Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.
The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.
Lobigs
M
M
Department of Molecular Biology, Karolinska Institute, Huddinge, Sweden.
Garoff
H
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012672:Semliki forest virus / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D015345:Oligonucleotide Probes; D011498:Protein Precursors; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D014357:Trypsin