Abstract/Summary

Coronaviruses infect many species causing a variety of diseases with a range of severities. Their members include zoonotic viruses with pandemic potential where therapeutic options are currently limited. Despite this diversity coronaviruses share some common features including the production, in infected cells, of elaborate membrane structures. Membranes represent both an obstacle and aid to coronavirus replication and in consequence virus encoded structural and nonstructural proteins have membrane binding properties. The structural proteins S, E and M encounter cellular membranes at both entry and exit of the virus while the nonstructural proteins nsp3, nsp4 and nsp6 reorganize cellular membranes to benefit virus replication. MERS CoV is responsible for sporadic infections in countries focused on the Middle East with occasional transfer elsewhere. A key step in the MERS CoV replication cycle is the fusion of the virus and host cell membranes mediated by the virus spike protein, S. The location of the fusion peptide within MERS S protein has not been precisely mapped. The coronavirus envelope protein by contrast has defined functions in virus assembly, production and release. It may also induce membrane curvature in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) leading to scission of budding virions. M is located among the S proteins in the virus envelope along with the small amounts of E and is the primary driver of the virus budding process. Nsp3, nsp4 and nsp6 may also have roles in the creation of double-membrane vesicles (DMVs) that are considered the site for viral RNA synthesis although their more precise role is not understood. Thus, S, E, M, nsp3, nsp4 and nsp6 potentially all contain membrane-modifying peptides. To search for such peptides, parameters such as amino acid conservation, and proximity to the membrane and/or Amphipaseek amphipathic helix prediction were used on the requisite open reading frames of both Mouse Hepatitis Virus (MHV) and Middle Eastern Respiratory Syndrome Virus. Peptides identified in silico were synthesised and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. A putative fusion peptide located near the N-terminus of the S2 domain was shown to change the shape and size of the GUVs membrane leading to extensive deformation. Key residues required for activity were mapped by amino acid replacement and their relevance in vitro tested by their introduction into recombinant MERS S protein expressed in mammalian cells. Mutations preventing membrane binding in vitro also abolished S mediated syncytium formation consistent with the identified peptide acting as the fusion peptide for the S protein of MERS CoV. Peptides from E, nsp3, nsp4 and nsp6 were also found to change the size and shape of vesicle membranes in a manner consistent with membrane insertion. Select peptides from nsp4 and nsp6 caused pore formation in GUVs. To assess the roles of the identified E in vivo, MHV E protein was expressed in insect cells using the baculovirus expression system and the relevant peptide sequence mutated. Mutant expression levels were modified compared to wild type with evidence for a redistribution within the expressing cell confirming a role for the MHV-E post transmembrane region in membrane binding in vitro and in vivo. The overall findings identify several conserved sequences as bona fide membrane binding motifs in the structural and non structural proteins of MERS-CoV and MHV. While some were validated by assay in physiologically relevant systems, the precise mechanism of action of others remains to be investigated.