Characterization of multiple nuclear localization signals in herpes simplex virus type 1 thymidine kinase.
We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.
Degrève
B
B
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, Leuven, B-3000, Belgium.
Esnouf
R
R
De Clercq
E
E
Balzarini
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology*; D003600:Cytosol / Q000821:virology; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase