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. 2008 Oct;82(20):9880-9.
doi: 10.1128/JVI.00909-08. Epub 2008 Aug 13.

NF-kappaB signaling differentially regulates influenza virus RNA synthesis

Naveen Kumar  1 , Zhong-Tao Xin, Yuhong Liang, Hinh Ly, Yuying Liang
Affiliations

NF-kappaB signaling differentially regulates influenza virus RNA synthesis

Naveen Kumar et al. J Virol. 2008 Oct.

Abstract

The NF-kappaB signaling pathway has previously been shown to be required for efficient influenza A virus replication, although the molecular mechanism is not well understood. In this study, we identified a specific step of the influenza virus life cycle that is influenced by NF-kappaB signaling by using two known NF-kappaB inhibitors and a variety of influenza virus-specific assays. The results of time course experiments suggest that the NF-kappaB inhibitors Bay11-7082 and ammonium pyrrolidinedithiocarbamate inhibited an early postentry step of viral infection, but they did not appear to affect the nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. Instead, we found that the levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by the inhibitors in virus-infected cells, indicating that NF-kappaB signaling is intimately involved in the vRNA synthesis. Furthermore, we showed that the NF-kappaB inhibitors specifically diminished influenza virus RNA transcription from the cRNA promoter but not from the vRNA promoter in a reporter assay, a result which is consistent with data obtained from virus-infected cells. The overexpression of the p65 NF-kappaB molecule could not only eliminate the inhibition but also activate influenza virus RNA transcription from the cRNA promoter. Finally, using p65-specific small interfering RNA, we have shown that p65 knockdown reduced the levels of influenza virus replication and vRNA synthesis. In summary, we have provided evidence showing, for the first time, that the NF-kappaB host signaling pathway can differentially regulate influenza virus RNA synthesis, which may also offer some new perspectives into understanding the host regulation of RNA synthesis by other RNA viruses.

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Figures

FIG. 1.
FIG. 1.
Influenza A virus infection strongly activates NF-κB signaling in human lung epithelial cells early in the infection and throughout the infection. A549 cells were infected with influenza A virus (WSN) at an MOI of 5. At different time points postinfection, nuclear extracts were prepared and subjected to gel shift assays with NF-κB-specific probes. c, oligonucleotide only; m, mock infection; 15′, 15 min.
FIG. 2.
FIG. 2.
NF-κB inhibitors decrease influenza A virus production and virus gene expression in virus-infected cells. (A) NF-κB inhibitors decrease influenza A virus production. A549 cells were infected with influenza A virus WSN at an MOI of 5 for 1 h, after which medium containing either a vehicle control (PBS or DMSO) or an NF-κB inhibitor (10 μM Bay11 or 50 μM PDTC) was added to the infected cells. The virus titers in the supernatants at 9 hpi were determined by plaque assays in three independent experiments. (B) Schematic illustration of the generation of the WSN-LUC virus. As detailed in Materials and Methods, 293T cells were transfected with eight plasmids required to generate infectious influenza A virus WSN, together with a plasmid carrying a LUC-encoding viral reporter PB2-LUC RNA segment. Viruses were harvested from the supernatants at 48 h posttransfection. (C) Ribavirin and NF-κB inhibitors inhibit LUC expression in the WSN-LUC-infected cells. WSN-LUC viruses were used to infect fresh MDCK cells at an MOI of 1 for 1 h. DMSO, different concentrations of ribavirin, or an NF-κB inhibitor (10 μM Bay11 or 50 μM PDTC) was applied at 1 hpi, and a LUC assay was conducted at 8 hpi. * represents statistical significance (P < 0.05).
FIG. 3.
FIG. 3.
Time course analysis of the effects of NF-κB inhibitors on virus production. A549 cells were infected with influenza A virus WSN at an MOI of 5. Chemical treatment (10 μM Bay11 or 50 μM PDTC) or a vehicle control (PBS or DMSO) was applied at different times postinfection. The virus titers in the supernatants at 9 hpi were determined by plaque assays. −1 h, 1 h before infection; 1, 2, 3, 5, and 7 h, different times postinfection.
FIG. 4.
FIG. 4.
NF-κB inhibitors do not affect the nucleocytoplasmic trafficking of vRNPs. A549 cells were infected with influenza A virus WSN at an MOI of 5 in the presence of either a vehicle control (PBS or DMSO) or a chemical compound (10 μM U0126, 10 μM Bay11, or 50 μM PDTC). At different times postinfection, cells were stained with anti-NP antibody and observed via microscopy.
FIG. 5.
FIG. 5.
NF-κB inhibitors differentially decrease influenza virus RNA transcription from the cRNA promoter compared to that from the vRNA promoter. As described in Materials and Methods, 293T cells were transfected with the five plasmids to recreate the LUC-based influenza virus RNA transcription assay, along with a reporter construct based on either the cRNA (cRNA-LUC) or the vRNA (vRNA-LUC) promoter. Treatment with a control (PBS) or chemical (50 μM PDTC or 10 ng of ribavirin/μl) was applied at 8 h posttransfection. LUC activity was determined at 24 h posttransfection, normalized by the β-gal level, and compared to the activity in the cells subjected to the control PBS treatment (set as 100%). * represents statistical significance (P < 0.05).
FIG. 6.
FIG. 6.
Effect of individual NF-κB molecules on influenza virus RNA transcription. (A) The overexpression of p65 abolishes the inhibition of influenza virus RNA transcription from the cRNA promoter by the NF-κB inhibitors. 293T cells were transfected with five plasmids to recreate influenza virus RNA transcription from the cRNA-LUC reporter, together with increasing amounts of either an empty vector or a vector expressing p50, p65, or c-Rel. Control treatment (PBS, indicated by −) or chemical treatment (50 μM PDTC, indicated by +) was applied at 8 h posttranfection. LUC activity was measured at 24 h posttransfection and normalized by the β-gal level. (B) The overexpression of p65 increases influenza virus RNA transcription from the cRNA promoter. 293T cells were transfected with the five plasmids to recreate influenza virus RNA transcription from the cRNA promoter, together with increasing amounts (indicated by triangles) of either an empty vector or a vector expressing p50, p65, or c-Rel. LUC activity was measured at 24 h posttransfection and normalized by the β-gal enzymatic activity. (C) A549 cells were transfected with p65 or control siRNA as described in Materials and Methods. Cell lysates were analyzed by Western blotting with antibodies against p65 and β-actin. (D) A549 cells were transfected with p65 or control siRNA and then infected with influenza A virus (WSN). The virus titer was determined at 9 hpi. (E) A549 cells were transfected with p65 or control siRNA and then infected with influenza A virus. Viral RNA was prepared at 5 hpi and quantified by real-time RT-PCR as described in Materials and Methods. The differences in CT values [ΔC(t)] between control and p65 siRNA-treated samples and the results of the statistical analysis of significance (P values) are shown. * represents statistical significance (P < 0.05).
FIG. 7.
FIG. 7.
Diagram of the corkscrew structures of cRNA and vRNA promoters. The differences between the cRNA and vRNA promoters are highlighted by circles and arrows.
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