IDENTIFICATION AND CHARACTERIZATION OF A SMALL MODULAR DOMAIN IN THE HERPES-SIMPLEX VIRUS HOST SHUTOFF PROTEIN SUFFICIENT FOR INTERACTION WITH VP16

The herpes simplex virus transactivator VP16 and the virion host shuto ff protein vhs are viral structural components that direct the activat ion of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infe cted cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affin ity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residue s 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing comp lex formation with VP16 in vivo and in vitro when fused to a heterolog ous protein. Site-directed mutagenesis of this region identified trypt ophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable comp lex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.