Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies

Abstract

Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWT from which cenicriviroc-resistant strain KK652-67 was obtained was resistant to these NAbs. The V3 region of KK652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other.

FIG 1

The CVC-resistant virus KK_652-67 was resistant to CVC but sensitive to the other CCR5 antagonists tested. Infectious viruses KK_WT and CVC-resistant variant KK_652-67 were examined for their sensitivity to the CCR5 antagonists CVC, TAK-779, TAK-220, and MVC using TZM-bl cells (first row). Data are presented as the mean and standard deviation from three independent experiments. Sensitivities to these CCR5 antagonists were also examined using pseudovirus clones with Env from KK_WT (second row) and KK_652-67 (third row). Inhibition assays using pseudovirus clones were performed at least twice, and representative results are shown with the mean plot for infectious viruses KK_WT and KK_652-67 (dashed lines).

FIG 2

The CVC-resistant virus KK_652-67 was sensitive to neutralization by antibodies against V3, CD4i, and CD4bs. Infectious viruses KK_WT and CVC-resistant variant KK_652-67 were examined for their sensitivity to NAbs 0.5γ (V3), 4E9C (CD4i), 49G2 (CD4bs), VRC01 (CD4bs), and 10E8 (gp41 MPER) using TZM-bl cells (first row). Data are presented as the mean and standard deviation from three independent experiments. Sensitivities to these NAbs were also examined using pseudovirus clones with Env from KK_WT (second row) and KK_652-67 (third row). Inhibition assays using pseudovirus clones were performed at least twice, and representative results are shown with the mean plot for infectious viruses KK_WT and KK_652-67 (dashed lines).

FIG 3

Amino acid substitutions required for CVC resistance and NAb sensitivity. (A) Schematic of Env recombinants constructed between KK_WT (clone T174A) and KK_652-67 (clone CS). The regions from KK_WT and KK_652-67 are shown in light blue and red, respectively. Recombinants with the C2-V5 amino acid substitutions of KK_652-67 in the backbone of WCW are shown at the bottom. (B) The sensitivities of recombinant pseudoviruses CWC and WCW to CVC and NAbs 0.5γ (V3), 4E9C (CD4i), and 49G2 (CD4bs) along with those of KK_WT (clone T174A) and KK_652-67 (clone CS) are shown. The C2-V5 region was found to be responsible for CVC resistance and sensitivity to NAbs. (C) The locations of amino acid substitutions in the C2-V5 region of KK_652-67 (clone CS) are shown by red residues with side chains in the gp120 structure (PDB accession number 2QAD). The substitutions were separated into four groups, V3, V4, C4, and C2/V4/V5, according to the regions in gp120. (D) The sensitivities of mutant pseudoviruses CR-V3V4C4, V3, and V4C4 to CVC and NAbs 0.5γ (V3), 4E9C (CD4i), and 49G2 (CD4bs) along with those of KK_WT (clone T174A) and KK_652-67 (clone CS) are shown. The substitutions in the V3 region from CVC-resistant KK_652-67 determined the sensitivity to NAbs, but additional substitutions in the C2-V5 region were required for CVC resistance.

FIG 4

Acquisition of resistance to anti-V3 NAb 0.5γ resulted in reversion to the CVC-sensitive phenotype. Anti-V3 NAb 0.5γ-resistant variant KK_{652-67/0.5γ-7} was obtained from CVC-resistant strain KK_652-67 by in vitro passages in the presence of 0.5γ. (A) The sensitivities of KK_{652-67/0.5γ-7} and KK_652-67 to CVC and 0.5γ are shown (top row). Data are presented as the mean and standard deviation from three independent experiments. The sensitivities of pseudovirus clones of KK_{652-67/0.5γ-7} to CVC and NAb 0.5γ are also shown (bottom row). Inhibition assays using pseudovirus clones were performed at least twice, and representative results are shown along with the mean plot for infectious viruses KK_{652-67/0.5γ-7}, KK_652-67, and KK_WT. (B) The reactivity of NAb 0.5γ to KK_{652-67/0.5γ-7} clones 86 (0.5γ sensitive) and 89 (0.5γ resistant) was examined by flow cytometry. Clone 89 did not react with 0.5γ at all. Anti-CD4bs NAb 49G2 was used as a positive control. none, no antibody.

