The adeno-associated virus type 2 regulatory proteins Rep78 and Rep68 interact with the transcriptional coactivator PC4

The adeno-associated virus type 2 (AAV-2) Rep78/Rep68 regulatory proteins a re pleiotropic effecters of viral and cellular DNA replication, of cellular transformation by viral and cellular oncogenes, and of homologous and hete rologous gene expression. To search for cellular proteins involved in media ting these functions, we used Rep68 as bait in the yeast two hybrid system and identified the transcriptional coactivator PC4 as a Rep interaction par tner. PC4 has been shown to mediate transcriptional activation by a variety of sequence-specific transcription factors in vitro. Rep amino acids 172 t o 530 were sufficient and amino acids 172 to 224 were absolutely necessary for the interaction with PC4, The PC4 domains required for interaction were mapped to the C-terminal single-stranded DNA-binding domain of PC4. In glu tathione S-transferase (GST) pull-down assays, in vitro-transcribed and -tr anslated Rep78 or Rep68 proteins were bound specifically by GST-PC4 fusion proteins. Similarly, PC4 expressed in Escherichia coli was bound by CST-Rep fusion proteins, confirming the direct interaction between Rep and PC4 in vitro. Rep was found to have a higher affinity for the nonphosphorylated, t ranscriptionally active form of PC4 than for the phosphorylated, transcript ionally inactive form. The latter is predominant in nuclear extracts of HeL a or 293 cells, In the yeast system, but not in vitro, Rep-PC4 interaction was disrupted by a point mutation in the putative nucleotide-binding site o f Rep68, suggesting that a stable interaction between Rep and PC4 in vivo i s ATP dependent. This mutation has also been shown to impair Rep function i n AAV-2 DNA replication and in inhibition of gene expression and inducible DNA amplification. Cytomegalovirus promoter-driven overexpression of PC4 le d to transient accumulation of nonphosphorylated PC4 with concomitant downr egulation of all three AAV-2 promoters in the absence of helper virus. In t he presence of adenovirus, this effect was relieved. These results imply an involvement of the transcriptional coactivator PC4 in the regulation of AA V-2 gene expression in the absence of helper virus.