Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain.
The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.
Ye
Rong
R
Wadsworth Center, New York State Department of Health, New Scotland Ave., P.O. Box 22002, Albany, NY 12201-2002, USA.
Montalto-Morrison
Cynthia
C
Masters
Paul S
PS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics; D017930:Genes, Reporter; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D017384:Sequence Deletion; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics*; D019065:Virus Assembly / Q000235:genetics* / Q000502:physiology*
D004279:DNA, Viral; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D003545:Cysteine