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. 2009 Dec;83(23):12151-63.
doi: 10.1128/JVI.01351-09. Epub 2009 Sep 23.

Structure-function analysis of human immunodeficiency virus type 1 gp120 amino acid mutations associated with resistance to the CCR5 coreceptor antagonist vicriviroc

Robert A Ogert  1 , Lei Ba, Yan Hou, Catherine Buontempo, Ping Qiu, Jose Duca, Nicholas Murgolo, Peter Buontempo, Robert Ralston, John A Howe
Affiliations

Structure-function analysis of human immunodeficiency virus type 1 gp120 amino acid mutations associated with resistance to the CCR5 coreceptor antagonist vicriviroc

Robert A Ogert et al. J Virol. 2009 Dec.

Abstract

Vicriviroc (VCV) is a small-molecule CCR5 coreceptor antagonist currently in clinical trials for treatment of R5-tropic human immunodeficiency virus type 1 (HIV-1) infection. With this drug in development, identification of resistance mechanisms to VCV is needed to allow optimal outcomes in clinical practice. In this study we further characterized VCV resistance in a lab-adapted, VCV-resistant RU570 virus (RU570-VCV(res)). We show that K305R, R315Q, and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from RU570 passage control gp120. The K305R amino acid change primarily impacted the degree of resistance, whereas K319T contributed to both resistance and virus infectivity. The P437S mutation in C4 had more influence on the relative degree of virus infectivity, while the R315Q mutation contributed to the virus concentration-dependent phenotypic resistance pattern observed for RU570-VCV(res). RU570-VCV(res) pseudovirus entry with VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A, and Y15A mutations in the N terminus of CCR5, whereas these mutations had less impact on entry in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown region of the V3 loop enhanced resistance to VCV, resulting in a stronger dependence on the N terminus for viral entry. By fitting the envelope mutations to a molecular model of a recently described docked N-terminal CCR5 peptide consisting of residues 2 to 15 in complex with HIV-1 gp120 CD4, potential new interactions in gp120 with the N terminus of CCR5 were uncovered. The cumulative results of this study suggest that as the RU570 VCV-resistant virus adapted to use the drug-bound receptor, it also developed an increased reliance on the N terminus of CCR5.

