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Mutations in variable domains of the HIV-1 envelope gene can have a significant impact on maraviroc and vicriviroc resistance.

ABSTRACT: BACKGROUND: Resistance to CCR5 inhibitors, such as maraviroc and vicriviroc is characterized by reduction of maximal percent inhibition which indicates the use of an inhibitor-bound conformation of CCR5 for human immunodeficiency virus-1(HIV-1) entry. It is accompanied by substitutions in gp120 and gp41. Variable domain 3 (V3) plays the most important role, but substitutions outside V3 could also be involved in phenotype resistance. In this work, we investigated how mutations in variable regions of the viral envelope protein gp120 can contribute to CCR5 inhibitor resistance. METHODS: Resistant isolates were selected by passaging CC1/85 and BaL viruses with sub-inhibitory MVC and VCV concentrations. Mutations in gp160 were identified and mutants containing V2 (V169M), V3 (L317W) and V4 (I408T) were constructed. RESULTS: MVC and VCV susceptibility and viral tropism were assessed by single cycle assay. Mutant I408T showed 4-fold change (FC) increase in the half maximal inhibitory concentration (IC50) to MVC, followed by L317W (1.52-FC), V169M (1.23-FC), V169M/I408T (4-FC) L317W/I408T (3-FC), V169M/L317W (1.30-FC), and V169M/L317W/I408T (3.31-FC). MPI reduction was observed for mutants I408T (85%), L317W (95%), V169M/I408T (84%), L317W/I408T (85%) and V169M/L317W/I408T (83%). For VCV, I408T increased the IC50 by 2-FC and few mutants showed MPI reduction less than 95%: I408T (94%), L317W/I408T (94%) and V169M/L317W/I408T (94%). All mutants remained R5-tropic and presented decreased infectivity. CONCLUSIONS: These results suggest that mutations in the V4 loop of HIV-1 may contribute to MVC and VCV resistance alone or combined with mutations in V2 and V3 loops.

SUBMITTER: Asin-Milan O

PROVIDER: S-EPMC3700831 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Mutations in variable domains of the HIV-1 envelope gene can have a significant impact on maraviroc and vicriviroc resistance.

Asin-Milan Odalis O Chamberland Annie A Wei Yi Y Haidara Alpha A Sylla Mohamed M Tremblay Cécile L CL

AIDS research and therapy 20130607 1

<h4>Background</h4>Resistance to CCR5 inhibitors, such as maraviroc and vicriviroc is characterized by reduction of maximal percent inhibition which indicates the use of an inhibitor-bound conformation of CCR5 for human immunodeficiency virus-1(HIV-1) entry. It is accompanied by substitutions in gp120 and gp41. Variable domain 3 (V3) plays the most important role, but substitutions outside V3 could also be involved in phenotype resistance. In this work, we investigated how mutations in variable ...[more]

PMID: 23758814

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Project description:The CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC.Envs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively "weak" and "strong" resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus.Clinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.

Project description:A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.

Project description:Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.

Dataset Information

Mutations in variable domains of the HIV-1 envelope gene can have a significant impact on maraviroc and vicriviroc resistance.

Publications

Mutations in variable domains of the HIV-1 envelope gene can have a significant impact on maraviroc and vicriviroc resistance.

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