Mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion.
The spike (S) protein of murine coronavirus strain A59 (MHV-A59) is a type I membrane protein that induces membrane fusion. In this study we have analyzed the role of two domains in the S protein on fusion. The 180-kDa mature S protein is partially cleaved into two 90-kDa subunits during transport to the plasma membrane. We have identified several amino acids that are important for cleavage of S, and we show that cleavage is not strictly required for fusion. However, the level of cleavage seems to influence the fusion kinetics. After introduction of an arginine at position P2 to mimick the MHV-JHM cleavage site, full cleavage of the spike protein was obtained. Further, we analyzed the effect of mutations in the transmembrane (TM) domain of the S protein. Maturation and cell surface expression of the mutant proteins were not affected, and all proteins became acylated. The mutant in which the predicted transmembrane domain was shortened did not induce syncytia. From a group of mutants in which several conserved cysteines in the TM domain had been replaced by serines, one was unable to induce syncytia, another showed delayed syncytia formation, and the third mutant induced syncytia as did the wild-type protein. The potential role of the transmembrane domain in fusion is discussed.
Bos
E C
EC
Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
Heijnen
L
L
Luytjes
W
W
Spaan
W J
WJ
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002459:Cell Fusion; D002460:Cell Line; D017261:Glycosylphosphatidylinositols / Q000502:physiology; D007739:L Cells; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D010169:Palmitic Acids; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D019308:Palmitic Acid