The Impact of Individual Human Immunodeficiency Virus Type 1 Protease Mutations on Drug Susceptibility Is Highly Influenced by Complex Interactions with the Background Protease Sequence

ABSTRACT The requirement for multiple mutations for protease inhibitor (PI) resistance necessitates a better understanding of the molecular basis of resistance development. The novel bioinformatics resistance determination approach presented here elaborates on genetic profiles observed in clinical human immunodeficiency virus type 1 (HIV-1) isolates. Synthetic protease sequences were cloned in a wild-type HIV-1 background to generate a large number of close variants, covering 69 mutation clusters between multi-PI-resistant viruses and their corresponding genetically closely related, but PI-susceptible, counterparts. The vast number of mutants generated facilitates a profound and broad analysis of the influence of the background on the effect of individual PI resistance-associated mutations (PI-RAMs) on PI susceptibility. Within a set of viruses, all PI-RAMs that differed between susceptible and resistant viruses were varied while maintaining the background sequence from the resistant virus. The PI darunavir was used to evaluate PI susceptibility. Single sets allowed delineation of the impact of individual mutations on PI susceptibility, as well as the influence of PI-RAMs on one another. Comparing across sets, it could be inferred how the background influenced the interaction between two mutations, in some cases even changing antagonistic relationships into synergistic ones or vice versa. The approach elaborates on patient data and demonstrates how the specific mutational background greatly influences the impact of individual mutations on PI susceptibility in clinical patterns.

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Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.2.495-501.2001 ◽

2001 ◽

Vol 45 (2) ◽

pp. 495-501 ◽

Cited By ~ 65

Author(s):

Atsuko Hachiya ◽

Saori Aizawa-Matsuoka ◽

Mari Tanaka ◽

Yukiko Takahashi ◽

Setsuko Ida ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cell Clone ◽

Drug Susceptibility ◽

Phenotypic Resistance ◽

Phenotypic Assay ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

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Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness

Journal of General Virology ◽

10.1099/vir.0.045492-0 ◽

2012 ◽

Vol 93 (12) ◽

pp. 2625-2634 ◽

Cited By ~ 6

Author(s):

Elena Capel ◽

Glòria Martrus ◽

Mariona Parera ◽

Bonaventura Clotet ◽

Miguel Angel Martínez

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Replication Capacity ◽

Recombinant Viruses ◽

Subtype B ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.

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Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

Journal of Virology ◽

10.1128/jvi.01614-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2405-2417 ◽

Cited By ~ 168

Author(s):

Vineela Chukkapalli ◽

Ian B. Hogue ◽

Vitaly Boyko ◽

Wei-Shau Hu ◽

Akira Ono

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Membrane Binding ◽

Particle Assembly ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P2 depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P2 depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P2-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P2. To examine a putative Gag interaction with PI(4,5)P2, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P2. Using this assay, we observed that PI(4,5)P2 significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P2 for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P2 binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P2-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P2 on the membrane and that the MA basic domain mediates this interaction.

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Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5555 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5555-5562 ◽

Cited By ~ 15

Author(s):

J D DeZazzo ◽

J M Scott ◽

M J Imperiale

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

A Site ◽

The Impact ◽

Hiv 1 ◽

Rna Levels

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

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The intra-host evolutionary and population dynamics of human immunodeficiency virus type 1: a phylogenetic perspective

Infectious Disease Reports ◽

10.4081/idr.2013.s1.e3 ◽

2013 ◽

Vol 5 (1S) ◽

pp. 3 ◽

Cited By ~ 25

Author(s):

Marco Salemi

Keyword(s):