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Figures

FIG. 1.
FIG. 1.
The RU570 chimeric envelope (ADA C2-V5pc) was generated by replacing the C2-V5 region of gp120 in the ADA envelope with the RU570 passage control sequence. (A) Schematic diagram depicting the RU570 C2-V5pc region encompassing amino acids 270 to 468 of gp120 (hatched bars) within the background of ADA gp160 (black bars). The four primary amino acid changes in the coreceptor binding region of RU570 gp120 associated with VCV resistance are delineated (in V3, K305R, R315Q, K319T; in C4, P437S). (B) Effect of forward mutations generated in the ADA C2-V5pc chimeric envelope on the relative infectivity of HIV-1 pseudoviruses. Pseudovirus stocks were normalized by p24, and 25 ng of p24 of each pseudovirus was used to infect U87-CD4-CCR5 cells. Data are representative of at least three independent experiments and represent the average ± standard deviations of four replicates. The relative change in expression is in relation to ADA C2-V5res pseudovirus. (C) Western blot detection of gp120 present in HIV-1 pseudoviruses generated with the ADA C2-V5pc envelope and ADA C2-V5pc envelopes containing forward mutations. Lane 1, ADA; lane 2, ADA C2-V5pc; lane 3, ADA C2-V5pc P437S; lane 4, ADA C2-V5pc K319T; lane 5, ADA C2-V5pc K319T/P437S; lane 6, ADA C2-V5pc K305R/K319T/P437S; lane 7, ADA C2-V5pc K305R/R315Q/K319T/P437S; and lane 8, ADA C2-V5res.
FIG. 2.
FIG. 2.
VCV dose-response curves for drug susceptibility assays performed in U87-CD4-CCR5 cells with HIV-1 pseudoviruses (amounts of p24 are as indicated by the symbols on the figure) generated with the following envelopes: ADA (A), ADA C2-V5pc K319T (B), ADA C2-V5pc K319T/P437S (C), ADA C2-V5pc K305R/K319T/P437S (D), ADA C2-V5pc K305R/R315Q/K319T/P437S (E), and ADA C2-V5res (G). In these assays, pseudovirus stocks were normalized by p24 levels, and equivalent amounts of pseudovirus per well were added to U87-CD4-CCR5 cells. Data were analyzed using nonlinear regression, four-parameter logistic curve fit analysis with GraphPad Prism software, version 4.0, and are representative of at least three independent assays. Data represent the average ± standard deviation of four replicates. The insets for panels A to E and G depict the linear dose response observed with increasing amounts of HIV-1 pseudovirus input using the p24-normalized stocks. (F) Dose-response curves for VCV susceptibility assays performed in U87-CD4-CCR5 cells using pseudovirus stocks normalized by p24 and cells infected with 25 ng of p24 of each pseudovirus stock per well. Data represent the average ± standard deviation of four replicates. (H) VCV dose-response curves generated using the laboratory-adapted RU570-VCVres and RU570 passage control virus from the original PM-1 cultures. Replicating virus was analyzed in a single-cycle virus infection assay as described in the Materials and Methods section using RU570-VCVres virus and RU570 passage control virus (inocula are as indicated on the figure and are given as RNA copies). The 50% effective concentration and MPI values for the passaged viruses were determined using nonlinear regression, four-parameter logistic curve fit analysis with GraphPad Prism software. Data are representative of the average ± standard deviation of three replicates and three independent experiments.
FIG. 3.
FIG. 3.
The effect of alanine substitutions in the N terminus of CCR5 (Y3A, Y10A, D11A, N13A, Y14A, and Y15A and a Δ2-17 N-terminal mutant) and alanine substitutions in ECL2 (R168A, K171A, E172A, L174A, and C178A) on HIV-1 pseudovirus entry. HIV-1 pseudovirus infection is expressed as a percentage of wt CCR5 infection in the absence (left panel) and presence of 1.0 μM VCV (right panel). (A) ADA HIV-1 pseudovirus. (B) ADA C2-V5pc K319T HIV-1 pseudovirus. (C) ADA C2-V5res HIV-1 pseudovirus. (D) ADA C2-V5pc K305R/R315Q/K319T/P437S HIV-1 pseudovirus. Data represent the average ± standard deviation of at least five independent assays.
FIG. 4.
FIG. 4.
RU570-VCVres pseudoviruses are more sensitive to neutralization with a MAb specific to the N terminus of CCR5 in the presence of VCV. The sensitivity of ADA C2-V5res (A) and ADA C2-V5pc K305R/R315Q/K319T/P437S (B) HIV-1 pseudoviruses to neutralization by MAb CTC5 in the absence (−) or presence (+) of VCV was determined in U87-CD4-CCR5 cells. Data are representative of two independent assays and represent the percent inhibition of control infection without MAb (mean ± standard deviation; n = 4). Ab, antibody.
FIG. 5.
FIG. 5.
VCV dose-response curves for drug susceptibility assays performed in U87-CD4-CCR5 cells with 5 ng of p24 HIV-1 pseudovirus (A) and 25 ng of HIV-1 pseudovirus (B). HIV-1 pseudoviruses were generated with the envelopes indicated on the figure. In these assays, pseudovirus stocks were normalized by p24 levels, and equivalent amounts of pseudovirus per well were added to U87-CD4-CCR5 cells. Data were analyzed using nonlinear regression, four-parameter logistic curve fit analysis with GraphPad Prism software, version 4.0, and are representative of at least three independent assays.
FIG. 6.
FIG. 6.
The effect of alanine substitutions in the N terminus of CCR5 (Y3A, Y10A, D11A, N13A, Y14A, and Y15A) and of the Δ2-17 N-terminal CCR5 mutant transiently expressed with CD4 in 293T cells on pseudovirus entry for HIV-1 ADA C2-V5res Q315E/I317F pseudoviruses. Pseudoviruses were analyzed for infectivity in the presence (right panel) or absence (left panel) of 1.0 μM VCV, and data are expressed as a percentage of wt CCR5 infection. Data represent the average ± standard deviation of at least 3 independent assays.
FIG. 7.
FIG. 7.
Molecular model of the V3 loop and C4 domain of RU570-VCVres gp120 and the N-terminal CCR5 peptide based on coordinates obtained from Huang et al. (22) depicting the interaction between D11 in the N terminus of CCR5 with K305 in RU570 passage control gp120 (A) or R305 in RU570-VCVres gp120 (B). (C) The modeled interaction for Asn 13 in the N terminus of CCR5 and RU570-VCVres gp120.
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References

    1. Anastassopoulou, C. G., T. J. Ketas, P. J. Klasse, and J. P. Moore. 2009. Resistance to CCR5 inhibitors caused by sequence changes in the fusion peptide of HIV-1 gp41. Proc. Natl. Acad. Sci. USA 106:5318-5323. - PMC - PubMed
    1. Baba, M., H. Miyake, X. Wang, M. Okamoto, and K. Takashima. 2007. Isolation and characterization of human immunodeficiency virus type 1 resistant to the small-molecule CCR5 antagonist TAK-652. Antimicrob. Agents Chemother. 51:707-715. - PMC - PubMed
    1. Blanpain, C., B. Lee, J. Vakili, B. J. Doranz, C. Govaerts, I. Migeotte, M. Sharron, V. Dupriez, G. Vassart, R. W. Doms, and M. Parmentier. 1999. Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity. J. Biol. Chem. 274:18902-18908. - PubMed
    1. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273. - PubMed
    1. Chan, D. C., and P. S. Kim. 1998. HIV entry and its inhibition. Cell 93:681-684. - PubMed