Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

Abstract

SC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors. T20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

“…Refolding of the gp41 NHR and CHR into a 6-HB conformation drives the viral and cellular membranes in close apposition and releases energy to overcome the kinetic barrier required for virus-cell membrane fusion. A body of evidence demonstrates that HIV-1 resistance to various peptide-based fusion inhibitors is mediated by genetic mutations in the NHR sequence, especially within the inhibitor-binding sites [13,15,[23][24][25][26][27][28][29]33,34]. Notably, some CHR mutations were frequently observed during escape selection and in T20-containing clinical therapy, such as N126K, E136G, E137K, S138A, and E154K [27][28][29][30]33,[35][36][37][38][39][40][41][42].…”

“…While other CHR mutations were often induced by a specific peptide, the N126K mutation was routinely selected by various fusion inhibitors. In this regard, we previously characterized the resistance profiles of the C34 derivatives SFT and SC29EK as well as the small inhibitors SC22EK and MTSC22EK, and all the resistant HIV-1 mutant viruses developed the N126K mutation [23][24][25][26]. For the primary NHR mutations, Eggink and coworkers described several resistance pathways, including large amino acid-mediated steric obstruction, small amino acid-mediated reduced contact, basic amino acid-mediated electrostatic attraction, and acidic amino acid-mediated electrostatic repulsion [43]; we also proposed the other two NHR-mediated resistance pathways: the disruption of hydrogen bonds and hydrophobic contacts would severely impair the binding of inhibitors thus determining the resistance [44].…”

“…By adding the M-T hook structure to the PBD sequence, we previously generated several highly potent short-peptide fusion inhibitors, including MTSC22EK, HP23, and 2P23, which mainly target the deep pocket sites rather than the T20 resistance sites [20][21][22]. As noted, the resistance profiles of both the C34 derivatives and M-T hook-based short peptides were previously characterized by the in vitro selection of HIV-1 variants that were resistant to the inhibitors, and interestingly, the secondary N126K mutation was universally found to accompany with the diverse primary resistance mutations [23][24][25][26][27][28][29][30]. In the CHR sequence, Asn126 locates immediately at the downstream of the PBD sequence and it is a highly conserved residue among all the HIV-1, HIV-2, and even simian immunodeficiency virus (SIV) isolates (www.hiv.lanl.gov).…”

“…In the previous studies [23][24][25][26][27][28][29][30], laboratory-adapted HIV-1 strains were exclusively used to characterize the resistance profiles for various fusion inhibitors, and in which the secondary N126K mutation was universally identified. In order to verify its biological relevance, here we generated a single N126K mutation on a large panel of HIV-1 Envs with divergent subtypes and phenotypes, including NL4-3 (subtype B, CXCR4-tropic), 92RW020 (subtype A, CCR5-tropic), JRFL (subtype B, CCR5-tropic), REJO4541 (subtype B, CCR5-tropic), AC10.29 (subtype B, CCR5-tropic), QH0692 (subtype B, CCR5-tropic), ZM53M.PB12 (subtype C, CCR5-tropic), and ZM109F.PB4 (subtype C, CCR5-tropic).…”

“…We next focused on characterizing the effect of N126K mutation on the expression and processing of viral Env. Firstly, a capture ELISA-based method with the human anti-gp120 monoclonal antibody VRC01 and anti-gp41 monoclonal antibody 10E8 was applied as described previously [23,25,26]. It is known that VRC01 can react with both the uncleaved gp160 and cleaved gp120 proteins in the lysates of transfected cells and with the secreted gp120 protein in the cell culture supernatants, whereas 10E8 reacts only with the gp160 and cleaved gp41 in the cell lysates but not with gp120 in the culture supernatants, thus their reactivity can theoretically reflect the expression profiles of gp160/gp120/gp41.…”