Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2.
A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.
Chang
D
D
Division of Biology, Kansas State University, Manhattan 66506.
Haynes
J I
JI
Brady
J N
JN
Consigli
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002451:Cell Compartmentation / Q000235:genetics*; D002467:Cell Nucleus / Q000648:ultrastructure*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D011120:Polyomavirus / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D014162:Transfection
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D009842:Oligopeptides; D021382:Protein Sorting Signals; C442543:VP2 protein, Polyomavirus