AMINO-ACID SUBSTITUTIONS IN THE HERPES-SIMPLEX VIRUS TRANSACTIVATOR VP16 UNCOUPLE DIRECT PROTEIN-PROTEIN INTERACTION AND DNA-BINDING FROM COMPLEX ASSEMBLY AND TRANSACTIVATION

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the asse mbly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNA-bound Oct-1 in the absence of VCAF-1, In ord er to probe the requirements of these individual interactions in the f unctional asembly of VIC, we mutated selected charged amino acids in t wo subdomains of VP16 previously shown to be important in protein-DNA complex formation, Purified VP16 proteins were analyzed for their abil ity to direct protein-DNA complex formation and to interact directly w ith VCAF-1. Several classes of mutants that were differentially compro mised in VCAF-1 interaction, direct DNA binding, and/or association wi th DNA-bound Oct-1 were obtained. Interestingly, all of the derivative s were still capable of generating the VIC complex in vitro and activa ting transcription in vivo, Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathway s that do not necessarily require the prior interaction of VP16 with V CAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.