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. 2000 Sep;74(18):8358-67.
doi: 10.1128/jvi.74.18.8358-8367.2000.

Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120

C A Derdeyn  1 , J M Decker, J N Sfakianos, X Wu, W A O'Brien, L Ratner, J C Kappes, G M Shaw, E Hunter
Affiliations

Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120

C A Derdeyn et al. J Virol. 2000 Sep.

Abstract

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC(50)) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log(10) higher than the mean IC(50) for CXCR4 (X4) isolates (P = 0. 0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC(50) for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log(10) higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC(50) for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log(10) lower than the IC(50) for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.

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Figures

FIG. 1
FIG. 1
Sensitivity of HIV-1 primary isolates to inhibition by T-20. JC53-BL HIV-1 indicator cells were infected with 2,000 infectious units of each primary isolate in the absence (control) or presence of increasing doses of T-20. Cells were lysed at 48 h postinfection, and luciferase activity was measured. Virus infectivity, calculated by dividing the infectivity at each concentration of T-20 by the infectivity of the control, is plotted along the vertical axis on a linear scale; T-20 concentration is plotted along the horizontal axis on a log10 scale. Each curve represents the inhibition profile of an individual viral isolate. X4 viruses are represented by filled symbols, and R5 viruses are represented by open symbols. Each point represents the mean infectivity calculated from two independent experiments, each with duplicate wells. The range of T-20 IC50 for all isolates was 0.029 to 0.982 μg/ml (1.5 log10).
FIG. 2
FIG. 2
Organization of the HIV-1 envelope gene and the panel of chimeric envelope constructs used to define determinants of T-20 sensitivity. (A) Two envelope glycoproteins, gp120 and gp41, are proteolytically processed from a precursor molecule, gp160. V3, one of five variable loops in gp120, contains the major determinants of coreceptor specificity. “CD4” indicates a conserved region involved in binding to the CD4 receptor. gp41 contains an extracellular domain, an MSD, and an intracellular cytoplasmic tail. The extracellular domain contains several conserved regions that are important for fusion and entry: the fusion peptide, the N-terminal heptad repeat (HR1), and the C-terminal heptad repeat (HR2). The 36-amino-acid T-20 peptide sequence corresponds to residues within the C-terminal half of HR2. (B) All chimeras were constructed in an NL4.3 proviral background. “V3” indicates the gp120 V3 loop; “GIV or DIV” represents the critical amino acid sequence of the T-20 interaction site in the N-terminal heptad repeat (HR1). NLHX-based constructs contain the HXB2-derived envelope in an NL4.3 proviral background. Wild-type NL4.3 represents two constructs: NL4.3 and NL(Stu)Xba. NL4.3 sequences are represented by gray boxes, and the chimeric envelope sequences of JRFL, HXB2, ADA, SF2, and SF162 are indicated in the key.
FIG. 3
FIG. 3
V3 loop and HR1 amino acid sequence alignments. HIV-1 NL4.3 is used as the reference sequence. Conserved amino acids are indicated as dashed lines, and residues that differ from the NL4.3 sequence are indicated by amino acid letter. Underscores represent gaps in the sequence. (A) Predicted amino acid sequences for the V3 loops (residues 294 to 329 in NL4.3 gp120) used in this study. HXB2 and NL4.3 are X4 specific; ADA, ADA V3B, JFRL, SF162, and SF2 are R5 specific. (B) Differences in the predicted amino acid sequences for the highly conserved HIV-1 HR1 regions (residues 32 to 75 of NL4.3 gp41) used in this study are shown. The critical T-20 interaction site (residues 36 to 38) is shown in boldface.
FIG. 4
FIG. 4
Contribution of coreceptor specificity to inhibition by T-20. Cells were infected with 2,000 infectious units of each chimeric virus stock in the absence (control) or presence of increasing doses of T-20. Virus infectivity is plotted along the vertical axis on a linear scale, and T-20 concentration is plotted along the horizontal axis on a log10 scale. Each curve represents the inhibition profile of an individual virus. (A) Luciferase activity was measured at 48 h in infected JC53-BL cells. Each point represents the mean relative infectivity calculated from two independent experiments, each with duplicate wells. The results were confirmed by one experiment in which blue foci were counted (data not shown). The range of IC50s of T-20 for the NLHX-based chimeras was 0.005 to 0.0.032 μg/ml. (B) Luciferase activity was measured at 48 h in infected JC53-BL cells. Each point represents the mean infectivity calculated from four independent experiments, each with duplicate wells. The results were confirmed by two experiments in which blue foci were counted (data not shown). The range of IC50s of T-20 for the NL4.3/JRFL V3 loop chimeras was 0.120 to 0.443 μg/ml. (C) PBMC were infected with a subset of the NL4.3/JRFL envelope chimeras, and supernatant p24 was measured on day 7 by ELISA. Each point represents the mean infectivity calculated from one experiment with duplicate wells. The range of IC50s of T-20 for the NL4.3/JRFL V3 loop chimeras was 0.024 to 0.109 μg/ml. (D) Chimeric constructs used in this analysis.
FIG. 5
FIG. 5
Contribution of HR1 to T-20 sensitivity. JC53-BL indicator cells were infected with 2,000 infectious units of each chimeric virus stock in the absence (control) or presence of increasing doses of T-20. At 48 h postinfection, cells were lysed and luciferase activity was measured. Virus infectivity is plotted along the vertical axis on a linear scale, and T-20 concentration is plotted along the horizontal axis on a log10 scale. (A) Each point represents the mean infectivity calculated from two independent experiments, each with duplicate wells. The results were confirmed by one experiment in which blue foci were counted (data not shown). The range of IC50s of T-20 for the NL4.3/HXB2/JRFL chimeras was 0.005 to 0.130 μg/ml. (B) Each point represents the mean infectivity calculated from four independent experiments, each with duplicate wells. The results were confirmed by two experiments in which blue foci were counted (data not shown). The range of IC50s of T-20 for these NL4.3/JRFL chimeras was 0.008 to 0.056 μg/ml. (C) Chimeric constructs used in this analysis.
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