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Journal of Virology, December 1998, p. 9535-9543, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Differential Intracellular Compartmentalization of Herpetic Thymidine Kinases (TKs) in TK Gene-Transfected Tumor Cells: Molecular Characterization of the Nuclear Localization Signal of Herpes Simplex Virus Type 1 TK

Bart Degrève,1,2 Magnus Johansson,2 Erik De Clercq,1 Anna Karlsson,2 and Jan Balzarini1,*

Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium,1 and Division of Clinical Virology, Department of Immunology, Microbiology, Pathology, and Infectious Diseases, Karolinska Institute, S-141 86 Stockholm, Sweden2

Received 24 June 1998/Accepted 14 August 1998

The thymidine kinases (TKs) of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) were expressed in human osteosarcoma cells as fusion proteins with the green fluorescent protein (GFP), and their intracellular localizations were determined. The three TK-GFP fusion products were localized in different subcellular compartments of the transfected tumor cells. HSV-1 TK-GFP was localized exclusively in the nucleus, HSV-2 TK-GFP was predominantly found in the cytosol, while VZV TK-GFP was localized in both the nucleus and the cytosol. In support of these findings, we identified a nuclear localization signal (NLS) in the N-terminal arginine-rich region of HSV-1 TK that was absent in HSV-2 and VZV TK. The first 34 amino acids proved necessary for the specific nuclear localization of HSV-1 TK and, when added to the VZV TK-GFP gene construct, also sufficed to specifically target VZV TK-GFP to the nucleus. Further analysis of this NLS through site-directed mutagenesis revealed that the basic amino acid-rich nonapeptide 25R-R-T-A-L-R-P-R-R33 is of crucial importance in the nuclear targeting of HSV-1 TK. In particular, we revealed that the presence of the arginine residues at positions 25, 26, 30, 32, and 33 is obligatory for efficient NLS functioning, whereas arginine and histidine residues outside of the nonapeptide (i.e., residues R18, R20, and H22) did not change the functional properties of the NLS.

* Corresponding author. Mailing address: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Phone: 32-16-337352. Fax: 32-16-337340. E-mail: jan.balzarini{at}rega.kuleuven.ac.be.

Journal of Virology, December 1998, p. 9535-9543, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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