Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2.
About 50% of Semliki Forest virus-specific nonstructural protein nsP2 is associated with the nuclear fraction in virus-infected BHK cells. Transport into the nucleus must be specific, since only trace amounts of nsP3 and nsP4 and about 13% of nsP1, all derived from the same polyprotein, were found in the nucleus. Subfractionation of [35S]methionine-labeled Semliki Forest virus-infected cells showed that 80 to 90% of the nuclear nsP2 was associated with the nuclear matrix. Indirect immunofluorescence, with anti-nsP2 antiserum, showed the most intensive staining of structures which by Nomarski optics appeared to be nucleoli. In the presence of 1 to 5 micrograms of dactinomycin per ml the nuclei were stained evenly and no nucleoli could be found. Transport of nsP2 into the nucleus occurred early in infection and was fairly rapid. A cDNA encoding the complete nsP2 was isolated by the polymerase chain reaction technique and ligated into a simian virus 40 expression vector derivative. When BHK cells were transfected with this pSV-NS2 vector by the lipofection procedure, nsP2 was expressed in about 1 to 5% of the cells, as shown by indirect immunofluorescence. In positively transfected cells the immunofluorescence stain was most intensive in the nucleoli. Thus, Semliki Forest virus-specific nsP2 must have information which directs it into the nuclear matrix and, more specifically, into the nucleoli.
Peränen
J
J
Institute of Biotechnology, University of Helsinki, Finland.
Rikkonen
M
M
Liljeström
P
P
Kääriäinen
L
L
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis* / Q000648:ultrastructure; D002472:Cell Transformation, Viral; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D012672:Semliki forest virus / Q000235:genetics*; D014758:Viral Core Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins
D015345:Oligonucleotide Probes; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins