Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

ABSTRACT The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.

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Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

Journal of Virology ◽

10.1128/jvi.01955-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11557-11571 ◽

Cited By ~ 18

Author(s):

Aniqa Shahid ◽

Alex Olvera ◽

Gursev Anmole ◽

Xiaomei T. Kuang ◽

Laura A. Cotton ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Hla Class I ◽

Replication Capacity ◽

Target Cells ◽

Fitness Costs ◽

Ctl Escape ◽

The Impact ◽

Hiv 1

ABSTRACTHLA-B*13 is associated with superiorin vivoHIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24Gag(GQMVHQAIGag140–147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107Rwere recognized better by HIV-specific T cells than those infected with HIV-1NL4.3or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13.IMPORTANCEProtective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

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Potential Associations of Mutations within the HIV-1 Env and Gag Genes Conferring Protease Inhibitor (PI) Drug Resistance

Microbiology Research ◽

10.3390/microbiolres12040071 ◽

2021 ◽

Vol 12 (4) ◽

pp. 967-977

Author(s):

Ntombikhona F. Maphumulo ◽

Michelle L. Gordon

Keyword(s):

Protease Inhibitor ◽

Viral Fitness ◽

Site Directed Mutagenesis ◽

Envelope Gene ◽

Subtype C ◽

Exact Test ◽

Number Of Patients ◽

Drug Naïve ◽

The Impact ◽

Hiv 1

An increasing number of patients in Africa are experiencing virological failure on a second-line antiretroviral protease inhibitor (PI)-containing regimen, even without resistance-associated mutations in the protease region, suggesting a potential role of other genes in PI resistance. Here, we investigated the prevalence of mutations associated with Lopinavir/Ritonavir (LPV/r) failure in the Envelope gene and the possible coevolution with mutations within the Gag-protease (gag-PR) region. Env and Gag-PR sequences generated from 24 HIV-1 subtype C infected patients failing an LPV/r inclusive treatment regimen and 344 subtype C drug-naïve isolates downloaded from the Los Alamos Database were analyzed. Fisher’s exact test was used to determine the differences in mutation frequency. Bayesian network probability was applied to determine the relationship between mutations occurring within the env and gag-PR regions and LPV/r treatment. Thirty-five mutations in the env region had significantly higher frequencies in LPV/r-treated patients. A combination of Env and Gag-PR mutations was associated with a potential pathway to LPV/r resistance. While Env mutations were not directly associated with LPV/r resistance, they may exert pressure through the Gag and minor PR mutation pathways. Further investigations using site-directed mutagenesis are needed to determine the impact of Env mutations alone and in combination with Gag-PR mutations on viral fitness and LPV/r efficacy.

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Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Journal of Virology ◽

10.1128/jvi.01086-10 ◽

2010 ◽

Vol 84 (22) ◽

pp. 11937-11949 ◽

Cited By ~ 94

Author(s):

Mark A. Brockman ◽

Zabrina L. Brumme ◽

Chanson J. Brumme ◽

Toshiyuki Miura ◽

Jennifer Sela ◽

...

Keyword(s):

T Cell ◽

Chronic Infection ◽

Statistical Power ◽

Viral Fitness ◽

Replication Capacity ◽

Hla Alleles ◽

Subtype B ◽

Compensatory Mutations ◽

The Impact ◽

Hiv 1

ABSTRACT Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.

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Changes in HIV-1 Capsid Stability Induced by Common Cytotoxic-T-Lymphocyte-Driven Viral Sequence Mutations

Journal of Virology ◽

10.1128/jvi.00867-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7579-7586 ◽

Cited By ~ 2

Author(s):

P. Schommers ◽

G. Martrus ◽

U. Matschl ◽

M. Sirignano ◽

M. Lütgehetmann ◽

...

Keyword(s):

Viral Replication ◽

Cytotoxic T Lymphocyte ◽

Hla Class I ◽

Viral Fitness ◽

Class I ◽

Replication Capacity ◽

Ctl Escape ◽

Capsid Stability ◽

Hiv 1 ◽

Ctl Responses

ABSTRACTHIV-1-infected individuals with protective HLA class I alleles exhibit better control of viremia and slower disease progression. Virus control in these individuals has been associated with strong and potent HIV-1-specific cytotoxic-T-lymphocyte (CTL) responses restricted by protective HLA alleles, but control of viremia also occurs in the presence of selected CTL escape mutations. CTL escape mutations restricted by protective HLA class I molecules are frequently located in the conserved p24 Gag sequence of HIV-1 that encodes the conical capsid core and have been suggested to reduce viral replication capacity. In this study, the consequences of well-described CTL-associated p24 Gag sequence mutations for HIV-1 capsid stability were assessed using a cyclosporine (CsA) washout assay. The frequently occurring HLA-B57- and HLA-B27-associated CTL escape mutations T242N and R264K resulted in delayed capsid uncoating, suggesting modulation of capsid stability. The described compensatory mutations L268M and S173A observed in R264K viruses reconstituted the capsid-uncoating half-time. Interestingly, capsid stability was correlated with infectivity. Taken together, these data demonstrate that CTL-driven escape mutations within p24 Gag restricted by protective HLA class I alleles have a significant impact on capsid stability that might contribute to the persistent control of viral replication observed despite viral escape from CTL responses.IMPORTANCESequence mutations within p24 Gag selected by CTL responses restricted by protective HLA class I alleles have been associated with reduced viral fitness. However, the precise mechanisms underlying the reduced viral replication capacity and lower viral loads associated with these mutations remain unclear. Here, we demonstrate that dominant HLA-B27-associated CTL escape mutations within HIV-1 capsid lead to enhanced capsid rigidity, providing a possible mechanism for the reduced viral fitness of these variants.

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Viral Characteristics Associated with the Clinical Nonprogressor Phenotype Are Inherited by Viruses from a Cluster of HIV-1 Elite Controllers

mBio ◽

10.1128/mbio.02338-17 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 16

Author(s):

Concepción Casado ◽

Sara Marrero-Hernández ◽

Daniel Márquez-Arce ◽

María Pernas ◽

Sílvia Marfil ◽

...

Keyword(s):

Viral Replication ◽

Clinical Phenotype ◽

Functional Characterization ◽

Viral Fitness ◽

Elite Controller ◽

Replication Capacity ◽

Viral Fusion ◽

Elite Controllers ◽

Hiv 1

ABSTRACTA small group of HIV-1-infected individuals, called long-term nonprogressors (LTNPs), and in particular a subgroup of LTNPs, elite controllers (LTNP-ECs), display permanent control of viral replication and lack of clinical progression. This control is the result of a complex interaction of host, immune, and viral factors. We identified, by phylogenetic analysis, a cluster of LTNP-ECs infected with very similar low-replication HIV-1 viruses, suggesting the contribution of common viral features to the clinical LTNP-EC phenotype. HIV-1 envelope (Env) glycoprotein mediates signaling and promotes HIV-1 fusion, entry, and infection, being a key factor of viral fitnessin vitro, cytopathicity, and infection progressionin vivo. Therefore, we isolated full-lengthenvgenes from viruses of these patients and from chronically infected control individuals. Functional characterization of the initial events of the viral infection showed that Envs from the LTNP-ECs were ineffective in the binding to CD4 and in the key triggering of actin/tubulin-cytoskeleton modifications compared to Envs from chronic patients. The viral properties of the cluster viruses result in a defective viral fusion, entry, and infection, and these properties were inherited by every virus of the cluster. Therefore, inefficient HIV-1 Env functions and signaling defects may contribute to the low viral replication capacity and transmissibility of the cluster viruses, suggesting a direct role in the LTNP-EC phenotype of these individuals. These results highlight the important role of viral characteristics in the LTNP-EC clinical phenotype. These Env viral properties were common to all the cluster viruses and thus support the heritability of the viral characteristics.IMPORTANCEHIV-1 long-term nonprogressor elite controller patients, due to their permanent control of viral replication, have been the object of numerous studies to identify the factors responsible for this clinical phenotype. In this work, we analyzed the viral characteristics of the envelopes of viruses from a phylogenetic cluster of LTNP-EC patients. These envelopes showed ineffective binding to CD4 and the subsequent signaling activity to modify actin/tubulin cytoskeletons, which result in low fusion and deficient entry and infection capacities. These Env viral characteristics could explain the nonprogressor clinical phenotype of these patients. In addition, these inefficientenvviral properties were present in all viruses of the cluster, supporting the heritability of the viral phenotype.