FIG 5

The V3 region was responsible for the phenotypic change of the 0.5γ-resistant variant. Clones containing Env from the anti-V3 NAb 0.5γ-resistant variant KK_{652-67/0.5γ-7} and recombinants between KK_652-67 and KK_{652-67/0.5γ-7} were analyzed for their sensitivities to CVC and 0.5γ to determine the amino acid substitutions responsible for the phenotypic change to 0.5γ resistance and CVC sensitivity. (A) Amino acid substitutions in KK_{652-67/0.5γ-7} compared with the sequence of KK_652-67 and sensitivities to CVC and 0.5γ are summarized. Amino acid substitutions in the KK_{652-67/0.5γ-7} clones which are different from the consensus sequence of KK_652-67 and their frequencies in KK_WT, KK_652-67, and KK_{652-67/0.5γ-7} are shown. Substitutions are shown by one-letter amino acid codes, and the major substitutions in KK_652-67 and KK_{652-67/0.5γ-7} are shown in pink and green, respectively. S315K is shown in yellow. Sensitivities to CVC and 0.5γ are shown as MPI and IC₅₀ (in micrograms per milliliter) values, respectively. The results indicating a resistant phenotype are shown in orange. (B) The sensitivities of pseudovirus clones consisting of recombinants between KK_652-67 (clone CS) and KK_{652-67/0.5γ-7} (clone 89) to CVC and NAb 0.5γ are shown. Recombinant R616, which contained the V3-C4 region of KK_{652-67/0.5γ-7} in the backbone of KK_652-67, was sensitive to CVC and resistant to 0.5γ, similar to the findings for the KK_{652-67/0.5γ-7} clone 89 (KK_{652-67/0.5γ-7#89}; top row). The sensitivities of pseudovirus clones with point mutations are shown in the bottom row. Inhibition assays were performed at least twice, and representative results are shown. Amino acid substitutions R315K and G324R in the V3 region contributed to the 0.5γ resistance and the reversion to CVC sensitivity.

FIG 6

Acquisition of resistance to anti-CD4i NAb 4E9C resulted in reversion to the CVC-sensitive phenotype. Anti-CD4i NAb 4E9C-resistant variant KK_{652-67/4E9C-11} was obtained from CVC-resistant strain KK_652-67 by in vitro passages in the presence of 4E9C. (A) The sensitivities of KK_{652-67/4E9C-11} (left) and the two KK_{652-67/4E9C-11} clones (right) to CVC and 4E9C along with those of KK_WT and KK_652-67 are shown. Data for uncloned viruses are presented as the mean and standard deviation from three independent experiments. Inhibition assays using pseudovirus clones were performed at least twice, and representative results are shown. The mean plots for uncloned viruses KK_WT, KK_652-67, and KK_{652-67/4E9C-11} are shown as dashed lines. (B) The sensitivities of pseudovirus clones consisting of recombinants between KK_652-67 (clone CS) and KK_{652-67/4E9C-11} (clone 10) to CVC and 4E9C are shown. Inhibition assays were performed at least twice, and representative results are shown. Recombinant R646, which contained the C2-V5 region of KK_{652-67/4E9C-11} in the backbone of KK_652-67, was sensitive to CVC and resistant to 4E9C, similar to the findings for KK_{652-67/4E9C-11} (left). The sensitivities of pseudovirus clones with point mutations are shown in the right column. The amino acid substitutions G324R and E381K contributed to 4E9C resistance and reversion to CVC sensitivity. (C) Amino acid substitutions in KK_{652-67/4E9C-11} compared with the sequence of KK_652-67 and sensitivities to CVC and 4E9C are summarized. Amino acid substitutions in the KK_{652-67/4E9C-11} clones which are different from the consensus sequence of KK_652-67 and their frequencies in KK_WT, KK_652-67, and KK_{652-67/4E9C-11} are shown. Substitutions are shown by one-letter amino acid codes, and the major substitutions in KK_{652-67/4E9C-11} are shown in magenta. Sensitivities to CVC and 4E9C are shown as MPI and IC₅₀ (in micrograms per milliliter) values, respectively. The results indicating a resistant phenotype are shown in orange. NA, not applicable.

FIG 7

Resistance to CVC is incompatible with resistance to antibodies. (A) A schematic of the relationship between CVC and NAb resistance. Acquisition of CVC resistance rendered the virus sensitive to NAbs, and further selection with NAbs resulted in reversion to a CVC-sensitive phenotype. (B) The locations of amino acid substitutions in the gp120 structure (PDB accession number 2QAD) critical to NAb resistance and reversion to the CVC-sensitive phenotype are shown. Two substitutions in the V3 region, G324R and R315K, were responsible for 0.5γ resistance and the reversion of CVC sensitivity in KK_{652-67/0.5γ-7}. Two substitutions, G324R and E381K, contributed to both 4E9C resistance and CVC sensitivity in KK_{652-67/4E9C-11}. (C) Alignment of the amino acid sequences of the V3 loop of KK_WT, KK_652-67, KK_{652-67/0.5γ-7}, and KK_{652-67/4E9C-11}. Only changes from the KK_WT sequence are shown, and dots indicate residues that are the same as those in KK_WT.