Human Immunodeficiency Virus ◽

Population Dynamics ◽

Disease Progression ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1 ◽

Over Time

The intra-host evolutionary and population dynamics of the human immunodeficiency virus type 1 (HIV-1), the cause of the acquired immunodeficiency syndrome, have been the focus of one of the most extensive study efforts in the field of molecular evolution over the past three decades. As HIV-1 is among the fastest mutating organisms known, viral sequence data sampled over time from infected patients can provide, through phylogenetic analysis, significant insights about the tempo and mode of evolutionary processes shaped by complex interaction with the host milieu. Five main aspects are discussed: the patterns of HIV-1 intra-host diversity and divergence over time in relation to different phases of disease progression; the impact of selection on the temporal structure of HIV-1 intra-host genealogies inferred from longitudinally sampled viral sequences; HIV-1 intra-host sub-population structure; the potential relationship between viral evolutionary rate and disease progression and the central evolutionary role played by recombination occurring in super-infected cells.

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Immunogenicity of Mutations Induced by Nucleoside Reverse Transcriptase Inhibitors for Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Cells

Journal of Virology ◽

10.1128/jvi.74.19.9306-9312.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9306-9312 ◽

Cited By ~ 47

Author(s):

Assia Samri ◽

Gaby Haas ◽

Joerg Duntze ◽

Jean-Marc Bouley ◽

Vincent Calvez ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Mononuclear Cells ◽

Immune Therapy ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

ABSTRACT The impact of drug resistance mutations induced by nucleoside reverse transcriptase (RT) inhibitors (NRTI) on cytotoxic T-lymphocyte (CTL) recognition of human immunodeficiency virus type 1 strain LAI (HIV-1LAI) RT was addressed in 35 treated or untreated patients. Two HIV-1LAI RT regions encompassing mutation M41L, L74V, M184V, and T215Y/F were recognized in 75 and 83% mutated and in 33 and 42% unmutated samples, respectively. A total of 41 new CTL epitopes overlapping these mutations were predicted. Mutations enhanced HLA-binding scores of 17 epitopes, decreased scores of 5, and had no effect in 19. Four predicted epitopes containing mutations 41, 74, and 184 were tested and recognized by CD8 cells from mutated or unmutated samples, with frequencies up to 270 gamma interferon spot-forming cells per 106 peripheral blood mononuclear cells. Therefore, RT mutations induced by NRTI can increase the immunogenicity of RT for CTL and might allow a better immune control of resistant viruses in vivo, suggesting that specific immune therapy might help prevent these mutations.

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Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5555-5562.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5555-5562

Author(s):

J D DeZazzo ◽

J M Scott ◽

M J Imperiale

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

A Site ◽

The Impact ◽

Hiv 1 ◽

Rna Levels

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The role of viral coreceptors and enhanced macrophage tropism in human immunodeficiency virus type 1 disease progression

Sexual Health ◽

10.1071/sh03006 ◽

2004 ◽

Vol 1 (1) ◽

pp. 23 ◽

Cited By ~ 14

Author(s):

Paul R. Gorry ◽

Jasminka Sterjovski ◽

Melissa Churchill ◽

Kristie Witlox ◽

Lachlan Gray ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Disease Progression ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Coreceptor Usage ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

Despite numerous studies on the impact of viral diversity, human immunodeficiency virus type 1 (HIV-1)-specific immune responses and host factors on disease progression, we still do not have a firm understanding of the long-term pathogenesis of HIV-1 infection. Rapid depletion of CD4+ T-lymphocytes has been associated with a switch in viral coreceptor usage from CCR5 to CXCR4 in ~40 to 50% of infected individuals. However, the majority of infected individuals who progress to AIDS harbour only CCR5-dependent (R5) viral strains. The progression HIV-1 disease is associated with an enhanced tropism of R5 viral strains for monocyte/macrophage lineage cells (enhanced M-tropism). However, the underlying molecular mechanisms contributing to enhanced M-tropism by R5 HIV-1 strains, and how HIV-1 variants with enhanced M-tropism cause CD4+ T-cell depletion in vivo are unknown. This review examines the relationship between viral coreceptor usage, M-tropism, and pathogenicity of HIV-1. We highlight evidence supporting the hypothesis that enhanced M-tropism of R5 HIV-1 results from adaptive viral evolution, resulting in HIV-1 variants that have increased ability to utilise relatively low levels of CCR5 expressed on macrophages, by way of increased CCR5 affinity. The evidence also suggests that these late-emerging, R5 viral strains have reduced sensitivity to entry inhibitors, and increased ability to cause CD4+ T-lymphocyte loss. These variants are likely to impact HIV-1 disease progression, especially in patients who persistently harbour only R5 viral strains.

