Viral Strategies to Regulate Transcription and Pre-mRNA Splicing

Abstract: A typical DNA virus encodes for a few key regulatory proteins that modify the host cell biosynthetic machinery to achieve a selective viral gene statement. One such example is illustrated by the adenovirus E4-ORF4 protein, which form an enzymatically active complex with the serine and threonine-specific protein phosphatase 2A (PP2A). Almost all biological machines in a cell are regulated through reversible phosphorylation, and the E4-ORF4-PP2A complex has been shown to regulate diverse processes such as transcription, RNA splicing and apoptosis.The adenovirus E1A gene encodes for a potent transcriptional activator protein, which stimulates transcription from all early viral promoters. Here we show that the E4-ORF4-PP2A complex autoregulates its own statement by blocking E1A-induced activation of the E4 promoter. Furthermore, the inhibitory activity of E4-ORF4 was specific for E1A-CR3-dependent transactivation. The E4-ORF4-PP2A complex also blocked E1A-induced activation of the E2 promoter, by reducing the stability of E2F/DNA complexes formed on the E2 promoter. The inhibitory effect of E4-ORF4, on both promoters, was relieved by inhibiting PP2A activity with okadaic acid, suggesting that E4-ORF4 regulates transcription by controlling the phosphorylated status of transcription factors important for E2 and E4 promoter activity.The polyoma virus small T antigen ( py-ST) also interacts with PP2A. Previous results from our laboratory have shown that E4-ORF4 activates adenovirus IIIa splicing by inducing PP2A dephosphorylation of the SR family of splicing factors. The results presented here demonstrate that py-ST also activates IIIa pre-mRNA splicing. However, py-ST modulation of PP2A activity did not correlate with its IIIa splicing enhancer activity. Interestingly, py-ST activated IIIa splicing through a genetic element previously shown to be functional only in the context of an adenovirus infection. Thus, different mammalian viruses may have evolved a common strategy to take control of the RNA splicing machinery in the infected cell.During an adenovirus infection the adenovirus E1B-55K and E4-ORF6 proteins form a stable complex that has been implicated in the selective nuclear to cytoplasmic transport of viral mRNAs, late after infection. Here we show that E4-ORF6 functions as an RNA splicing factor, specifically stimulating IIIa pre-mRNA splicing, both in vitro and in transient transfection assays. Interestingly, E4-ORF6 and E4-ORF4 both enhances IIIa splicing through the same genetic element; the IIIa splicing repressor element (3RE). Moreover, the E1B-55K protein augmented the stimulatory effect of E4-ORF6 on IIIa splicing. Collectively, these results may suggest that the E1B-55K/E4-ORF6 complex regulates RNA export by a direct effect on the efficiency of viral pre-mRNA splicing.