Date Published: June 7, 2013
Publisher: BioMed Central
Author(s): Odalis Asin-Milan, Annie Chamberland, Yi Wei, Alpha Haidara, Mohamed Sylla, Cécile L Tremblay.
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.
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.
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.
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.
HIV-1 entry into target cells is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4. It triggers conformational changes in gp120, forming the co-receptor binding site [1-3]. gp120 interaction with C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4) induces other conformational changes in gp120, which evoke structural re-arrangement of gp41 and enables the viral and cellular membrane fusion, permitting viral entry . CCR5 inhibitors, including maraviroc (MVC), vicriviroc (VCV), aplaviroc, TAK-779 and TAK-220, antagonize this process and have strong anti-viral activity against HIV-1 in vitro[5,6]. Although they bind the hydrophobic pocket within transmembrane domains of CCR5 with high affinity, they occupy different sub-cavities by interacting with different amino acids . MVC is the first CCR5 inhibitor approved for the treatment of R5-tropic HIV-1 infection in both naïve and treatment-experienced adult patients. VCV development was stopped because of suboptimal efficacy . Since MCV and VCV are allosteric inhibitors of virus entry, resistance to these drugs is evidenced by reduction in the plateau of virus inhibition curves rather than by increases in 50 percent inhibitory concentration (IC50) [7,8]. The magnitude of this decrease can be expressed as maximum plateau inhibition (MPI) . Plateau height depends on the relative affinity of HIV-1 for inhibitor-bound versus free CCR5, the greater the affinity for inhibitor-bound CCR5, the lower the height of the plateau . MOTIVATE clinical trials of MVC revealed that the MPI of most MVC-resistant viruses in subjects failing therapy ranged from 80 to 95% . In VICTOR-E1 clinical trials of VCV, phenotypic resistance was manifested by reductions in relative MPI. The cut-off value was 0.94 . Changes in susceptibility to CCR5 inhibitors are usually accompanied by substitutions in gp120, with V3 domain appearing to play a critical role. However, substitutions outside this region also contribute to the resistance phenotype . The aim of this study is to investigate how mutations in other variable loops of the HIV-1 Env can contribute to MVC and VCV resistance.
Our findings indicate that viruses resistant to MVC can retain the use of CCR5 coreceptor as reported previously [14-16]. CCR5 inhibitors are associated with mutations in the Env V3 region of R5 isolates [14,17]. Our passage experiments revealed only 1 polymorphism in the V3 loop crown, L317W, which was associated with reduced infectivity, but not with resistance to CCR5 inhibitors or changes in V3 net charge (Tables 1 and 2). The selection of HIV-1 resistance to CCR5 inhibitors is relatively difficult [14,18], the V3 loop being the least variable of the HIV-1 Env variable regions . Marozsan et al. found no amino acid changes in the V3 loop of CC1/85 resistant to VCV generated in vitro. Wesby et al. reported a MVC-resistant CC1/85 virus generated in passage experiments with only 2 changes in amino acid positions 316 and 323 in the V3 loop . Anastassopoulou et al. described D1/86.16, a VCV escape mutant that has no mutations in V3 .
Several mutations outside the V3 loop were shown to contribute to CCR5 inhibitor resistance. Our results showed that I408T, L317W/I408T and V169M/L317W/I408T mutants had the highest impact on MVC susceptibility, mostly due to I408T in V4. This mutation could lower the activation energy needed to enable gp41 to undergo the next conformational changes and acquire a more stable low-energy state. All mutants retained the CCR5 co-receptor, supporting the concept that resistant viruses maintained the ability to use inhibitor-bound CCR5, depending on co-receptor density on the cellular surface and the degree of CCR5 co-receptor occupancy by drugs.
(HIV-1): Human immunodeficiency virus-1 entry; (V3): Variable domain 3; (IC50): Half maximal inhibitory concentration; (Env): Envelope protein; (CCR5): C-C chemokine receptor 5; (CXCR4): C-X-C chemokine receptor 4; (MVC): Maraviroc; (VCV): Vicriviroc; (MPI): As maximum percent inhibition; (FC): Measuring fold-change; (env): Envelope gene.
CT is the Pfizer/University of Montreal Chair in HIV Translational Research and a scholar from Fonds de la recherche en santé du Québec. The other authors declare no conflicts of interest.
OA-M participated in study conception and design, data collection, analysis and interpretation as well as manuscript drafting; AC supervised the study, analyzed and interpreted the data, and reviewed the manuscript; YW participated in study conception and design; AH participated in data collection; MS participated in data collection, analysis and interpretations; CT participated in study conception and design, data analysis and interpretation, study supervision, and manuscript review. All authors read and approved the final manuscript.