Processing of the p62 envelope precursor protein of Semliki Forest virus.
The spike protein of Semliki Forest virus is composed of three subunits, E1, E2, and E3, which mediate the fusion of the virus membrane with that of the host cell. E2 and E3 are synthesized as a precursor, p62, which is cleaved post-translationally after an Arg-His-Arg-Arg sequence. In vitro mutagenesis of a cDNA clone of the spike proteins was used to specifically alter amino acids in this cleavage site. Cleavage of p62 was completely blocked by mutation of the proximal Arg residue to Phe, without affecting transport or surface expression of the spike protein. The cleavage mutation resulted in the loss of spike protein fusion activity within the physiological pH range. Fusion activity was restored by cleavage with exogenous chymotrypsin and showed the same low pH dependence as that of wild type. The cleavage sensitivity of newly synthesized p62 was investigated by pulse-chase analysis and chymotrypsin treatment in detergent solution. p62 was sensitive to cleavage immediately following its synthesis. Protein trapped in the rough endoplasmic reticulum or Golgi apparatus by carbonyl cyanide m-chlorophenylhydrazone, monensin, or Brefeldin A treatment was also fully sensitive to cleavage. These results suggest that p62 does not require an organelle-mediated conformational change for processing. Thus, in vivo, the site of p62 processing is probably controlled by the location or activity of the cleavage enzyme, rather than the sensitivity of the p62 substrate.
Jain
S K
SK
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
DeCandido
S
S
Kielian
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012672:Semliki forest virus / Q000378:metabolism*; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*
D011498:Protein Precursors; D014759:Viral Envelope Proteins; D002918:Chymotrypsin