Localization and Phosphorylation of Semliki Forest Virus Non-structural Protein nsP3 Expressed in COS Cells from a Cloned cDNA

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Abstract
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.

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