Role of spike protein endodomains in regulating coronavirus entry.
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.
Shulla
Ana
A
Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153, USA.
Gallagher
Tom
T
D000595:Amino Acid Sequence; D000818:Animals; D017934:Coronavirus / Q000472:pathogenicity*; D003545:Cysteine / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000502:physiology*
D008055:Lipids; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D019308:Palmitic Acid; D003545:Cysteine