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Activation of NF-kB pathway by virus infection requires Rb expression.

Garcia MA, Gallego P, Campagna M, González-Santamaría J, Martínez G, Marcos-Villar L, Vidal A, Esteban M, Rivas C - PLoS ONE (2009)

Bottom Line: Besides these roles, additional functions in the control of immune response have been suggested.Here we show that virus replication is increased by the absence of Rb, and that Rb is required for the activation of the NF-kB pathway in response to virus infection.These results reveal a novel role for tumor suppressor Rb in viral infection surveillance and further extend the concept of a link between tumor suppressors and antiviral activity.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Madrid, Spain.

ABSTRACT
The retinoblastoma protein Rb is a tumor suppressor involved in cell cycle control, differentiation, and inhibition of oncogenic transformation. Besides these roles, additional functions in the control of immune response have been suggested. In the present study we investigated the consequences of loss of Rb in viral infection. Here we show that virus replication is increased by the absence of Rb, and that Rb is required for the activation of the NF-kB pathway in response to virus infection. These results reveal a novel role for tumor suppressor Rb in viral infection surveillance and further extend the concept of a link between tumor suppressors and antiviral activity.

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Rb expression is required for NF-kB pathway activation in response to VSV infection.A, Kinetics of IkBα phosphorylation on serine 32 and serine 36 and total IkB upon VSV infection at M.O.I. of 5 PFU/cell in wild type (left panel) or Rb−/− (right panel) primary MEFs were assessed by Western-blotting analysis. B, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 8 h after infection cells were fixed, immunostained for p65 (green) and DAPI (blue). C, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 12 h after infection total RNA was isolated. After reverse transcription the samples were amplified by TaqMan-based QRT-PCR using taqman probes for TNF-α, IFN-β, and GADPDH, and analyzed. The expression levels were determined relative to GAPDH and represented as fold change in Rb−/− cells relative to the expression detected in WT cells. All errors bars indicate mean+/−SE. D, MEFs were infected with VSV at M.O.I. of 5 and 24 h after infection IFN-β production in the cell culture supernatants was measured by ELISA. All errors bars indicate mean+/−SE. ND, not detected. E, Rb−/− or WT MEFs were infected with VSV at M.O.I. of 5 and 7 h after infection, Western-blotting analysis using the indicated antibodies was performed.
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pone-0006422-g002: Rb expression is required for NF-kB pathway activation in response to VSV infection.A, Kinetics of IkBα phosphorylation on serine 32 and serine 36 and total IkB upon VSV infection at M.O.I. of 5 PFU/cell in wild type (left panel) or Rb−/− (right panel) primary MEFs were assessed by Western-blotting analysis. B, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 8 h after infection cells were fixed, immunostained for p65 (green) and DAPI (blue). C, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 12 h after infection total RNA was isolated. After reverse transcription the samples were amplified by TaqMan-based QRT-PCR using taqman probes for TNF-α, IFN-β, and GADPDH, and analyzed. The expression levels were determined relative to GAPDH and represented as fold change in Rb−/− cells relative to the expression detected in WT cells. All errors bars indicate mean+/−SE. D, MEFs were infected with VSV at M.O.I. of 5 and 24 h after infection IFN-β production in the cell culture supernatants was measured by ELISA. All errors bars indicate mean+/−SE. ND, not detected. E, Rb−/− or WT MEFs were infected with VSV at M.O.I. of 5 and 7 h after infection, Western-blotting analysis using the indicated antibodies was performed.

