Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion

Virus Res. 1988 May;10(2-3):177-91. doi: 10.1016/0168-1702(88)90014-7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bromelains / metabolism*
  • Cell Line
  • Centrifugation, Density Gradient
  • Electrophoresis, Polyacrylamide Gel
  • Glycoproteins / analysis
  • Glycoproteins / isolation & purification*
  • Influenzavirus C / analysis*
  • Influenzavirus C / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Orthomyxoviridae / analysis*
  • Trypsin / metabolism
  • Viral Proteins / analysis
  • Viral Proteins / isolation & purification*
  • Virion / analysis
  • Virion / metabolism

Substances

  • Glycoproteins
  • Viral Proteins
  • influenza C virus glycoprotein gpII
  • Bromelains
  • Trypsin