The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.
A cDNA fragment coding for poliovirus capsid polypeptide VP1 was inserted into a simian virus 40 (SV40) genome in the place of the SV40 VP1 gene and fused in phase to the 3' end of the VP2-VP3 genes. Simian cells were infected with the resulting hybrid virus in the presence of an early SV40 mutant used as a helper. Indirect immunofluorescence analysis of the infected cells using anti-poliovirus VP1 immune serum revealed that the SV40/poliovirus fusion protein was located inside the cell nucleus. Deletions of various lengths were generated in the SV40 VP2-VP3 portion of the hybrid gene using BAL31 nuclease. The resulting virus genomes expressed spliced fusion proteins whose intracellular location was either intranuclear or intracytoplasmic, depending on the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed by site-directed mutagenesis of the Lys residue at position 320. Modification of Lys-320 into either Thr or Asn abolished the nuclear accumulation of the fusion protein. It is concluded that at least part of the sequence of VP2 amino acids 317 to 323 allows VP2 and VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cell cytoplasm to the cell nucleus by interaction, with VP1 acting as a carrier.
Wychowski
C
C
Unité de Virologie Moléculaire (CNRS UA 545), Institut Pasteur, Paris, France.
Benichou
D
D
Girard
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D010455:Peptides / Q000032:analysis; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis*; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes