Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor

Abstract

T-20 is a synthetic peptide that corresponds to 36 amino acids within the C-terminal heptad repeat region (HR2) of human immunodeficiency virus type 1 (HIV-1) gp41. T-20 has been shown to potently inhibit viral replication of HIV-1 both in vitro and in vivo and is currently being evaluated in a Phase III clinical trial. T-649 is an inhibitory peptide that also corresponds to 36 amino acids within HR2. This sequence overlaps the T-20 sequence but is shifted 10 residues toward the N terminus of gp41. Both inhibitors are thought to exert their antiviral activity by interfering with the conformational changes that occur within gp41 to promote membrane fusion following gp120 interactions with CD4 and coreceptor molecules. We have shown previously that coreceptor specificity defined by the V3 loop of gp120 modulates sensitivity to T-20 and that a critical region within the N-terminal heptad repeat (HR1) of gp41 is the major determinant of sensitivity (C. A. Derdeyn et al., J. Virol. 74:8358-8367, 2000). This report shows that (i) regions within gp41 distinct from those associated with T-20 sensitivity govern the baseline sensitivity to T-649 and (ii) T-649 sensitivity of chimeric viruses that contain sequences derived from CXCR4- and CCR5-specific envelopes is also modulated by coreceptor specificity. Moreover, the pattern of sensitivity of CCR5-specific chimeras with only minor differences in their V3 loop was consistent for both inhibitors, suggesting that the individual affinity for coreceptor may influence accessibility of these inhibitors to their target sequence. Finally, an analysis of the sensitivity of 55 primary, inhibitor-naive HIV-1 isolates found that higher concentrations of T-20 (P < 0.001) and T-649 (P = 0.016) were required to inhibit CCR5-specific viruses compared to viruses that utilize CXCR4. The results presented here implicate gp120-coreceptor interactions in driving the complex conformational changes that occur in gp41 to promote fusion and entry and suggest that sensitivity to different HR1-directed fusion inhibitors is governed by distinct regions of gp41 but is consistently modulated by coreceptor specificity.

FIG. 1

The HIV-1 env gene. (A) The HIV-1 env gene produces surface (gp120) and transmembrane (gp41) glycoprotein subunits from a precursor molecule through posttranslational proteolytic cleavage. The five variable regions of gp120 and a conserved CD4 binding site are indicated. The enlarged inset depicts the major structural domains of gp41 (not to scale). Amino acid alignments of the N-terminal (B) and C-terminal (C) heptad repeat regions are shown for HXB2 (used as the reference sequence), NL4.3, and JRFL. Residues that are identical to the HXB2 sequence are indicated by dashes, and substitutions are indicated by the amino acid letter. (B) The T-20 interaction site in HR1, residues 36 to 38, is shown in bold. (C) The corresponding positions of T-20 and T-649 in HR2 are indicated above the alignments. Both peptides are identical to the HXB2 sequence.

FIG. 2

Chimeric proviruses containing substitutions in env. All proviruses were created in an NL4.3 proviral background. Constructs designated NLHX contain HXB2-derived env sequences. V3, the gp120 V3 loop; GIV, DIV, or SIM, the residues comprising the critical T-20 interaction site. Restriction sites used for construction are shown. Shown are NL4.3 sequences, HXB2 sequences, JRFL sequences, and sequences from CCR5-specific V3 loop donors ADA, SF2, and SF162.

FIG. 3

Effect of coreceptor utilization on sensitivity to fusion inhibitors. JC53-BL indicator cells were infected with each chimeric virus in the presence of increasing concentrations of T-649 (left) or T-20 (right). Inhibitor concentrations are plotted along the horizontal axes. Luciferase activities in the infected cell lysates were measured at 48 h postinfection and were used to calculate virus infectivity relative to that of the control (vertical axes) and IC₅₀s for each inhibitor. (A and B) Comparison of the infectivity of NL4.3 (DIV) and the JRFL-derived CCR5 V3 loop chimeras in the presence of T-649 and T-20, respectively. (C and D) Comparison of the infectivity of NLHX and the four CCR5-specific V3 loop chimeras in the presence of T-649 and T-20, respectively. (E and F) Comparison of the infectivity of two additional NL4.3-derived viruses that differed only in substitution of JRFL V3 loop in the presence of T-649 and T-20, respectively. (G) Chimeric constructs that correspond to the infectivity graphs.

FIG. 4

Effect of coreceptor utilization in primary cells and in the presence of nevirapine. PBMC were infected with two isogenic CXCR4-CCR5 pairs of chimeric viruses in the presence of increasing concentrations of T-649 (A) or T-20 (B), which are plotted along the horizontal axes. Production of HIV-1 p24 in the supernatant was measured 7 days later and was used to calculate relative virus infectivity (vertical axes) and IC₅₀s for T-649 and T-20. (C) JC53-BL cells were infected with the same virus pairs in the presence of increasing concentrations of the nonnucleoside reverse transcriptase inhibitor nevirapine (horizontal axis), which targets the pol gene. Luciferase activity was measured 48 h later and was used to calculate virus infectivity (plotted along the vertical axes). (D) Pairs of chimeric constructs shown in the infectivity graphs.

FIG. 5

Effect of changes within the GIV motif on inhibition by T-649 and T-20. JC53-BL cells were infected with each chimeric virus in the presence of increasing concentrations of T-649 (A) or T-20 (B), which are plotted along the horizontal axes. Luciferase activity was measured at 48 h postinfection and was used to calculate the virus infectivity relative to the control, which is plotted along the vertical axes. Shown are the infectivities of NL4.3-based isogenic viruses containing DIV, GIV, or SIM in the T-20 interaction site (residues 36 to 38) in the presence of each inhibitor.

FIG. 6

Sensitivity of CCR5 NSI and CXCR4 SI primary isolates to fusion inhibitors. JC53-BL cells were infected with 29 CCR5 NSI (A) and 26 CXCR4 SI (B) patient isolates in the presence of increasing doses of T-20 or T-649. Luciferase activity was measured at 48 h postinfection, and the relative infectivities for each virus were used to calculate the IC₅₀s for T-20 and T-649 (vertical axis). The mean (±SD) IC₅₀ for T-20 was 0.20 ± 0.18 μg/ml for CCR5 viruses and 0.10 ± 0.15 μg/ml for CXCR4 viruses. The mean (±SD) IC₅₀ for T-649 was 0.10 ± 0.06 μg/ml for CCR5 viruses and 0.07 ± 0.05 μg/ml for CXCR4 viruses.