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) th ymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degreve et al. (199 8) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or s elective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-te rminal region of HSV-1 TK, resulted in the loss of the specific nuclear loc alization of HSV-1 TK. Utilizing information on the crystallographic struct ure of HSV-1 TK, we have now identified three additional putative nuclear l ocalization signals and evaluated their potential role in the nuclear traff icking of HSV-1 TK by site-directed mutagenesis. We found that the sites co ntaining the amino acids R236-R237 and K317-R318 are absolutely required fo r 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, c ould act as a nuclear localization signal for monomeric HSV-1 TK. Alternati vely, crystallographic data indicate that R318 might be essential for the f ormation of the TK dimer, and therefore it is required if HSV-1 TK is trans ported as a dimer, (C) 1999 Academic Press.