Covalent Trimeric Coiled Coils of the HIV gp41 HR1 Region are Extremely Potent and Broadly Neutralizing Inhibitors of Viral Infection

Abstract

Introduction The key players in HIV viral entry are the envelope glycoprotein receptor-binding gp120 and transmembrane fusogenic gp41 subunits. The mechanism of fusion involves two helical regions of gp41, an N-terminal heptad repeat (HR1) and a Cterminal heptad repeat (HR2). The HR1 and HR2 helical regions form a fusogenic structure, a six-helix-bundle, in which three α-helices formed by HR2 peptides pack in an antiparallel manner against a central three stranded coiled coil formed by the HR1 peptides. It is generally accepted that fusion progresses via the formation of a fusion intermediate, in which both the HR1 coiled coil and the HR2 regions are exposed. The fusion intermediate is the target of both synthetic Cand N-peptides, that inhibit viral infection by preventing formation of the 6-helix bundle. HR2 peptides are potent inhibitors of viral fusion, and the peptide DP178 has become the first fusion inhibitor approved as a human therapeutic [1]. Peptides from the HR1 region of gp41 protein can also inhibit viral fusion, but their potency is limited by the low tendency to form a trimeric coil-coil. Accordingly, chimeric peptides, consisting of a designed trimeric coiled coil (IZ) fused to gp41 HR1 sequences are potent inhibitors of fusion as reported for IZN17 [2] (Fig. 1). Based on the hypothesis that antiviral potency of IZN17 is limited by the selfassociation equilibrium, we designed a new construct, CCIZN17, with the aim of stabilizing the homotrimeric coiled coil structure to a covalent trimer via formation of intermolecular disulfide bonds. To this purpose, two cysteine residues were introduced at the N-terminus of the IZN17 sequence (Fig. 1). Also, two glycine residues were added between the pair of cysteines and the IZ scaffold sequence to ensure a high conformational freedom of the cysteines from the coiled coil domain and thus to enhance the likelihood of spontaneous formation of the intermolecular disulfide bridges among the three peptide chains.