Role of spike protein endodomains in regulating coronavirus entry

J Biol Chem. 2009 Nov 20;284(47):32725-34. doi: 10.1074/jbc.M109.043547. Epub 2009 Sep 30.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Coronavirus / pathogenicity*
  • Cysteine / chemistry
  • Fibroblasts / metabolism
  • HeLa Cells
  • Humans
  • Lipids / chemistry
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / physiology*
  • Mice
  • Molecular Sequence Data
  • Palmitic Acid / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins / chemistry*
  • Viral Envelope Proteins / physiology*

Substances

  • Lipids
  • Membrane Glycoproteins
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins
  • spike glycoprotein, SARS-CoV
  • spike protein, mouse hepatitis virus
  • Palmitic Acid
  • Cysteine