Unscheduled expression of capsid protein IIIa results in defects in adenovirus major late mRNA and protein expression.
ABSTRACT Adenovirus gene expression is to a large extent regulated at the level of alternative RNA splicing. For example, in the major late region 1 (L1) unit, a common 5' splice site can be joined to two alternative 3' splice sites, resulting in the formation of the so-called 52,55K (proximal 3' splice site) or the IIIa (distal 3' splice site) mRNAs. Whereas, the 52,55K mRNA is expressed both early and late during infection, the IIIa mRNA is strictly confined to the late phase of the infectious cycle. We have previously shown that IIIa mRNA splicing is subjected to a tight viral control of IIIa 3 splice site usage. In an attempt to determine why adenovirus uses elaborate mechanisms to confine IIIa mRNA production to the late phase of infection, we characterized the phenotype of a recombinant adenovirus expressing the IIIa protein from an inducible tetracycline regulated gene cassette. The results show that expression of the IIIa protein during the early phase of infection results in a significant reduction in late viral protein synthesis and a moderate block to viral DNA replication. Interestingly, unscheduled IIIa protein expression resulted in a perturbation of the accumulation of alternatively spliced L1 mRNAs. Thus, 52,55K mRNA accumulation was inhibited while no effects on endogenous IIIa mRNA expression was detected.
Article: Cellular splicing and transcription regulatory protein p32 represses adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II.[show abstract] [hide abstract]
ABSTRACT: The cellular protein p32 is a multifunctional protein, which has been shown to interact with a large number of cellular and viral proteins and to regulate several important activities like transcription and RNA splicing. We have previously shown that p32 regulates RNA splicing by binding and inhibiting the essential SR protein ASF/SF2. To determine whether p32 also functions as a regulator of splicing in virus-infected cells, we constructed a recombinant adenovirus expressing p32 under the transcriptional control of an inducible promoter. Much to our surprise the results showed that p32 overexpression effectively blocked mRNA and protein expression from the adenovirus major late transcription unit (MLTU). Interestingly, the p32-mediated inhibition of MLTU transcription was accompanied by an approximately 4.5-fold increase in Ser 5 phosphorylation and an approximately 2-fold increase in Ser 2 phosphorylation of the carboxy-terminal domain (CTD). Further, in p32-overexpressing cells the efficiency of RNA polymerase elongation was reduced approximately twofold, resulting in a decrease in the number of polymerase molecules that reached the end of the major late L1 transcription unit. We further show that p32 stimulates CTD phosphorylation in vitro. The inhibitory effect of p32 on MLTU transcription appears to require the CAAT box element in the major late promoter, suggesting that p32 may become tethered to the MLTU via an interaction with the CAAT box binding transcription factor.Journal of Virology 06/2006; 80(10):5010-20. · 5.40 Impact Factor