A. HEK293-TLR3 cells were cotransfected with pIFN-β-Luc and pCMVβgal plasmids, and a vector encoding SARS PLpro-Sol or SARS PLpro-TM, BVDV Npro, or A20, or an empty vector. Twenty-four hours later, cells were either mock-treated (empty bars), or incubated with 50 μg/ml poly (I-C) in culture medium for 6h (hatched bars, left panel), or infected with SeV at 100 HAU/ml for 16 h (solid bars, right panel) prior to cell lysis for both luciferase and β-galactosidase assays. Bars show relative luciferase activity normalized to β-galactosidase activity, i.e, IFN-β promoter activity. B and C. IFN-β promoter activity in cells transfected with increasing amounts of WT or C1651A mutant PLpro-TM-expressing plasmid (B) or equivalent amounts of WT or D1826A mutant PLpro-TM plasmid (C). D and E. IFN-β promoter activity in cells transfected with increasing amounts of WT or C1651A mutant PLpro-Sol-expressing plasmid (D) or equivalent amounts of WT, C1651A or D1826A mutant PLpro Sol plasmids (E).

A. Immunofluorescence staining of PLpro-TM in HeLa PLpro-4 cells cultured in the presence (left) or absence (right) of 2 μg/ml tetracycline for 3 days followed by confocal microscopy. PLpro-TM showed green fluorescence staining (detected with a V5 tag antibody), while nuclei were counterstained blue with DAPI. B. The left panels show HeLa PLpro-4 and PLpro-10 cells that were cultured with and without tetracycline for 3 days and subsequently mock-infected or challenged with SeV for 16 h prior to cell lysis and immunoblot blot analysis of PLpro (using a V5 tag antibody), ISG56, actin and SeV. The right panels show HeLa PLpro-4 cells that were cultured in the indicated concentrations of tetracycline for 3 days prior to mock infection (lanes 9 through 13) or infection of SeV (lanes 14 through 17) for 16 h. Expression of PLpro-TM, ISG56, and actin were determined by immunoblot analysis. C. Real-time RT-PCR analysis of IFN-β (left), A20 (middle), and IL-6 (right) mRNA transcripts in HeLa PLpro-4 cells repressed or induced for PLpro-TM expression, and mock treated (empty bars), treated with 50 μg/ml poly(I-C) in culture medium (hatched bars), or infected with SeV (solid bars) for 16 h. mRNA abundance was normalized to cellular 18S ribosomal RNA. Fold changes were calculated by dividing the normalized mRNA abundance under various treatment conditions by that of the mock-treated cells without PLpro expression.

A. HeLa PLpro-4 cells were cultured to repress or induce PLpro-TM expression and subsequently mock-infected or infected with SeV for 16 h. In left panels, expression of p396-IRF-3, total IRF-3, PLpro and actin proteins in cell lysates were determined by immunoblot analysis. In right panels, cell lysates were subjected to immunoprecipitation with a rabbit anti-IRF-3 antibody (lanes 1 through 4) or a control rabbit serum (lanes 5 through 8). The immunoprecipitates were separated on SDS-PAGE, followed by immunoblot analysis of IRF-3 (using an mAb anti-IRF-3) and PLpro (using an mAb anti-V5 tag). B. HEK293 cells were transfected with WT (lanes 1 through 3), C1651A (lanes 4 through 6), or D1826A (lanes 7 through 9) PLpro-TM, respectively, and where indicated, along with an empty vector (lanes 1, 4, and 7), or a vector expressing Flag-IRF-3 (lanes 2, 5, and 8) or untagged IRF-3 (lanes 3, 6, and 9). Cell lysates were immunprecipitated with anti-V5 (PLpro-TM), followed by immunodetection of Flag-IRF-3 (anti-Flag) or PLpro-TM (anti-V5) (right panels). * denotes IgG heavy chain. Immunoblot analysis of input for Flag-IRF-3 and PLpro-TM is shown in left panels. All 3 forms of PLpro-TM interacted with IRF-3. C. HEK293 cells were transfected with WT (lanes 1 and 2), C1651A (lanes 3 and 4), or D1826A (lanes 5 and 6) PLpro-Sol, respectively, and where indicated, along with an empty vector (lanes 1, 3, and 5) or a vector expressing Flag-IRF-3 (lanes 2, 4, and 6). Cell lysates were immunoprecipitated with anti-V5 (PLpro-Sol), followed by immunodetection of Flag-IRF-3 (anti-Flag) or PLpro-Sol (anti-V5) (lower panels). Immunoblot analysis of input for Flag-IRF-3 and PLpro-Sol is shown in upper panels. Note that WT and D1826A PLpro-Sol interacted with IRF-3, while C1651A PLpro-Sol did not. D. HeLa PLpro-4 cells induced for PLpro-TM expression were transfected with GFP, GFP-IRF-3, or GFP-IRF3-5D, respectively. Cell lysates were subjected to Co-IP experiments using either GFP antibody or V5 antibody for immunoprecipitation, followed by immunoblot analysis of the immunoprecipitates using anti-GFP or anti-V5 antibodies. Note that both GFP-IRF3 and GFP-IRF3-5D were associated with PLpro-TM (detected by anti-V5), while free GFP was not.

