Localization and phosphorylation of Semliki Forest virus non-structural protein nsP3 expressed in COS cells from a cloned cDNA.
The nsP3 protein of Semliki Forest virus is a phosphoprotein, which is processed from a large nonstructural polyprotein. The nsP3 gene was isolated from the large coding region by the polymerase chain reaction technique and cloned into a eukaryotic expression vector. Using the constructed pSVNS3 expression vector it was shown that nsP3 could be phosphorylated in the absence of other virus-specific proteins. This suggests that the formation of a complex with the other non-structural proteins is not required for the phosphorylation of nsP3. About half of the synthesized nsP3, in pSVNS3-transfected COS cells, could be fractionated into the mitochondrial pellet fraction indicating that nsP3 is associated with large intracellular structures. Immunofluorescence microscopy of pSVNS3-transfected COS cells showed that nsP3 was found in the cytoplasm localized to vesicle-like structures. These results suggest that nsP3 contains information for specific interaction with large intracellular vesicular structures.
Peränen
J
J
Institute of Biotechnology, University of Helsinki, Finland.
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012672:Semliki forest virus / Q000235:genetics*; D014162:Transfection; D014758:Viral Core Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins