TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5

Abstract

Here we address the role of RIG-I and TLR3 in differential cytokine responses against Simian Virus 5 (SV5) and two distinct cytokine inducing SV5 mutants. IFN-beta and IL-6 secretion was induced by infection with P/V-CPI-, an SV5 mutant with P/V substitutions, and were reduced by either siRNA-mediated knockdown of RIG-I expression or by expression of a dsRNA-binding protein. TLR3 overexpression did not alter cytokine secretion induced by P/V-CPI- or by Le-(U5C, A14G), an SV5 promoter mutant. TLR3 signaling by addition of exogenously added dsRNA was not blocked by WT SV5 or either SV5 mutant. Unexpectedly, TLR3 activation in infected cells led to enhanced IL-8 secretion, which correlated with increased RIG-I expression. Dominant negative RIG-I and TRIF supported a model whereby TLR3 activation upregulates RIG-I expression and in turn hypersensitizes cells to RIG-I-mediated cytokine secretion. Implications for crosstalk between different innate immunity pathways in mounting antiviral responses to paramyxoviruses are discussed.

Figure 1. RIG-I contributes to cytokine induction by P/V-CPI-

A) Reporter Gene Assay. A549 cells were co-transfected for 16 h with a plasmid expressing luciferase under control of the IFN-beta promoter and a either a RIG-I DN plasmid or its empty vector (control), and pSV-beta-galactosidase for normalization. Sixteen hrs pt, cells were infected at an moi of 10 with the indicated viruses. At 24 h pi lysates were harvested and analyzed for levels of luciferase activity. Results are expressed as fold over mock and error bars indicate standard deviation of triplicate samples. B) siRNA knockdown of RIG-I. A549 cells were left untreated (lanes 1 and 4) or transfected with 100 uM of siRNA targeting RIG-I (lanes 2 and 5) or GAPDH (lanes 3 and 6) as a control. At 48 and 72 h pt, levels of RIG-I, GAPDH, and actin were assayed by Western blotting. C) Cytokine secretion. A549 cells were transfected with siRNA targeting GAPDH or RIG-I and at 48 h pt cells were infected at an moi of 10 with SV5-GFP or P/V-CPI-. At 24 h pi (72 h pt), levels of IL-6 (left panel) or IFN-beta (right panel) were measured by ELISA. Results are expressed as mean values from triplicate samples with error bars representing standard deviation and values are normalized to 10⁶ cells. *, p value less than 0.008 compared to corresponding samples from GAPDH siRNA treated control cells.

Figure 2. Role of dsRNA in cytokine induction by P/V-CPI-

A) Virus growth. Control or Sigma3-expressing A549 cells were infected at an moi of 0.05 with WT SV5-GFP or P/V-CPI- and virus titers were determined at the indicated times post infection. Results are representative of two independent experiments. B) Cytokine induction. Control or sigma3-expressing A549 cells were infected at an moi of 10 with SV5-GFP or P/V-CPI-. At 24 h pi, media was analyzed for levels of IL-6 by ELISA (left panel) or Type I IFN by bioassay (right panel). Results are expressed as mean values from triplicate samples with error bars representing standard deviation. For IL-6, values are normalized to 10⁶ cells. *, p value less than 0.004 compared to corresponding control cells. C) IRF3 nuclear translocation. Control or sigma3 A549 cells were infected at an moi of 10 with the indicated viruses. At 24 h pi, cells were stained with DAPI and with antibodies specific for IRF-3.

Figure 3. WT and mutant SV5 do not activate TLR3 pathways, but they are also not able to block dsRNA-mediated TLR3 signaling

A) Overexpression of TLR3. A549 cells were co-transfected with a plasmid expressing TLR3 or its empty vector (as a control) along with a plasmid encoding luciferase under the control of the IFN-beta promoter. At 16 h pt, cells were infected with indicated viruses at an moi of 10. At 24 h pi, lysates were harvested and luciferase activity was determined. B) Infection of 293 cells. 293 cells stably expressing TLR3 or LacZ (as a control) were mock infected or infected at an moi of 10 with WT SV5-GFP, or the mutants P/V-CPI-. At 24 h pi, cells were examined for GFP expression. C) 293 cells stably expressing TLR3 or LacZ (as a control) were mock infected or infected at an moi of 10 with WT SV5-GFP, or the mutants P/V-CPI- or Le-(U5C, A14G). As a positive control, cells were treated with exogenous dsRNA (PolyI:C; 5 ug/ml). At 24 h pi, media were analyzed by ELISA for levels of IL-8. D) 293-TLR3 were mock infected or infected at an moi of 10 with the indicated viruses and 6 h later were left untreated or challenged with exogenous dsRNA (PolyI:C; 5 ug/ml). At 24 h pi media was harvested and analyzed by ELISA for levels of IL-8. For all panels, results are expressed as mean values from triplicate samples with error bars representing standard deviation. IL-8 values are normalized to 10⁶ cells. Le Mut; Le-(U5C, A14G).

