The Semliki-Forest-virus-specific nonstructural protein nsP4 is an autoproteinase.
The Semliki-Forest-virus-specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either P123 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcription-translation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3-nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3-nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino-terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.
Takkinen
K
K
Institute of Biotechnology, University of Helsinki, Finland.
Peränen
J
J
Keränen
S
S
Söderlund
H
H
Kääriäinen
L
L
D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics*; D003062:Codon / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012672:Semliki forest virus / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins
D003062:Codon; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D010447:Peptide Hydrolases