Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion

Abstract

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity

“…5 B) indicating that the binding was specific of the HR2 target region. Moreover, other chimeric proteins that mimic HIV-1 gp41 HR1 [42] , [45] did not show significant binding. Residual binding in presence of competing V39E peptide varied in the order L3C > L3B > L3A, consistently with their relative stabilities.…”

“…These proteins imitate accurately an exposed HR1 groove and tightly bind to gp41 HR2 peptides with identical HR1-HR2 interactions to the post-fusion gp41 structure [42] , [43] . Moreover, the proteins have a potent and broad inhibitory activity against a variety of HIV-1 strains [22] , [40] , [42] , [44] , [45] . Because of the structural similarities between the post-fusion structures of gp41 and S2, we hypothesized that similar single-chain chimeric proteins could be designed to mimic an exposed SARS-CoV-2 HR1 and that these proteins could also have antiviral activity.…”

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins

“…In support of this inhibition mechanism, vaccination with stabilized gp41 HR1 trimers can elicit neutralizing antibodies against HIV-1 [ 40 ] and HIV-1 in infected patients to elicit neutralizing antibodies that target exposed epitopes of trimeric HR1 [ 41 ]. Moreover, a strong correlation between conformational stability and HIV-1 inhibitory activity has been observed in gp41 HR1-mimetic proteins [ 31 , 32 , 33 ]. Other researchers have suggested an alternative model, in which both HR1 and HR2 regions transiently interact with the membranes, destabilizing them and facilitating fusion [ 42 ].…”

“…Among the available approaches to stabilize HR1-based polypeptides in a stable coiled-coil trimeric structure, we previously reported that organizing three HR1 helices in an antiparallel orientation connected with short loops is very effective to create highly accurate structural mimics of an exposed HR1 region and that these mimics can potently and broadly inhibit HIV-1 [ 24 , 30 , 33 ]. Moreover, these mimetic proteins constitute very useful models to investigate the structural and thermodynamic determinants of the HR1–HR2 interaction [ 32 , 43 , 44 ].…”

“…These chimeric proteins folded spontaneously and stably as expected, accurately imitated an exposed HR1 groove between the two parallel HR1 helices and tightly bound to gp41 HR2 peptides [ 30 , 31 ]. Moreover, these mimetic proteins showed potent and broad inhibitory activity against a variety of HIV-1 strains [ 24 , 30 , 31 , 32 , 33 ].…”