Determinants of human immunodeficiency virus type 1 baseline susceptibility to the fusion inhibitors enfuvirtide and T-649 reside outside the peptide interaction site

Abstract

The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-naïve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional env genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log(10) and 1.2 to 1.8 log(10) greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo.

FIG. 1.

PFI susceptibility of patient isolates and Env pseudotypes. The mean IC₅₀ for the virus isolates includes the original patient isolate, a regrown stock, and infected PBMC culture supernatants from days 5 and 14 postinfection. The mean IC₅₀ for the Env pseudotypes includes between 6 and 10 individual Envs produced by PCR amplification of genomic DNA from day 5 of a PBMC infection. Error bars indicate the standard error of the mean for each bar. All experiments were performed in triplicate in at least two independent experiments.

FIG. 2.

PFI susceptibility of patient-derived Envs relative to NLHX. The standard errors of the means ranged from 0.04 to 0.15 μg/ml for enfuvirtide and 0.12 to 0.24 μg/ml for T-649. The points on each curve represent the mean infectivity of between 6 and 10 individual Env pseudotypes at each drug concentration. Patient-derived Env pseudotypes are represented by dashed lines, while the Env pseudotypes derived from the lab strains NL4-3 and NLHX are represented by solid lines and filled symbols.

FIG. 3.

Amino acid sequence of the gp41 ectodomain of patient-derived Envs. A predicted amino acid alignment for the ectodomain of gp41 was generated for each virus isolate by using a consensus sequence representing the majority sequence for each set of patient-derived Envs. Parentheses indicate the number of individual Env clones that were sequenced and included in the consensus. The sequences of CONSENSUS_B (www.hiv.lanl.gov, 2002 HIV and SIV alignments) NL4-3, and NLHX are included for reference. The amino acid sequence of each patient-derived consensus sequence was analyzed for variation relative to NLHX. A dot indicates that the residue was conserved with NLHX, while the residues that are shown differed from NLHX in more than 50% of the patient-derived gp41 sequences. The amino acid sequence of each patient derived consensus sequence was also analyzed for variation relative to CONSENSUS_B. Letters in gray indicate residues that are conserved relative to the subtype B consensus but differ from NLHX. For consensus B, the residues in lowercase vary in subtype B viruses and the residues in uppercase are invariant in subtype B viruses. The heptad repeats (HR-1 and HR-2) are shown in bold, and the amino acids that compose the fusion peptide are underlined. The position of residues in HR-2 that corresponds to enfuvirtide and T-649 are indicated by dashed lines above the alignment. The numbering system is based on HXB2 gp41.

FIG. 4.

PFI susceptibility of gp120-gp41 chimeric Env pseudotyped viruses. Log₁₀ inhibitor concentration is shown on the x axis. Four types of Env pseudotypes were analyzed for each virus: the patient-derived Env, the NLHX Env, a patient-derived gp120 with the NLHX gp41 (PD gp120-NL gp41), and the NLHX gp120 with a patient-derived gp41 (NL gp120-PD gp41). Pseudotyped viruses containing the chimeric Env combination gp120/X23-gp41/NLHX could not be produced at a sufficiently high titer to be analyzed in these studies.