Mentions: VSV infection induces several cellular immunomodulatory pathways with antiviral activity such as the NF-kB pathway. The initial key segment of the NF-kB activation pathway involves IKK activation, IKK phosphorylation of IkBα, and consequent IkBα degradation [22]. Analysis of the activation of this pathway was then carried out in WT or Rb−/− cells. Cells were infected with VSV at M.O.I. of 5 and at different times after infection, IkB phosphorylation and IkB degradation were assessed by Western-blotting. As shown in Figure 2A, IkB phosphorylation was detected by 2 h after VSV infection in WT cells. In agreement with these results IkBα degradation was evident by 3 h after VSV infection of WT cells and was complete by 5 hpi (Figure 2A). In parallel, analysis of Rb−/− cells revealed a basal level of phosphorylated IkB that was slightly increased between 1 and 4 hpi. However, no degradation of IkB was detected at any time after VSV infection of Rb−/− cells, indicating Rb-dependent IkB degradation in response to VSV infection. The second key segment of the NF-kB activation pathway is nuclear translocation of NF-kB subunits and their dimerization and binding to the kB enhancer. Nuclear translocation of NF-kB in response to VSV infection, as a consequence of IkB degradation, was then tested in both WT and Rb−/− MEFs by immunofluorescence. As shown in Figure 2B, nuclear translocation of the p65 subunit of NF-kB was clearly detected at 8 h after infection in WT cells. However, most of the Rb−/− cells showed cytoplasmic staining for p65 at 8 hpi, confirming that VSV-induced nuclear translocation of p65 was also dependent on Rb. The NF-kB transcription factor promotes the expression of many different genes implicated in the antiviral response. To explore the correlation between the nuclear translocation of NF-kB and the induction of NF-kB targets, upregulation of TNF-α or IFN-β genes at 12 h after VSV infection, in WT and Rb−/− MEFs was measured. Semiquantitative RT-PCR showed that relative TNF-α gene transcription levels in non-infected Rb−/− cells was similar to that detected in WT cells (Figure 2C, left panel). Interestingly, a downregulation of the steady-state levels of IFN-β mRNA in Rb−/− cells in comparison with WT cells was found (Figure 2C, left panel), as it has been reported for the interferon-induced Ifi202 gene [23]. Reduced transactivation of both TNF-α and IFN-β in Rb−/− cells in response to VSV infection was observed (Figure 2C, right panel). The impairment of IFN-β production in the absence of Rb after VSV infection was confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) (Figure 2D). These data indicate that a defect in the activation of the NF-kB pathway and, consequently, in the production of IFN-β in Rb−/− MEFs may explain their increased susceptibility to VSV infection. Activation of NF-kB and eIF2-α by VSV occur in a dsRNA-dependent protein kinase (PKR)-dependent manner [24], [25]. Analysis of PKR activation in Rb−/− and WT MEFs in response to VSV infection revealed decreased levels of both phospho-PKR and phospho-eI2F-α proteins in Rb−/− cells (Figure 2E). Taken together, these results demonstrate the requirement of Rb to activate NF-kB in response to VSV infection and point to PKR as a possible mediator.


Activation of NF-kB pathway by virus infection requires Rb expression.

Garcia MA, Gallego P, Campagna M, González-Santamaría J, Martínez G, Marcos-Villar L, Vidal A, Esteban M, Rivas C - PLoS ONE (2009)

Rb expression is required for NF-kB pathway activation in response to VSV infection.A, Kinetics of IkBα phosphorylation on serine 32 and serine 36 and total IkB upon VSV infection at M.O.I. of 5 PFU/cell in wild type (left panel) or Rb−/− (right panel) primary MEFs were assessed by Western-blotting analysis. B, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 8 h after infection cells were fixed, immunostained for p65 (green) and DAPI (blue). C, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 12 h after infection total RNA was isolated. After reverse transcription the samples were amplified by TaqMan-based QRT-PCR using taqman probes for TNF-α, IFN-β, and GADPDH, and analyzed. The expression levels were determined relative to GAPDH and represented as fold change in Rb−/− cells relative to the expression detected in WT cells. All errors bars indicate mean+/−SE. D, MEFs were infected with VSV at M.O.I. of 5 and 24 h after infection IFN-β production in the cell culture supernatants was measured by ELISA. All errors bars indicate mean+/−SE. ND, not detected. E, Rb−/− or WT MEFs were infected with VSV at M.O.I. of 5 and 7 h after infection, Western-blotting analysis using the indicated antibodies was performed.
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Related In: Results  -  Collection

