Structure of the Fusion Core and Inhibition of Fusion by a Heptad Repeat Peptide Derived from the S Protein of Middle East Respiratory Syndrome Coronavirus

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical ␣-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design.

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

“…In the S1 subunit, the receptor binding domain (RBD, also called the C terminal domain, CTD) is localized in the C-terminal region, spanning ∼200 amino acids, and structural studies have revealed that the RBD consists of two subdomains: the core and external subdomains14151617. In the S2 subunit, the heptad repeat (HR) regions are also well characterized181920, and as expected, the HR1 and HR2 of MERS-CoV fold into an intra-hairpin helical structure that can assemble trimerically into a six-helix bundle (a trimer of the HR1/HR2 heterodimer), demonstrating a classical type I membrane fusion process21. Peptide inhibitors have been designed targeting these HR regions and been proven to be effective in vitro and in vivo 1819222324.…”

Single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal Middle East respiratory syndrome (MERS)-coronavirus infection

“…To identify the epitopes on the MERS-CoV RBD recognized by hMS-1, we utilized a panel of mutant RBD proteins generated by mutating the key residues at the MERS-CoV RBD and the hDPP4 binding interface to alanine (Du et al, 2014); the binding between hMS-1 and the respective mutant RBD proteins was then measured by ELISA as described (Gao et al, 2013; Lu et al, 2013). hMS-1 was not capable of recognizing the RBD of the R511A mutant and reduced its binding to RBDs with mutations at D510A and W553A, respectively (Fig.…”

“…Its spike protein, consisting of S1 and S2 subunits, plays important roles in viral entry into host cells (Bonavia et al, 2003; Gao et al, 2013; Xu et al, 2004). MERS-CoV depends on the receptor-binding domain (RBD) in the S1 subunit to bind to its viral receptor, human dipeptidyl peptidase 4 (hDPP4), on the host cell surface, following which the S2 subunit undergoes a dramatic conformational change to allow for membrane fusion and subsequent MERS-CoV penetration into the cellular membrane (Gao et al, 2013; Lu et al, 2013; Raj et al, 2013; Wang et al, 2013). Therefore, the entry of MERS-CoV into target cells might be prevented by either blocking RBD/DPP4 viral receptor binding or by inhibiting MERS-CoV/host cell membrane fusion.…”

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

“…The CoV S protein is a class I fusion protein involved in attachment of the CoV surface to the host aminopeptidase N [2,58]. The S protein is presented as a trimer and mediates receptor binding, membrane fusion, and virus entry [59,60,61]. The S protein is the major target for neutralizing antibodies [62,63].…”