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.
Highlights
By the acidic, proteolytic environments encountered after viruses are endocytosed [3,4,5]
It is notable that many viral fusion protein ectodomain fragments lacking TM and ENDO domains fold into postfusion states [17, 18], suggesting that membrane-anchoring parts help maintain functional metastable high energy conformations
We and others consider it plausible that changes in the fusion protein endodomain impact refolding rates, which in turn control the route and timing of virus entry. This is because the transitions from prehairpin intermediate to postfusion states require large scale transit of TM-ENDO domains across lipid stalks [19], which may be a rate-limiting step in the process
Summary
By the acidic, proteolytic environments encountered after viruses are endocytosed [3,4,5]. It is notable that many viral fusion protein ectodomain fragments lacking TM and ENDO domains fold into postfusion states [17, 18], suggesting that membrane-anchoring parts help maintain functional metastable high energy conformations It is not entirely clear how the intravirion parts of the fusion protein influence reactions that are carried out by the much. We and others consider it plausible that changes in the fusion protein endodomain impact refolding rates, which in turn control the route and timing of virus entry This is because the transitions from prehairpin intermediate to postfusion states require large scale transit of TM-ENDO domains across lipid stalks [19], which may be a rate-limiting step in the process. Interference with S endodomain palmitoylation, either by engineered mutations or pharmacologic agents, diminishes or eliminates S-mediated membrane fusion activities [28, 29, 31, 34], but the mechanisms by which these endodomain alterations influence membrane fusion activities are unknown
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