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

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.

Figure 1. Rb^−/− MEFs are more susceptible to virus infection.

A, Rb^−/− or WT MEFs were infected in duplicate with VSV at M.O.I. of 5 and a quantification of the virus yield after a total destruction of the monolayers was assessed (left panel). The same results were obtained in at least three different experiments and using MEFs derived from three different wild type or transgenic embryos. Data represent means+/−SE for one experiment. *, P<0.05, compared with WT cells, Student's test. Right panel, Rb^−/− or WT MEFs were infected with VSV at the indicated M.O.I. and 24 h after infection, cell viability was quantified as described in Methods. Indicated values are means+/−SE of triplicate wells for one experiment. The same results were obtained in at least three different experiments and using MEFs derived from three different wild type or transgenic embryos. *, P<0.05, **, P<0.005, compared with WT cells, Student's test. B, Rb^−/− or WT MEFs were infected with VSV at a M.O.I. of 0.5 and at the indicated times, Western-blotting analysis using antibodies against VSV proteins was performed. C, Rb^−/− or WT MEFs were infected with EMCV, sindbis virus or vaccinia virus at M.O.I. of 5 and when the viability of all the cells was lost quantification of the intracellular virus yield (vaccinia virus) or the virus present in the supernatant (EMCV or sindbis virus) was measured. Data represents means+/−SE for four experiments. *, P<0.05 compared with WT cells, Student's test.

Figure 2. 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.

Figure 3. Rb-independent activation of NF-kB in response to TNF-α or poly-IC treatment.

A, MEFs derived from WT or Rb^−/− mice were treated with 20 ng/ml TNF-α (left panel) or transfected with poly-IC (right panel) and at the indicated times Western-blotting analysis of the indicated proteins was carried out. B, WT or Rb^−/− MEFs were transfected with poly-IC and 4 h after transfection 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 GADPH and analyzed. The expression levels were determined relative to GADPH and represented as fold change in Rb^−/− relative to the expression detected in WT cells. C, WT or Rb^−/− MEFs were transfected with poly-IC and 8 h after transfection IFN-β production in the cell culture supernatants was measured by ELISA. Error bars indicate mean+/−SE. ND, not detected.

Figure 4. Reconstitution of Rb^−/− MEFs increases resistance to VSV infection.

A, MEFs WT, Rb^−/−, and Rb^−/− MEFs transduced with retroviral vectors encoding for Rb or GFP were infected with VSV at the indicated M.O.I. After 24 h of infection, cell viability was determined and represented as means+/−SE of triplicate wells (upper panel). **, P<0.005, ***, P<0.0005, Rb-transduced cells compared with GFP-transduced cells, Student's test. Whole cell extracts from Rb WT and transduced Rb^−/− cells were immunoblotted with anti-Rb antibody (lower panel). i, inespecific band. B, Reduction in viral yield after Rb reconstitution of Rb^−/− MEFs. Virus yield assay of the supernatant of the Rb^−/− MEFs transduced with the indicated retroviral vectors after the viability of all the cells was lost as a result of VSV infection (5 M.O.I.). *, P<0.05 Rb-reconstituted cells compared with GFP-transduced Rb^−/− cells, Student's test. C, Rb^−/− MEFs transduced with the indicated retroviral vectors were infected with VSV at M.O.I. of 5 PFU/cell and at the indicated times after infection, cells were collected, extracts prepared and immunoblot analysis using antibodies against total IkB or actin were performed.