Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by double-stranded RNA

Abstract

Mouse hepatitis virus (MHV) does not induce interferon (IFN) production in fibroblasts or bone marrow-derived dendritic cells. In this report, we show that the essential IFN-beta transcription factors NF-kappaB and IFN regulatory factor 3 are not activated for nuclear translocation and gene induction during infection. However, MHV was unable to inhibit the activation of these factors and subsequent IFN-beta production induced by poly(I:C). Further, MHV infection did not inhibit IFN-beta production mediated by known host pattern recognition receptors (PRRs) (RIG-I, Mda-5, and TLR3). These results are consistent with the notion that double-stranded RNA, produced during MHV infection, is not accessible to cellular PRRs.

FIG. 1.

IFN is not induced in MHV-infected cells, as measured by bioassay. 17Cl-1 cells were infected with MHV-A59 or MHV-JHM for 12 h. As controls, cells were transfected with poly(I:C) or infected with SenV for the same period of time. The amount of IFN was measured by a VSV-based bioassay as described in Materials and Methods. Data are representative of two independent experiments. ND, not detectable.

FIG. 2.

MHV does not induce IFN-β production, as measured by luciferase assay. 17Cl-1 fibroblasts were transfected with pLuc-IFN-β for 16 to 18 h and then infected with MHV-A59 at an MOI of 100 or MHV-JHM at an MOI of 10 for 12 h (A) or 24 h (B). 17Cl-1 cells were also tested after MHV infection at an MOI of 0.1 or 1 (C). 293T cells were transfected with pcDNA-CEACAM1a and pLuc-IFN-β and then infected with MHV-A59 or MHV-JHM for 12 h (D). As positive controls, cells were transfected with poly(I:C) or infected with SenV for the same period of time. As an internal control, a Renilla luciferase construct was transfected concomitantly with pLuc-IFN-β. Cells were harvested and subjected to a dual-luciferase assay as described in Materials and Methods. Data were analyzed, and the ratio of firefly luciferase expression to Renilla luciferase activity is shown. Each sample was assayed in triplicate. Levels of luciferase in poly(I:C)-transfected or SenV-infected cells were significantly greater than levels in mock- or MHV-infected cells (P < 0.005). Data are representative of at least three independent experiments.

FIG. 3.

MHV does not activate NF-κB or IRF-3. (A) 17Cl-1 fibroblasts were transfected with pLuc-(PRDII)₂ or pLuc-(PRDIII-I)₃ and then infected with MHV-A59 or MHV-JHM for 12 h prior to harvest for luciferase analysis. MHV did not induce luciferase activity. Levels of luciferase in poly(I:C)-transfected or SenV-infected cells were significantly greater than levels in mock- or MHV-infected cells (P < 0.005). (B) Cells were infected with MHV-A59 or MHV-JHM for 8 h. Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100-phosphate-buffered saline. Samples were stained with anti-MHV (green) and anti-NF-κB p65 or anti-IRF-3 (red) antibodies. In uninfected (negative control) and in MHV-infected cells, NF-κB and IRF-3 remained in the cytoplasm. NF-κB and IRF-3 localized to the nucleus after poly(I:C) transfection (positive control). Initial magnification, ×40. Data are representative of at least three independent experiments.

FIG. 4.

MHV does not inhibit poly(I:C)-induced IRF-3 or NF-κB activation and IFN-β production. (A) Cells transfected with pLuc-IFN-β (a) or pLuc-(PRDIII-I)₃ (b) were infected with MHV-A59 or MHV-JHM for 5 h and then transfected with poly(I:C) for an additional 6 h. Cells were harvested and subjected to luciferase activity analysis at 11 h p.i. MHV did not inhibit poly(I:C)-induced activation of these reporter genes (P > 0.05). (B) Six hours after MHV infection, cells were transfected with poly(I:C) for an additional 2 h prior to fixation as described above. Cells were then stained with anti-N (green) and anti-IRF-3 (a) or anti-NF-κB p65 (b) (red) antibodies. MHV infection did not inhibit IRF-3 and NF-κB nuclear accumulation occurring after stimulation with poly(I:C). Of note, NF-κB and IRF-3 appear to remain in the cytoplasm in some infected cells, but it is likely that these cells were not successfully transfected with poly(I:C). Initial magnification, ×40. Data are representative of at least three independent experiments.

FIG. 5.

MHV does not inhibit poly(I:C)-induced IRF-3 or NF-κB activation at 10 h p.i. 17Cl-1 cells were infected with MHV-A59 or MHV-JHM for 10 h and then transfected with poly(I:C) for an additional 2 h. Cells were stained with anti-N (green) and anti-IRF3 (A) or anti-NF-κB p65 (B) (red) antibodies. Poly(I:C) transfection resulted in nuclear accumulation of IRF-3 and NF-κB, even at 10 h p.i.

FIG. 6.

IFN-β production mediated by intracellular helicases is not inhibited by MHV. Cells were transiently transfected with pLuc-IFN-β together with empty vector pEF-BOS (vector control), pEF-RIG, or pEF-Mda5. The ratio of plasmids was 1:1. These cells were then infected with MHV-A59 for 5 h prior to transfection with poly(I:C) for an additional 6 h. Cells were harvested at 11 h p.i. and subjected to luciferase assays. Data are representative of at least three independent experiments.

FIG. 7.

TLR3-mediated IFN-β production is not inhibited by MHV. Cells were transfected with pLuc-IFN-β together with empty vector pEF-BOS (vector control), pEF-TRIF, or pEF-TLR3 for 6 to 10 h prior to MHV-A59 infection. Cells were harvested at 12 h p.i. and then subjected to luciferase assays. Data are representative of at least three independent experiments.