Pseudorabies virus early protein 0 trans-activates the TATA-associated promoter by stimulating the transcription initiation

Pseudorabies virus (PRV) early protein 0 (EP0) is a transactivator containing a RING finger domain. To assess the transactivation mechanism of PRV EP0, we performed the in vitro transcription by combining HeLa nuclear extract, purified recombinant EP0 and simple promoter constructs, and evaluated the results by primer extension. The data showed that EP0 could significantly activate the TATA-containing synthetic promoters. Moreover, EP0 activated transcription by stabilizing the formation of transcription initiation complex instead of enhancing the elongation rate. To further understand the role of EP0 on assembling the transcription initiation complex, we performed the pull-down assay using affinity precipitation of proteins from HeLa nuclear extracts and bacterially expressed glutathione-S-transferase EP0 RING finger fusion. The data showed that at least six nuclear proteins physically interacted with the EP0 RING finger. Overall, the transactivation of PRV EP0 is accomplished by enhancing the transcription initiation and is associated with at least six nuclear proteins.