PROTEIN INTERACTIONS IN THE HERPES-SIMPLEX VIRUS TYPE-1 VP16-INDUCED COMPLEX - VP16 PEPTIDE INHIBITION AND MUTATIONAL ANALYSIS OF HOST-CELLFACTOR REQUIREMENTS

The herpes simplex virus VP16 protein functions as a potent transcript ional activator and targets DNA sites with the consensus TAATGARAT pre sent in all the viral immediate-early gene promoters, To do so VP16 di rects assembly of a multiprotein complex involving two cellular protei ns, host cell factor (HCF) and the Oct-1 DNA-binding transcription fac tor, To investigate the importance of specific protein-protein interac tions to formation of this VP16-induced complex (VIC), we used oligope ptides to prevent VIC assembly, Linear and cyclic peptides correspondi ng to a region of VP16 previously implicated in complex formation were potent inhibitors of VIC assembly, To further characterize the protei n interactions involved, we cloned a human cDNA encoding the minimal V P16 interaction domain of HCF, containing amino acids I to 380 [HCF (1 -380)]. The REHAYS-based peptides active in preventing VIC assembly we re found to specifically block binding of VP16 to HCF (1-380), without affecting VP16-Oct-1 binding, The inhibitory activity of these VP16 p eptides was strictly sequence specific for the EHAY residues, Site-dir ected mutagenesis of the HCF (1-380) domain revealed residues E102 and K105 to be critical determinants in support of VIC formation, Alterat ion of a single residue in HCF, K105, was shown to virtually abolish c omplex assembly, Interestingly however, none of the HCF mutants that w ere impaired in their ability to support complex formation exhibited d efects in direct VP16 binding, supporting loss of function at a higher order in complex assembly.