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pone-0006422-g002: Rb expression is required for NF-kB pathway activation in response to VSV infection.A, Kinetics of IkBα phosphorylation on serine 32 and serine 36 and total IkB upon VSV infection at M.O.I. of 5 PFU/cell in wild type (left panel) or Rb−/− (right panel) primary MEFs were assessed by Western-blotting analysis. B, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 8 h after infection cells were fixed, immunostained for p65 (green) and DAPI (blue). C, Wild type or Rb−/− MEFs were infected with VSV at M.O.I. of 5 and 12 h after infection total RNA was isolated. After reverse transcription the samples were amplified by TaqMan-based QRT-PCR using taqman probes for TNF-α, IFN-β, and GADPDH, and analyzed. The expression levels were determined relative to GAPDH and represented as fold change in Rb−/− cells relative to the expression detected in WT cells. All errors bars indicate mean+/−SE. D, MEFs were infected with VSV at M.O.I. of 5 and 24 h after infection IFN-β production in the cell culture supernatants was measured by ELISA. All errors bars indicate mean+/−SE. ND, not detected. E, Rb−/− or WT MEFs were infected with VSV at M.O.I. of 5 and 7 h after infection, Western-blotting analysis using the indicated antibodies was performed.
Mentions: VSV infection induces several cellular immunomodulatory pathways with antiviral activity such as the NF-kB pathway. The initial key segment of the NF-kB activation pathway involves IKK activation, IKK phosphorylation of IkBα, and consequent IkBα degradation [22]. Analysis of the activation of this pathway was then carried out in WT or Rb−/− cells. Cells were infected with VSV at M.O.I. of 5 and at different times after infection, IkB phosphorylation and IkB degradation were assessed by Western-blotting. As shown in Figure 2A, IkB phosphorylation was detected by 2 h after VSV infection in WT cells. In agreement with these results IkBα degradation was evident by 3 h after VSV infection of WT cells and was complete by 5 hpi (Figure 2A). In parallel, analysis of Rb−/− cells revealed a basal level of phosphorylated IkB that was slightly increased between 1 and 4 hpi. However, no degradation of IkB was detected at any time after VSV infection of Rb−/− cells, indicating Rb-dependent IkB degradation in response to VSV infection. The second key segment of the NF-kB activation pathway is nuclear translocation of NF-kB subunits and their dimerization and binding to the kB enhancer. Nuclear translocation of NF-kB in response to VSV infection, as a consequence of IkB degradation, was then tested in both WT and Rb−/− MEFs by immunofluorescence. As shown in Figure 2B, nuclear translocation of the p65 subunit of NF-kB was clearly detected at 8 h after infection in WT cells. However, most of the Rb−/− cells showed cytoplasmic staining for p65 at 8 hpi, confirming that VSV-induced nuclear translocation of p65 was also dependent on Rb. The NF-kB transcription factor promotes the expression of many different genes implicated in the antiviral response. To explore the correlation between the nuclear translocation of NF-kB and the induction of NF-kB targets, upregulation of TNF-α or IFN-β genes at 12 h after VSV infection, in WT and Rb−/− MEFs was measured. Semiquantitative RT-PCR showed that relative TNF-α gene transcription levels in non-infected Rb−/− cells was similar to that detected in WT cells (Figure 2C, left panel). Interestingly, a downregulation of the steady-state levels of IFN-β mRNA in Rb−/− cells in comparison with WT cells was found (Figure 2C, left panel), as it has been reported for the interferon-induced Ifi202 gene [23]. Reduced transactivation of both TNF-α and IFN-β in Rb−/− cells in response to VSV infection was observed (Figure 2C, right panel). The impairment of IFN-β production in the absence of Rb after VSV infection was confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) (Figure 2D). These data indicate that a defect in the activation of the NF-kB pathway and, consequently, in the production of IFN-β in Rb−/− MEFs may explain their increased susceptibility to VSV infection. Activation of NF-kB and eIF2-α by VSV occur in a dsRNA-dependent protein kinase (PKR)-dependent manner [24], [25]. Analysis of PKR activation in Rb−/− and WT MEFs in response to VSV infection revealed decreased levels of both phospho-PKR and phospho-eI2F-α proteins in Rb−/− cells (Figure 2E). Taken together, these results demonstrate the requirement of Rb to activate NF-kB in response to VSV infection and point to PKR as a possible mediator.

Bottom Line: Besides these roles, additional functions in the control of immune response have been suggested.Here we show that virus replication is increased by the absence of Rb, and that Rb is required for the activation of the NF-kB pathway in response to virus infection.These results reveal a novel role for tumor suppressor Rb in viral infection surveillance and further extend the concept of a link between tumor suppressors and antiviral activity.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Madrid, Spain.

ABSTRACT
The retinoblastoma protein Rb is a tumor suppressor involved in cell cycle control, differentiation, and inhibition of oncogenic transformation. Besides these roles, additional functions in the control of immune response have been suggested. In the present study we investigated the consequences of loss of Rb in viral infection. Here we show that virus replication is increased by the absence of Rb, and that Rb is required for the activation of the NF-kB pathway in response to virus infection. These results reveal a novel role for tumor suppressor Rb in viral infection surveillance and further extend the concept of a link between tumor suppressors and antiviral activity.

Show MeSH
Related in: MedlinePlus