Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion.
The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.
Formanowski
F
F
Abteilung für Virologie, Technischen Universität München, F.R.G.
Meier-Ewert
H
H
D000595:Amino Acid Sequence; D001963:Bromelains / Q000378:metabolism*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification*; D009982:Gammainfluenzavirus / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009975:Orthomyxoviridae / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis / Q000302:isolation & purification*; D014771:Virion / Q000032:analysis / Q000378:metabolism
D006023:Glycoproteins; D014764:Viral Proteins; C052637:influenza C virus glycoprotein gpII; D001963:Bromelains; D014357:Trypsin