HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike

Abstract

The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells by using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be understood better in order to optimally design vaccines and therapeutics. We generated viruses with resistance to VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K, and H66R) that rendered these viruses dependent on the inhibitor. These viruses could infect target cells only when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors and provide new ways to stabilize Env trimer vaccines. IMPORTANCEBecause of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelopebased HIV vaccines. With over 35 million people currently infected, HIV-1 remains a major health problem. Although progress has been made in the development of antiviral drugs, the potential of the virus to acquire resistance remains an issue. Neither a cure nor an effective vaccine is available. HIV-1 enters target cells by using its envelope glycoprotein (Env) spikes on the virus surface. Env is the target for drugs that inhibit the viral entry process and for broadly neutralizing antibodies (bNAbs) that researchers aim to induce with Env-based vaccines.Env is a trimeric complex consisting of three gp41 transmembrane subunits and three noncovalently attached gp120 subunits. The entry process is initiated by binding of gp120 to the CD4 receptor. This interaction induces conformational changes in Env, revealing the binding site for a chemokine coreceptor, generally CXCR4 or CCR5. Additional conformational changes in gp41 involve two heptad repeat regions (HR1 and HR2) in gp41 and the fusion peptide (FP) (1). The FP might be partially exposed in the prefusion, native state of Env. In a com...

Reduced Susceptibility to VIRIP-Based HIV-1 Entry Inhibitors Has a High Genetic Barrier and Severe Fitness Costs

“…We observed that the alterations selected in the presence of VIR-353 also affect HIV-1 susceptibility to the antibody VRC34 that targets an epitope that includes the N-terminal part of the FP (20). Notably, Eggink et al also suggested that the abovementioned alterations in the gp120 C1 and gp41 HR1 regions alter VIR165 sensitivity because they change the kinetics of Env conformational changes and thus decrease the accessibility of the FP target region (27).…”

“…In addition, it is well known that alterations in the helical regions of gp41 affects fusion kinetics and susceptibility to HIV-1 entry inhibitors (33,34). The finding that alterations reducing HIV-1 susceptibility to VIR-165 and VIR-353 increase viral sensitivity to inhibition by T20 targeting the HR1 region of gp41 (13,27) also supports effects on fusion kinetics. Thus, the results of the previous studies are not in disagreement with the gp41 FP being the primary target of VIR-based inhibitors.…”

“…Similarly to M1 (V570I), M4 (L545M) is localized in the flexible loop B connecting the A and C helices in the prefusion structure ( Fig. 9), and a similar substitution (A558T) was identified in an independent study examining HIV-1 resistance to VIR-165 (27). This mutation in HR1 might interfere with six-helix bundle formation, thereby decreasing viral fitness but also susceptibility to VIR-353/576.…”

“…The results of Esté et al called this into question since none of the seven mutations is located in the FP region (13,14). Another study selected HIV-1 variants against the VIRIP analogue VIR165 and also identified alterations in the C1 domain of gp120 and the HR1 domain of gp41 instead of the FP (27). Notably, some C1 mutations rendered HIV-1 dependent on the inhibitor (27).…”

“…Another study selected HIV-1 variants against the VIRIP analogue VIR165 and also identified alterations in the C1 domain of gp120 and the HR1 domain of gp41 instead of the FP (27). Notably, some C1 mutations rendered HIV-1 dependent on the inhibitor (27). Further studies are required to fully clarify the mechanisms underlying VIRIP inhibition and viral evasion.…”