Mapping of key functions of the herpes simplex virus 1 U(S)3 protein kinase: the U(S)3 protein can form functional heteromultimeric structures derived from overlapping truncated polypeptides

Earlier studies have shown that the herpes simplex virus (HSV) U(S)3 encodes two transcriptional units directing the synthesis of the U(S)3 (residues 1 to 481) and U(S)3.5 (residues 77 to 481) protein kinases. Both kinases phosphorylate histone deacetylase 1 (HDAC1) and HDAC2 and enable the expression of genes cotransduced into U2OS cells by recombinant baculoviruses, an activity designated the "helper function." The two kinases differ with respect to antiapoptotic activity. In the studies reported here, we made a series of FLAG-tagged amino- and carboxyl-terminal truncations of U(S)3 and these were tested for antiapoptotic activity, phosphorylation of HDAC1, and the helper function. We report the following. (i) HDAC1 phosphorylation and the helper function were expressed in cells transduced by the truncation encoding residues 182 to 481 but not in cells transduced by the truncation encoding residues 189 to 481 or the amino-terminal polypeptides encompassing the first 188 amino acids. (ii) The self-posttranslational modification requires residues 164 to 481. (iii) The antiapoptotic activity requires both the amino-terminal and the carboxyl-terminal domains, of which the truncated protein containing residues 1 to 163 and that containing residues 164 to 481, respectively, were the smallest fragments tested to be effective. The two domains need not be on the same molecule, but they must overlap. The smallest overlapping pair tested was the fragment containing residues 1 to 181 and that containing residues 164 to 481. Consistent with the hypothesis that the effective overlapping truncations form a heteromultimeric structure, antibody to FLAG coprecipitated untagged U(S)3 from lysates of cells cotransduced with FLAG-tagged, truncated U(S)3 constructs. Although U(S)3 has been reported to be a monomeric enzyme, the results indicate that it can form enzymatically active multimeric structures.