RT, by drugs, and/or by expression of Vpr variants. Such altered virus mutant frequencies could impact HIV-1 dynamics and evolution in small population sizes.

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Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

Clinical Microbiology Reviews ◽

10.1128/cmr.00017-07 ◽

2007 ◽

Vol 20 (4) ◽

pp. 550-578 ◽

Cited By ~ 45

Author(s):

Carrie Dykes ◽

Lisa M. Demeter

Keyword(s):

Human Immunodeficiency Virus ◽

Clinical Outcomes ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Relative Fitness ◽

Drug Resistant ◽

Immunodeficiency Virus ◽

Replication Fitness ◽

Hiv 1

SUMMARY The relative fitness of a variant, according to population genetics theory, is that variant's relative contribution to successive generations. Most drug-resistant human immunodeficiency virus type 1 (HIV-1) variants have reduced replication fitness, but at least some of these deficits can be compensated for by the accumulation of second-site mutations. HIV-1 replication fitness also appears to influence the likelihood of a drug-resistant mutant emerging during treatment failure and is postulated to influence clinical outcomes. A variety of assays are available to measure HIV-1 replication fitness in cell culture; however, there is no agreement regarding which assays best correlate with clinical outcomes. A major limitation is that there is no high-throughput assay that incorporates an internal reference strain as a control and utilizes intact virus isolates. Some retrospective studies have demonstrated statistically significant correlations between HIV-1 replication fitness and clinical outcomes in some patient populations. However, different studies disagree as to which clinical outcomes are most closely associated with fitness. This may be in part due to assay design, sample size limitations, and differences in patient populations. In addition, the strength of the correlations between fitness and clinical outcomes is modest, suggesting that, at present, it would be difficult to utilize these assays for clinical management.

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