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The Semliki-Forest-virus-specific nonstructural protein nsP4 is an autoproteinase
K Takkinen1, J Peränen, S Keränen
1Institute of Biotechnology, University of Helsinki, Finland.
European Journal of Biochemistry
|April 20, 1990
Summary
Semliki Forest virus nonstructural protein 4 (nsP4) plays an autoproteolytic role in cleaving the nsP3-nsP4 junction. This cleavage is essential for releasing functional polymerase components and is inhibited by pepstatin A.
Area of Science:
- Virology
- Molecular Biology
- Protein Chemistry
Background:
- Semliki Forest virus nonstructural proteins are translated as a large polyprotein.
- Proteolytic cleavage releases mature polymerase components (nsP1, nsP2, nsP4).
- Two alternative cleavage pathways exist for the ns polyprotein (P1234).
Purpose of the Study:
- To investigate the autoproteolytic role of nsP4 in the nsP3-nsP4 cleavage.
- To map the region within nsP4 responsible for this cleavage.
- To understand the mechanism and inhibition of nsP3-nsP4 processing.
Main Methods:
- In vitro transcription-translation system using rabbit reticulocyte lysates.
- Cloning of cDNAs encoding P34 precursor and nsP4 segments under T7 promoter.
- Analysis of translational products by SDS-PAGE.
- Site-directed mutagenesis (deletion and truncation) of nsP4 cDNA.
Main Results:
- Precursor proteins containing nsP4 sequences were cleaved at the nsP3-nsP4 junction.
- Proteolytic activity was mapped to the N-terminal 102 amino acids of nsP4.
- Cleavage was inhibited by pepstatin A, suggesting aspartic protease activity.
- Cleavage likely occurs in cis, as exogenous nsP4 did not mediate it.
Conclusions:
- nsP4 possesses autoproteolytic activity essential for Semliki Forest virus replication.
- The N-terminal region of nsP4 is critical for its protease function.
- The nsP3-nsP4 cleavage is an intramolecular event, likely occurring in cis.