Figure 4. TLR3 signaling increases RIG-I expression, but is not dependent on IFN signaling

A and B) dsRNA increases RIG-I expression. 293 cells expressing TLR3 or LacZ (control) were treated without (−) or with (+) exogenous dsRNA (PolyI:C; 5 ug/ml). After 18 h, lysates were harvested and analyzed by Western blotting for levels of RIG-I and actin (panel A) or by RT-PCR for levels of RIG-I and GAPDH RNA. C) IFN-beta secretion. 293 cells expressing TLR3 or LacZ were treated without (−) or with (+) 5 ug/ml PolyI:C dsRNA and after 18 h media was collected and analyzed by ELISA for IFN-beta. Media from A549 cells infected with P/V-CPI- was used as a positive control. Data are from triplicate samples and error bars represent standard deviation. D) STAT1 levels. 293-TLR3 cells were mock infected or infected at an moi of 10 with the indicated viruses. At 6 h pi, cells were left untreated or treated with (+) dsRNA. Six hr later, cell lysates were analyzed by western blotting for STAT1 and actin.

Figure 5. Kinetics of RIG-I induction and cytokine secretion following infection and treatment with dsRNA

293-TLR3 cells were mock infected or infected at an moi of 10 with the indicated viruses. At 6 h pi, cells were left untreated or treated with (+) 5 ug/ml PolyI:C dsRNA. At the indicated times post challenge with dsRNA, cell lysates were analyzed for levels of RIG-I and actin (panel A) or media was analyzed for levels of IL-8 by ELISA (panel B). For panel B, data are from triplicate samples and error bars represent standard deviation from the mean.

Figure 6. dsRNA-induced enhancement of IL-8 synthesis in SV5 infected cells is dependent on RIG-I

293-TLR3 cells were co-transfected with plasmids encoding luciferase under control of the IL-8 promoter, a DN form of RIG-I or the empty vector (control), and pSV-beta-galactosidase for normalization. Sixteen hrs pt, cells were infected with the indicated viruses at an moi of 10. Six hours later, cells were left untreated or treated with dsRNA (PolyI:C; 5 ug/ml). Lysates were harvested at 24 h pi and analyzed for luciferase activity. Results are expressed as fold over that seen with mock infected cells not treated with dsRNA. Data are from triplicate samples and error bars represent standard deviation. *; p<0.05 comparing SV5-GFP infected control cells with infected cells expressing DN RIG-I; #, p<0.02 comparing P/V mutant infected control cells with infected cells expressing DN RIG-I. P/V Mut, P/V-CPI-; Le Mut, Le-(U5C, A14G).

Figure 7. dsRNA-induced enhancement of IL-8 synthesis in SV5 infected cells is dependent on TRIF

293-TLR3 cells were co-transfected with plasmids encoding luciferase under the control of the IL-8 promoter, a dominant negative form of TRIF or the empty vector (control), and pSV-beta-galactosidase for normalization. Sixteen hrs pt, cells were infected at an moi of 10 with the indicated viruses. Six hours later, cells were left untreated or treated with dsRNA (PolyI:C; 5 ug/ml). Lysates were harvested at 24 h pi and analyzed for luciferase activity. Results are expressed as fold over that seen with mock infected cells not treated with dsRNA. Data are from triplicate samples and error bars represent standard deviation. *; p<0.02 comparing SV5-GFP infected control cells with infected cells expressing DN TRIF; #, p<0.001 comparing P/V mutant infected control cells with infected cells expressing DN TRIF; ^, p<0.005 comparing Leader mutant infected control cells with infected cells expressing DN TRIF. P/V Mut, P/V-CPI-; Le Mut, Le-(U5C, A14G).