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Suppression of Viremia and Evolution of Human Immunodeficiency Virus Type 1 Drug Resistance in a Macaque Model for Antiretroviral Therapy

Journal of Virology ◽

10.1128/jvi.01301-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12145-12155 ◽

Cited By ~ 41

Author(s):

Zandrea Ambrose ◽

Sarah Palmer ◽

Valerie F. Boltz ◽

Mary Kearney ◽

Kay Larsen ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Drug Resistance ◽

Antiretroviral Therapy ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

ABSTRACT Antiretroviral therapy (ART) in human immunodeficiency virus type 1 (HIV-1)-infected patients does not clear the infection and can select for drug resistance over time. Not only is drug-resistant HIV-1 a concern for infected individuals on continual therapy, but it is an emerging problem in resource-limited settings where, in efforts to stem mother-to-child-transmission of HIV-1, transient nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy given during labor can select for NNRTI resistance in both mother and child. Questions of HIV-1 persistence and drug resistance are highly amenable to exploration within animals models, where therapy manipulation is less constrained. We examined a pigtail macaque infection model responsive to anti-HIV-1 therapy to study the development of resistance. Pigtail macaques were infected with a pathogenic simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT-SHIV) to examine the impact of prior exposure to a NNRTI on subsequent ART comprised of a NNRTI and two nucleoside RT inhibitors. K103N resistance-conferring mutations in RT rapidly accumulated in 2/3 infected animals after NNRTI monotherapy and contributed to virologic failure during ART in 1/3 animals. By contrast, ART effectively suppressed RT-SHIV in 5/6 animals. These data indicate that suboptimal therapy facilitates HIV-1 drug resistance and suggest that this model can be used to investigate persisting viral reservoirs.

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Predicting the Impact of Blocking Human Immunodeficiency Virus Type 1 Nef In Vivo

Journal of Virology ◽

10.1128/jvi.00821-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2349-2356 ◽

Cited By ~ 5

Author(s):

W. David Wick ◽

Peter B. Gilbert ◽

Otto O. Yang

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Antiretroviral Drugs ◽

Mhc I ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef is a multifunctional protein that confers an ability to evade killing by cytotoxic T lymphocytes (CTLs) as well as other advantages to the virus in vivo. Here we exploited mathematical modeling and related statistical methods to estimate the impact of Nef activity on viral replication in vivo in relation to CTLs. Our results indicate that downregulation of major histocompatibility complex class I (MHC-I) A and B by wild-type Nef confers an advantage to the virus of about 82% in decreased CTL killing efficiency on average, meaning that abolishing the MHC-I downregulation function of Nef would increase killing by more than fivefold. We incorporated this estimate, as well as prior estimates of replicative enhancement by Nef, into a previously published model of HIV-1 and CTLs in vivo (W. D. Wick, O. O. Yang, L. Corey, and S. G. Self, J. Virol. 79:13579-13586, 2005), generalized to permit CTL recognition of multiple epitopes. A sequence database analysis revealed that 92.9% of HIV-1 epitopes are A or B restricted, and a previous study found an average of about 19 epitopes recognized (M. M. Addo et al., J. Virol. 77:2081-2092, 2003). We combined these estimates in the model in order to predict the impact of inhibiting Nef function in the general (chronically infected) population by a drug. The predicted impact on viral load ranged from negligible to 2.4 orders of magnitude, depending on the effects of the drug and the CTL dynamical scenario assumed. We conclude that inhibiting Nef could make a substantial reduction in disease burden, lengthening the time before the necessity of undertaking combination therapy with other antiretroviral drugs.

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