EPSTEIN-BARR-VIRUS DNASE CONTAINS 2 NUCLEAR-LOCALIZATION SIGNALS, WHICH ARE DIFFERENT IN SENSITIVITY TO THE HYDROPHOBIC REGIONS

The DNase of Epstein-Barr virus (EBV) is a 470-amino-acid protein whic h possesses both endonuclease and exonuclease activities and accepts b oth double-stranded DNA and single-stranded DNA as substrates. It has been reported that this protein may be found in the nucleus and/or cyt oplasm of infected cells. In this study, using cell fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cel ls stimulated with anti-human immunoglobulin G antibody (anti-IgG), th e DNase was found to be located predominantly in the nucleus. To map t he signals in DNase which mediate its nuclear localization, we monitor ed the nuclear transport of fusion proteins consisting of various frag ments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (beta-Gal). The results demonstrated that two regions of the DNase wi th nuclear localization signal (NLS) activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independent ly to localize the beta-Gal to the nuclei of HEp-2 and HeLa cells. Fiv e basic residues (R or K) were found in each NLS and distributed diffe rently in primary structure. The basic domains and flanking residues o f NLS-A and NLS-B are (YKRPCKRSFIRFI262)-Y-260 and (294)LKDVRKRKLGPGH( 306), respectively. Further examination of these sequences revealed th at NLS-A contains bulky aromatic amino acids (Y and F) which may dimin ish its capacity to act as a strong NLS and lacks the typical proline and glycine helix-breakers. However, NLS-B contains typical proline an d glycine helix-breakers and the histidine residue at amino acid 306 i s required for NLS activity. In addition, two hydrophobic regions with in the DNase were found to inhibit the function of NLS-A but not NLS-B , suggesting that these two domains are different types of NLSs and di ffer in their sensitivity to hydrophobic regions in the context of pro tein structure. (C) 1998 Academic Press.