Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Abstract

Enveloped viruses enter cells by viral glycoprotein-mediated binding to host cells and subsequent fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. Palmitate adducts are generally required for protein-mediated fusions, but their precise roles in the process are unclear. To obtain additional insights into the S-mediated membrane fusion process, we focused on these acylated carboxyl-terminal intravirion tails. Substituting alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S-mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral heptad repeat peptides that bind only to folding intermediates in the S-mediated fusion process and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly 10 times longer than native S proteins. This slower refolding was also reflected in the paucity of postfusion six-helix bundle configurations among the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. These studies reveal a previously unappreciated role for covalently attached lipids on the endodomains of viral proteins eliciting membrane fusion reactions.Biological membranes are configured in large part by protein-mediated fission and fusion reactions. Enveloped viruses can reveal the principles of these processes because their assembly and budding from infected cells requires membrane fissions, and their entry into susceptible cells depends on membrane fusions. Glycoproteins extending from virion surfaces mediate the fusion process. These specialized integral membrane proteins are in metastable high energy configurations on virus surfaces, and they drive coalescence of opposing virus and cell membranes by undergoing a series of energy-releasing unfolding and refolding events (1). The structural rearrangements are triggered by virus binding to cellular receptors (2) and by the acidic, proteolytic environments encountered after viruses are endocytosed (3-5). These reactions begin with an unfolding process that reveals hydrophobic fusion peptides (FPs) 2 that dagger into cellular membranes. This is then followed by a refolding process that, in analogy to a closing hairpin, brings FPs and associated cellular membranes toward the virion membranes, driving formation of a lipid stalk connecting the opposing outer membrane leaflets (6) and culminating in complete cell-virion membrane coalescence (7,8). For viral fusion proteins in the so-called "class I" category, the arms...

Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell–cell fusion but not interaction with M protein

“…Coronavirus S proteins contain a cysteine-rich domain in their cytoplasmic tails with at least 7 cysteine residues (Hogue and Machamer, 2008). Coronavirus S protein palmitoylation has been implicated in cell-cell fusion, virus-cell fusion, virus assembly and infectivity; however, studies focusing on the role of S protein palmitoylation have so far mutated only some of the palmitoylated cysteines (Petit et al, 2007;Shulla and Gallagher, 2009) or used pharmacological inhibitors of protein palmitoylation (Thorp et al, 2006). These methods and results led to only a partial disruption of protein palmitoylation and partial phenotypes.…”

Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis

“…A short cytoplasmic tail or endodomain is located at the C-terminal end of S. It contains conserved stretches of cysteine residues, which can be palmitoylated. Palmitoylation of cysteine residues within the endodomain was found to be important for regulating fusogenicity of S (Petit et al, 2007; Shulla and Gallagher, 2009). Cryo-EM studies on single particles of the SARS-CoV have shed light on the overall architecture of S protein trimers and the conformational changes they undergo during fusion (Beniac et al, 2006; Beniac et al, 2007).…”

Supramolecular Architecture of the Coronavirus Particle

“…The more variable amino-terminal region of the spike protein (S1) has been demonstrated to contain the receptorbinding activity (Wong et al, 2004). The more conserved S2 region contains the transmembrane anchor, palmitic acid acylation site (Thorp et al, 2006) that is important for membrane fusion (McBride and Machamer, 2010;Shulla and Gallagher, 2009), and the coiled-coil fusion motor domain (Bosch et al, 2003;Duquerroy et al, 2005;Liu et al, 2004;Tripet et al, 2004;Xu et al, 2004a,b). One or more protease cleavage events are necessary to prime S for membrane fusion.…”