Influenza virus inhibits RNA polymerase II elongation

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Virology (Impact Factor: 3.32). 08/2006; 351(1):210-7. DOI: 10.1016/j.virol.2006.03.005
Source: PubMed


The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation (ChIP) analysis, that similar amounts of Pol II associate with Pol II promoter DNAs in influenza virus-infected and mock-infected cells. However, there is a statistically significant reduction in Pol II densities in the coding region of Pol II genes in infected cells. Thus, influenza virus specifically interferes with Pol II elongation, but not Pol II initiation. We propose that influenza virus RNA polymerase, by binding to the CTD of initiating Pol II and subsequent cleavage of the capped 5' end of the nascent transcript, triggers premature Pol II termination.

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    • "Alpha-amanitin inhibits both RNA polymerase II initiation and elongation (39) and thus, like ActD, prevents synthesis of viral mRNA, cRNA, and vRNA (33, 34). In contrast, the RNA polymerase II elongation inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) (40, 41) does not affect primary viral transcription but inhibits cRNA and vRNA synthesis (and therefore secondary viral transcription) (42) by preventing the nuclear export of viral transcripts (23, 43). Consistent with previous publications, no viral mRNA, cRNA, or vRNA synthesis can be detected in infected cells following treatment with either α-amanitin or ActD (Fig. 6A). "
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    ABSTRACT: Unlabelled: We have examined the requirements for virus transcription and replication and thus the roles of input and progeny genomes in the generation of interferon (IFN)-inducing pathogen-associated molecular patterns (PAMPs) by influenza A viruses using inhibitors of these processes. Using IFN regulatory factor 3 (IRF3) phosphorylation as a marker of activation of the IFN induction cascade that occurs upstream of the IFN-β promoter, we demonstrate strong activation of the IFN induction cascade in A549 cells infected with a variety of influenza A viruses in the presence of cycloheximide or nucleoprotein (NP) small interfering RNA (siRNA), which inhibits viral protein synthesis and thus complementary ribonucleoprotein (cRNP) and progeny viral RNP (vRNP) synthesis. In contrast, activation of the IFN induction cascade by influenza viruses was very effectively abrogated by treatment with actinomycin D and other transcription inhibitors, which correlated with the inhibition of the synthesis of all viral RNA species. Furthermore, 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole, an inhibitor that prevents viral RNA export from the nucleus, was also a potent inhibitor of IRF3 activation; thus, both viral RNA synthesis and nuclear export are required for IFN induction by influenza A viruses. While the exact nature of the viral PAMPs remains to be determined, our data suggest that in this experimental system the major influenza A virus PAMPs are distinct from those of incoming genomes or progeny vRNPs. Importance: The host interferon system exerts an extremely potent antiviral response that efficiently restricts virus replication and spread; the interferon response can thus dictate the outcome of a virus infection, and it is therefore important to understand how viruses induce interferon. Both input and progeny genomes have been linked to interferon induction by influenza viruses. However, our experiments in tissue culture cells show that viral RNA synthesis and nuclear export are required to activate this response. Furthermore, the interferon induction cascade is activated under conditions in which the synthesis of progeny genomes is inhibited. Therefore, in tissue culture cells, input and progeny genomes are not the predominant inducers of interferon generated by influenza A viruses; the major viral interferon inducer(s) still remains to be identified.
    Journal of Virology 01/2014; 88(8). DOI:10.1128/JVI.03109-13 · 4.44 Impact Factor
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    • "It has been suggested that this may increase the efficiency of the cap-snatching process, as well as linking viral transcription to cellular pathways of mRNA processing and nuclear export [16] [17] [18]. It has also been shown that this interaction increases the efficiency of host shutoff as the viral polymerase not only inhibits Pol II elongation but causes it to be degraded [15] [19] [20], a mechanism that appears to contribute to virulence [21]. As viral transcription, but not replication , is dependent on Pol II-generated capped transcripts, the suppression of Pol II synthesis as infection proceeds may contribute to the decline in viral mRNA synthesis late in infection [16]. "
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    ABSTRACT: Unusually for an RNA virus, influenza A viruses transcribe and replicate their genomes in the nuclei of infected cells. As a result the viral ribonucleoprotein complexes (RNPs), and their newly synthesised protein subunits, must interact with the host nuclear import machinery. In this review we discuss how the virus exploits nuclear import pathways to allow regulated and chaperoned assembly of RNPs in the nucleus, and describe how the import machinery itself can be a determinant of host tropism.
    Vaccine 05/2012; 30(51). DOI:10.1016/j.vaccine.2012.04.085 · 3.62 Impact Factor
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    • "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 [8], 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 [9]. "
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    ABSTRACT: 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 RNAPII(a) in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPII(a)) to hyperphosphorylated RNAPII (RNAPII(o)).
    Virology Journal 03/2011; 8:120. DOI:10.1186/1743-422X-8-120 · 2.18 Impact Factor
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