Characterization of resistance to a potent D-peptide HIV entry inhibitor

Abstract

Background: PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket.

Results: Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data.

Conclusions: These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.

Keywords: Compensatory mutation; D-peptide; HIV entry inhibition; Hydrophobic pocket; PIE12-trimer; Viral fitness; Viral passaging; Viral resistance mutation; gp120; gp41.

Fig. 1

Representative inhibitory curves of replication-competent NL4-3 (WT) and PIE12-trimer resistant viruses. The polyclonal viral pools (W1 and W2) show slightly enhanced resistance compared to clonal virus expressing Env with Q577 point mutations. Representative normalized data are shown from a single assay with triplicate measurements (error bars show standard error). IC₅₀ values of this representative data set are indicated in parentheses

Fig. 2

Protein interaction analysis of Q577R mutation. SPR equilibrium binding data (in triplicate) for interaction between immobilized IZN36 (WT or Q577R) and PIE12 monomer. The calculated K_D values are 0.031 µM for WT and 2.0 µM for Q577R

Fig. 3

Crystal structures of PIE12/IQN17 (Q577R) (PDB: 6PSA) and PIE12/IQN17 (WT PDB:3L37). Stereoview of the overlay of crystal structures of PIE12 binding to the WT (yellow) and a mutant (Q577R, turquoise) pocket showing the disruption of hydrogen bonding interactions mediated by Q577

Fig. 4

Viral Growth Assays. Comparison of growth kinetics between control clonal NL4-3 (WT), Q577 mutant viruses (R, N and K), and resistant polyclonal viral pools (W1 and W2). Viral titers were determined by qRT-PCR and compared to a standard curve of known HIV-1 titers (based on p24 antigen levels). Each culture was inoculated with virus containing 1 ng p24 (average of biological duplicate assays shown with s.d. error bars)

Fig. 5

Prominent mutations in gp120 and gp41 observed in PIE12-trimer resistant viruses. The primary structure of the Env precursor, gp160, is depicted, with HXB2 (UniProtKB P04578) numbering. The gp41 N- and C-peptide regions are numbered according to [45]. gp160 is processed into two non-covalently associated subunits, the surface gp120 (orange) and the membrane-spanning gp41 (blue). gp120 comprises five constant domains (C1–C5) and five variable domains (V1-V5). gp41 contains a fusion peptide (FP), an N-peptide region that forms the N-trimer coiled coil (N), a C-peptide region (C) that together with the N-trimer forms a 6-helix bundle in the post-fusion state, a membrane-proximal external region (MPER), a transmembrane domain (TM), and a cytoplasmic tail (CT). The primary resistance mutations (Q577R/N/K) are represented in red, and candidate resistance and fitness compensation mutations are shown in black. Mutations denoted by * are within the Rev Response Element (RRE)