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Drug susceptibility and replication capacity of a rare HIV-1 subtype C protease hinge region variant

Antiviral Therapy ◽

10.3851/imp3308 ◽

2019 ◽

Vol 24 (5) ◽

pp. 333-342

Author(s):

Jake Zondagh ◽

Adriaan E Basson ◽

Ikechukwu Achilonu ◽

Lynn Morris ◽

Heini W Dirr ◽

...

Keyword(s):

Drug Susceptibility ◽

Hinge Region ◽

Replication Capacity ◽

Subtype C ◽

Hiv 1

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Impact of HIV-1 Diversity on Its Sensitivity to Neutralization

Vaccines ◽

10.3390/vaccines7030074 ◽

2019 ◽

Vol 7 (3) ◽

pp. 74 ◽

Cited By ~ 3

Author(s):

Karl Stefic ◽

Mélanie Bouvin-Pley ◽

Martine Braibant ◽

Francis Barin

Keyword(s):

Public Health ◽

Envelope Protein ◽

Neutralizing Antibodies ◽

Continuous Process ◽

Population Level ◽

Global Public Health ◽

Broadly Neutralizing Antibodies ◽

The Many ◽

The Impact ◽

Hiv 1

The HIV-1 pandemic remains a major burden on global public health and a vaccine to prevent HIV-1 infection is highly desirable but has not yet been developed. Among the many roadblocks to achieve this goal, the high antigenic diversity of the HIV-1 envelope protein (Env) is one of the most important and challenging to overcome. The recent development of broadly neutralizing antibodies has considerably improved our knowledge on Env structure and its interplay with neutralizing antibodies. This review aims at highlighting how the genetic diversity of HIV-1 thwarts current, and possibly future, vaccine developments. We will focus on the impact of HIV-1 Env diversification on the sensitivity to neutralizing antibodies and the repercussions of this continuous process at a population level.

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The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission

PLoS ONE ◽

10.1371/journal.pone.0161596 ◽

2016 ◽

Vol 11 (9) ◽

pp. e0161596

Author(s):

Eveline Santos da Silva ◽

Martin Mulinge ◽

Morgane Lemaire ◽

Cécile Masquelier ◽

Cyprien Beraud ◽

...

Keyword(s):

Cytoplasmic Tail ◽

Replication Capacity ◽

Viral Infectivity ◽

Subtype C ◽

Cell Transmission ◽

Hiv 1

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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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Impact of Primary Elvitegravir Resistance-Associated Mutations in HIV-1 Integrase on Drug Susceptibility and Viral Replication Fitness

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02568-12 ◽

2013 ◽

Vol 57 (6) ◽

pp. 2654-2663 ◽

Cited By ~ 72

Author(s):

Michael E. Abram ◽

Rebecca M. Hluhanich ◽

Derrick D. Goodman ◽

Kristen N. Andreatta ◽

Nicolas A. Margot ◽

...

Keyword(s):

Viral Replication ◽

Drug Susceptibility ◽

Viral Fitness ◽

Cross Resistance ◽

Replication Capacity ◽

Strand Transfer ◽

Recombinant Viruses ◽

Transfer Inhibitor ◽

Replication Fitness ◽

Hiv 1

ABSTRACTElvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

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