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...