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Processing of the p62 envelope precursor protein of Semliki Forest virus
S K Jain1, S DeCandido, M Kielian
1Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Abstract:
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.
Insights
Mutating the Semliki Forest virus spike protein precursor p62 blocked its cleavage, halting viral fusion. This suggests enzyme activity, not substrate sensitivity, controls p62 processing in vivo.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Semliki Forest virus spike protein mediates host cell fusion.
- Spike protein subunits E1, E2, and E3 are derived from a p62 precursor.
- p62 cleavage occurs post-translationally at a specific Arg-His-Arg-Arg sequence.
Purpose of the Study:
- To investigate the role of the p62 cleavage site in Semliki Forest virus spike protein function.
- To determine the requirements for p62 processing and its impact on viral fusion.
- To elucidate the control mechanisms governing p62 post-translational modification.
Main Methods:
- In vitro mutagenesis of Semliki Forest virus spike protein cDNA.
- Analysis of p62 cleavage, transport, and surface expression.
- Assay of viral fusion activity at physiological pH.
- Pulse-chase analysis and exogenous chymotrypsin treatment.
Main Results:
- Mutation of the proximal Arg residue in the p62 cleavage site blocked processing without affecting protein transport or surface expression.
- The cleavage-defective mutant lost fusion activity at physiological pH.
- Fusion activity was restored upon exogenous chymotrypsin cleavage, retaining low pH dependence.
- p62 was sensitive to cleavage immediately after synthesis and in various cellular compartments.
Conclusions:
- p62 processing does not require organelle-mediated conformational changes.
- The location or activity of the cleavage enzyme, rather than p62 substrate sensitivity, likely controls in vivo processing.
- Proper cleavage of p62 is essential for Semliki Forest virus fusion activity.