Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry.
The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. S(VSV-Cyt), an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and S(MHV-TMDCyt), an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. S(VSV-TMDCyt), a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that S(VSV-TMDCyt) trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.
Broer
Rene
R
Department of Medical Microbiology, E4-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Boson
Bertrand
B
Spaan
Willy
W
Cosset
François-Loïc
FL
Corver
Jeroen
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics; D008561:Membrane Fusion / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D004279:DNA, Viral; D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D049410:Luciferases, Renilla