V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies

Abstract

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.

Figure 1. HIV-1 Env Constructs and the Effects of Env Variable Loop Deletions on Fusion Activity

(A) A schematic representation of the R3A, ΔV1/V2 (deletion of residues 126–196, but retains the cysteines at the base of the loops), ΔV3(9,9), and TA1 constructs is shown. Amino acid changes in the adapted TA1 Env are indicated, including R254K and T342A in gp120, a mutation in the three amino acid linker introduced into the truncated V3 loop (GAG to GVG), a deletion of residues 185–188 in the V2 loop, and an A to V mutation in the amino terminal residue of gp41. FP, fusion peptide. (B) The effects of Env variable loop deletions on coreceptor use was determined in cell–cell fusion assays, in which cells expressing the indicated Envs were incubated with cells expressing CD4 and CXCR4 (white bars) or CCR5 (black bars). The results are expressed as a percentage of parental R3A Env, and represent the average of 21 independent experiments.

Figure 2. Replication of Virus with R3A Parental, Mutant, and Adapted Envs in Supt1.CCR5.DCSIGNR Cells

Cells were infected with 5 or 50 ng of the indicated replication competent virus, and culture supernatants sampled at the times indicated for RT activity. Data are representative of two independent experiments.

Figure 3. Sensitivity of Parental, Mutant, and Adapted Envs to Neutralization by HIV Patient Sera

(A–D) Serum from four HIV-infected individuals who were not on antiretroviral therapy and had CD4 counts >400 were tested for neutralization activity by limiting dilution. Virus pseudotypes bearing the indicated Env were incubated with serial dilutions of patient sera for 1 h, added to 293.CD4.CCR5 cells, and the amount of luciferase activity measured 3 d later. The amount of luciferase activity obtained for each virus pseudotype in the absence of serum was set to 100%. The results shown are the average for three or four independent experiments, ± standard error of the mean.

Figure 4. Sensitivity of Viruses to Neutralization by MAbs

Luciferase reporter viruses pseudotyped with parental, mutant, or adapted Envs as indicated were incubated with various concentrations of the MAbs (A) IgG b12, (B) 4E10, or (C) 17b for 1 h prior to infection of 293.CD4.CCR5 cells. The amount of luciferase activity obtained for each virus pseudotype in the absence of any antibody was set to 100%. The results shown are the average for three independent experiments, ± standard error of the mean.

Figure 5. Relative Sensitivity of Parental, Mutant, and Adapted Envs to Entry Inhibitors

Pseudotyped luciferase reporter viruses were used to determine sensitivity to the fusion inhibitor (A) enfuvirtide, or the CCR5 inhibitors (B) aplaviroc, (D) AD101, and (F) CMPD167. Sensitivity of Envs to (C) aplaviroc, (E) AD101, and (G) CMPD167 was determined in a cell–cell fusion luciferase reporter assay, using QT6 effector cells expressing the indicated Env and QT6 target cells expressing CD4 and CCR5. Results are the average of three or four independent experiments, ± standard error of the mean.

Figure 6. Altered Dependence on CCR5 N-Terminus for Viral Entry

(A) Luciferase reporter viruses pseudotyped with R3A or TA1 were incubated with various concentrations of the MAb CTC5 for 1 h prior to infection of 293.CD4.CCR5 cells. The amount of luciferase activity obtained for each virus pseudotype in the absence of any antibody was set to 100%. The results shown are the average for three independent experiments, ± standard error of the mean. (B) Luciferase reporter viruses pseudotyped with R3A (white bars) or TA1 (black bars) Env were incubated with 293T cells expressing CD4 only, CD4 and CCR5, or CD4 and mutant CCR5, as indicated. Δ2–17 refers to a CCR5 construct lacking amino acids 2–17. Single amino acid mutants refer to changes in the CCR5 N-terminus. The amount of luciferase activity obtained for each virus pseudotype on WT CCR5 was set to 100%. The results shown are the average for three independent experiments, ± standard error of the mean.