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

Mario Cano-Muñoz,Li-Yun Lin,Julie Lucas,Christiane Moog,Samuele Cesaro,Francisco Conejero-Lara

doi:10.3390/ijms23052794

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.

Highlights

Introduction published maps and institutional affilThe HIV/AIDS pandemic is still very active and continues to be one of the world’s largest pandemics to date with more than 40 million people currently living with HIV still representing a worldwide health issue [1,2]
The initial designs of the parent molecules in this study, covNHR and covNHRN, contain, respectively, the four and two (NTP and middle pocket (MP)) of the pockets described in gp41 (Figure 1 and Figure S1)
We had achieved a considerable stabilization of over +20 ◦ C in covNHRN (Tm ≈ 41 ◦ C) by engineering a disulfide bridge that connected the first loop of the miniprotein with its C-terminal end (G33C/R94C mutations) resulting in a new variant called covNHR-N-SS [24]

Summary

Introduction

The HIV/AIDS pandemic is still very active and continues to be one of the world’s largest pandemics to date with more than 40 million people currently living with HIV still representing a worldwide health issue [1,2]. China’s students, where the number of newly diagnosed college students has seen an annual growth rate ranging from 30 to 50% over the past several years [3]. All this together with the fact that HIV newly infects 1.8 million people each year, makes the development of an HIV vaccine a global health priority [4]. Almost 40 years after the discovery of HIV as the causative agent of AIDS we still do not have a licensed vaccine. Progress has been hindered by the extensive genetic variability of HIV and our limited understanding of the immune responses required to protect against HIV acquisition [5]. iations.

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Results

Conclusion