- Short report
- Open Access
DNA intercalator stimulates influenza transcription and virus replication
© Li and Poon; licensee BioMed Central Ltd. 2011
- Received: 1 February 2011
- Accepted: 15 March 2011
- Published: 15 March 2011
Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPIIa in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPIIa) to hyperphosphorylated RNAPII (RNAPIIo).
- Virus Replication
- MDCK Cell
- Transcription Elongation
- Viral Polymerase
The C-terminal domain (CTD) of RNAPII is important for cellular mRNA transcription, and interacts with several post-transcriptional factors for RNA maturation and nuclear export. The phosphorylation status of CTD is known to be a critical regulatory checkpoint for RNAPII transcription . The hyperphosphorylated (transcriptionally engaged) form of RNAPII is designated as RNAPIIo, whereas its nonphosphorylated (transcriptionally inactive) form is designated as RNAPIIa. At the early stage of transcription, free RNAPIIa interacts with other general transcription factors on cellular DNA promoters to form a transcription pre-initiation complex, which is followed by transcription initiation . The newly initiated RNAPIIa then proceeds to the promoter-proximal pause region, and the paused RNAPIIa is subsequently hyperphosphorylated, preferably on the serine 5 (Ser5) positions, by cyclin-dependent kinase (Cdk) 7. As transcription elongation proceeds, the serine 2 (Ser2) and Ser5 positions in the CTD of RNAPII are hyperphosphorylated by Cdk9  and dephosphorylated by SCP1 , respectively. The Ser5-phosphorylation helps to recruit enzymes to cap the nascent RNA transcript, whereas the Ser2-phosphorylation facilitates the conversion of RNAPII into a productive elongating form.
Influenza viral RNA synthesis is dependent on its host transcription machinery. Various RNAPII inhibitors such as α-amantin and actinomycin D (ActD) have been shown to inhibit influenza virus replication [5–7]. Chan et al. demonstrated that the influenza viral polymerase complex can inhibit RNAPII transcription elongation, but not initiation , a phenomenon that is similar to the transcriptional arrest of RNAPII. This transcriptional arrest may be related to direct interaction between vRNP and Ser5-phosphorylated RNAPIIo . It has also been demonstrated that a robust polymerase complex is more capable of binding to RNAPIIo . Recently, influenza viral polymerase has been proposed to induce the direct degradation of RNAPIIa [11–13], thereby inhibiting host gene expression. The overall conclusion of these previous findings is that RNAPII plays a critical role in viral RNA transcription, although little is known about the mechanism responsible for RNAPIIa disappearance during infection. Moreover, the role played by the post-translation modification of RNAPII in viral RNA synthesis is yet to be determined. In this study, we would like to determine the effect of various RNAPII inhibitors on influenza viral polymerase functions and virus replications. In particular, the inhibitors used in this study are known to inhibit RNAPII via different mechanisms and have different effects on the phosphorylation status of RNAPII. It is of our interest to use these chemicals to understand how the influenza virus can utilize RNAPII to facilitate viral RNA synthesis.
Influenza A virus infection results in a significant loss of transcriptionally inactive RNAPII (RNAPIIa) [11–13]. However, as influenza polymerase requires capped primers snatched from the host nuclear RNA for its viral RNA transcription [20–22], a direct induction of RNAPIIa degradation via the viral polymerase may not favour such a transcription. In this study, it has been demonstrated here that the disappearance of RNAPIIa is related to a shift of RNAPIIa to RNAPIIo (Figure 3). In addition, this conversion of RNAPIIa to RNAPIIo is found to be important to viral RNA synthesis, which suggests that newly synthesized RNAPIIo may be a critical determinant of viral transcription. RNAPII can be subjected to various post-translational modifications . Further investigation of the post-translational modification of RNAPII in influenza virus-infected cells may help us to better understand the transcription and replication of influenza viruses.
This project is supported by National Institutes of Health (NIAID contract HHSN266200700005C, LLM), Research Grant Council of Hong Kong (HKU 7530/06M, LLM), Area of Excellence Scheme of the University Grants Committee (Grant AoE/M-12/06). We thank George Brownlee for plasmids, Ervin Fodor and Hui-Ling Yen for critical comments.
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