MAPPING AND CHARACTERIZATION OF A DOMAIN IN THE HERPES SIMPLEX VIRUS VHS PROTEIN THAT INTERACTS WITH VP16

Abstract

The herpes simplex virus has devised mechanisms to manipulate and apprehend the cell’s inherent processes for its own efficient replication. Two proteins brought into the cell upon infection are partially responsible for this action; VP16, a transactivator of immediate-early genes, and vhs, which is responsible for the shutoff of host protein synthesis and the degradation of host and viral mRNAs. The virus therefore, must be capable of integrating strategic means of temporal gene expression controls to effectively progress through its lytic cycle. Recent Studies have shown that vhs and VP16 interact with one another (Smibert et al., 1994), suggesting that this association might directly link their corresponding regulation. The project described herein was concerned with identifying regions of vhs required for interaction with VP16. Through the construction of deletion mutants and analysis of protein-protein interactions in vivo and in vitro, a minimal region of vhs spanning residues 310 to 330, was identified as being sufficient for mediating a selective interaction between these two proteins. This region was further characterized by the generation of 13 mutants; each of which were then analyzed by the yeast two hybrid system and in vitro solid-phase capture assays to identify requirements for association with VP16. The tryptophan residue at position 321 was discovered to be essential for maintaining this interaction. Mutating this residue to alanine rendered the two proteins completely incapable of associating with one another. Furthermore, additional residues located close to this residue affected complex formation in vitro, suggesting that these residues might constitute part of the binding interface and aid in stabilizing the interaction. Thus, the isolation of mutants which disrupt and abate the vhs and VP16 complex should facilitate further research identifying the functional significance of this interaction and the consequences when this interaction is abrogated.