Mapping resistance to the CCR5 co-receptor antagonist vicriviroc using heterologous chimeric HIV-1 envelope genes reveals key determinants in the C2-V5 domain of gp120

Schering-Plough Research Institute, Department of Biological Sciences-VIROLOGY, K-15-4945, Kenilworth, NJ 07033, USA.
Virology (Impact Factor: 3.32). 05/2008; 373(2):387-99. DOI: 10.1016/j.virol.2007.12.009
Source: PubMed


Several small molecule drugs that bind to the host CCR5 co-receptor and prevent viral entry have been developed for the treatment of HIV-1 infection. The innate variability found in HIV-1 envelope and the complex viral/cellular interactions during entry makes defining resistance to these inhibitors challenging. Here we found that mapping determinants in the gp160 gene from a primary isolate RU570-VCV(res), selected in culture for resistance to the CCR5 entry inhibitor vicriviroc, was complicated by inactivity of the cloned envelope gene in pseudovirus assays. We therefore recombined the envelope from RU570-VCV(res) into a highly active and susceptible ADA gp160 backbone. The chimeric envelopes generated robust signals in the pseudovirus assay and a 200 amino acid fragment, encompassing a C2-V5 region of the RU570-VCV(res) envelope, was required to confer resistance in both the single-cycle assay and in replicating virus. In contrast, a chimeric envelope that contained only the V3-loop region from this resistant virus was completely susceptible suggesting that the V3-loop changes acquired are context dependent.

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Available from: Robert Ralston
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    • "Even under the selection pressure of the same drug, different strains appear to escape by mutating different Env sequences. For instance, Ogert et al. (2008, 2009) recently reported that K305R, R315Q and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the vicriviroc resistance phenotype in a chimeric strain ADA envelope. Others, however, showed that the substitutions K305R, H308P, A316V and G321E in the V3 loop or changes of G516V, M518V and F519I in the gp41 fusion peptide conferred vicriviroc resistance (Anastassopoulou et al., 2009; Berro et al., 2009). "
    Dataset: Hu's JGV

    Full-text · Dataset · May 2015
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    • "Although MVC and another CCR5 inhibitor, vicriviroc (VCV), can efficiently suppress HIV-1 replication, resistant variants can arise both in vitro and in vivo, and these resistant viruses are adapted to use drug-bound CCR5 for entry (Berro et al., 2009; Kuhmann et al., 2004; Marozsan et al., 2005; Ogert et al., 2009, 2010; Ratcliff et al., 2013; Roche et al., 2011b; Tilton et al., 2010; Tsibris et al., 2008; Westby et al., 2007; Yuan et al., 2011; Yusa et al., 2005). Current models of gp120 binding to a coreceptor suggest that the crown of the gp120 V3 loop interacts principally with the second extracellular loop region of the coreceptor, whilst the gp120 bridging sheet, which is formed after CD4 binding, and the stem of the V3 loop interact with the N terminus of the coreceptor (Brelot et al., 1999; Cormier & Dragic, 2002; Farzan et al., 1999; Huang et al., 2005). "
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    ABSTRACT: The aim of this study was to generate maraviroc (MVC)-resistant viruses in vitro using a subtype B clinical isolate (HIV-1KP-5) to understand the mechanism(s) of resistance to MVC. To select HIV-1 variants resistant to MVC in vitro, we exposed the CCR5 high expressing PM1/CCR5 cells to the HIV-1KP-5 followed by serial passage in the presence of MVC. We also passaged HIV-1KP-5 in PM1 cells, which were low expressing CCR5 to determine low CCR5 adapted substitutions and compared the Env sequences of the MVC-selected variants. Following 48 passages with MVC (10 µM), HIV-1KP-5 acquired a resistant phenotype (maximal percent inhibition [MPI]: 24%), while the low CCR5 adapted variant had low sensitivity to MVC (IC50: around 200 nM), but not reduction of the MPI. The common substitutions observed in both the MVC selected and low CCR5 adapted variants were selected from the quasispecies, in V1, V3 and V5. After 14 passages, the MVC selected variants harbored substitutions around the CCR5 N-terminal binding site and V3 (V200I, T297I, K305R and M434I). The low CCR5 adapted infectious clone became sensitive to anti-CD4bs and CD4i monoclonal antibodies (mAbs), but not to anti-V3 mAb and autologous plasma IgGs. Conversely, the MVC selected clone became highly sensitive to the anti-envelope mAbs tested and the autologous plasma IgGs. These findings suggest that the four MVC-resistant mutations required for entry using MVC-bound CCR5 result in a conformational change of envelope protein that is associated with a phenotype sensitive to anti-envelope neutralizing antibodies.
    Full-text · Article · May 2014 · Journal of General Virology
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    • "Treatment failures can occur because of an increasing number of pre-existing CXCR4-using viruses [5], [6]. Alternatively, escape mutants can evade a CCR5 inhibitor by accumulating multiple mutations in gp120 and/or gp41 without switching their coreceptor usage [7]–[14]. Escape mutants can use the drug-bound form of CCR5 as a coreceptor, a property known as noncompetitive resistance [8], [9], [11]. "
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    ABSTRACT: Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4(+) cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1V3-M5 derived from HIV-1JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.
    Full-text · Article · Jun 2013 · PLoS ONE
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