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Abstract Background We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.

Background: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer. [ABSTRACT FROM AUTHOR]

The function of HIV-1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC-1, was accelerated by the presence of X4. The kinetics of these key post-Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemifusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env-mediated cell fusion event. [ABSTRACT FROM AUTHOR]

Abstract: Human immunodeficiency virus type 1 (HIV-1) infection is initiated by the binding of the viral envelope protein gp120 to the host cell CD4 receptor through a high-affinity interaction involving amino acids 39–60 within the CD4. We obtained evidence demonstrating functional importance of this region in CD4 for viral infectivity by showing that a synthetic peptide corresponding to this CD4 sequence exhibited competitive binding to gp120 and significantly reduced infection by diverse HIV-1 strains, including primary isolates. Treatment of HIV-1-infected cells with this CD4 peptide induced shedding of gp120 and exposure of the transmembrane protein gp41. Furthermore, we observed that deletion or substitution of arginine at position 59 (Arg59) within the CD4 peptide sequence abrogated its gp120-shedding property. These results indicate a critical role for Arg59 in the CD4 for conformational changes in gp120 during the sequential process of entry and infection by HIV-1. [Copyright &y& Elsevier]

An ideal vaccine against HIV-1 will specifically elicit bNAbs (broadly neutralizing antibodies) which can cross-neutralize a wide spectrum of circulating viral strains belonging to different clades. The current paradigm for developing such a vaccine is to generate HIV-1 envelope (Env)-based immunogens which can specifically elicit bNAbs. For this purpose, it is necessary to identify Envs, belonging to different clades, suitable for immunogen design. Efficient cleavage of the HIV-1 Env precursor gp160 polypeptide into its constituent subunits determines its ability to selectively bind to bNAbs and poorly to non-NAbs (non-neutralizing antibodies), properties desirable in Env-based immunogens. Thus, efficiently cleaved HIV-1 Envs with desirable antigenic properties can be good candidates for developing immunogens. Here we describe in detail a six step method we have used in our laboratory to identify such efficiently cleaved Envs. Some of these protocols are optimizations of previously reported assays such as FACS-based cell surface antibody binding assay, pseudovirus neutralization assay and gp120 shedding assay. Other protocols like biotinylation-neutravidin-agarose pull-down assay and plasma membrane protein immunoprecipitation assay have been developed by taking inputs from reagent/kit manufacturer's protocols and previous studies. These protocols will help the field in identifying more such Envs which can be used for immunogen development. •Six step process to identify efficiently cleaved, membrane-bound, functional HIV-1 Envs with high degree of repeatability.•Method applicable for characterizing any HIV-1 envelope protein.•New method of immunoprecipitation of plasma membrane fraction to validate efficiently cleaved HIV-1 envelopes.