RT Journal Article
SR Electronic
T1 Characterization of resistance to a potent D-peptide HIV entry inhibitor
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 748111
DO 10.1101/748111
A1 Amanda R. Smith
A1 Matthew T. Weinstock
A1 Amanda E. Siglin
A1 Frank G. Whitby
A1 J. Nicholas Francis
A1 Christopher P. Hill
A1 Debra M. Eckert
A1 Michael J. Root
A1 Michael S. Kay
YR 2019
UL http://biorxiv.org/content/early/2019/08/28/748111.abstract
AB 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 combat resistance via stepwise accumulation of modest affinity-disrupting mutations. 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. Identified candidate compensatory mutations in gp160 will be the focus of future mechanistic studies.