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, Oct 05, 2015
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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
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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.
    PLoS ONE 06/2013; 8(6):e65115. DOI:10.1371/journal.pone.0065115 · 3.23 Impact Factor
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    • "No signature pattern of common amino acid mutations has been identified that would predict resistance to CCR5 antagonists, such as that which has been noted for HIV-1 resistance to the fusion inhibitor enfuviritide. Rather, the V3 loop alterations responsible for resistance to CCR5 antagonists appear to be strain specific [31,37,44,46,47]. "
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    ABSTRACT: Background 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. Results 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. Conclusions 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.
    Retrovirology 04/2013; 10(1):43. DOI:10.1186/1742-4690-10-43 · 4.19 Impact Factor
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