A. MA104 cells were mock-infected (lanes 1 through 3) or infected with SARS-CoV (MOI=10) for 18 h (lanes 4 through 6), followed by mock-treatment (lanes 2 and 4), transfection of poly (I-C) (lanes 1 and 6) or superinfection of SeV (lanes 3 and 5) for 10 h, respectively (a total of 28 h after mock or SARS-CoV infection under these conditions). Immunoblot analysis of cell lysates for expression of ISG56, IRF-3, SeV and actin is shown. Asterisks (*) in ISG56 panels denote a nonspecific band. B. MA104 cells were mock-infected, infected with SeV for 10 h, infected with SARS-CoV (MOI=10) for 18 or 28 h, or transfected with poly (I-C) for 10 h, respectively, or pre-infected with SARS-CoV for 18 h and then superinfected with SeV or transfected with poly (I-C) for additional 10 h (a total of 28 h after SARS-CoV infection under these conditions). Cell-free culture supernatants were collected, irradiated, and subjected to IFN bioactivity assay against VSV as described in Experimental Procedures. Bars indicate IFN bioactivity (U/ml) in culture supernatants under the indicated conditions. C. MA104 cells were mock-infected, infected with SeV for 8 h, infected with SARS-CoV (MOI=10) for 16 or 24 h, or pre-infected with SARS-CoV for 16 h and then superinfected with SeV for additional 8 h. Cells were harvested for total RNA extraction, cDNA synthesis, and subsequent real-time RT-PCR detection of IFN-β mRNA. Data shown are representative of two independently conducted experiments.

A. Human lung/bronchial epithelial Calu-3 cells were mock-infected, or infected with 100 HAU/ml SeV (left panels) or SARS-CoV (right panels) at an MOI of 5 for the indicated times prior to cell lysis and immunoblot analysis of IRF-3, ISG56, and SARS-CoV nucleocapsid (NC) protein. B. The left panels show African green monkey kidney MA104 cells that were mock-infected or infected with SeV (100 HAU/ml) or with SARS-CoV (MOI=10) for the indicated times prior to cell lysis and immunoblot analysis of phospho-Ser396 IRF-3, total IRF-3, MxA, ISG56, SARS-CoV NC, and SeV proteins. A nonspecific band (*) detected by the anti-ISG56 antibody indicates equal loading. The arrowhead and hatch marks in the total IRF-3 panel denote phosphorylated (virus-activated) and inactive IRF-3 forms, respectively. In the right panel, MA104 cells grown in 35-mm dishes were mock-treated, infected with SARS-CoV (MOI=10) for 20 or 30 h, or transfected with 5 μg poly (I-C) for 10 h prior to total RNA extraction and real-time RT-PCR analysis of IFN-β mRNA abundance, which was subsequently normalized to cellular 18S ribosomal RNA. Bars show fold change in IFN-β mRNA levels (relative to mock-treated cells).

A. Activation of p55C1B, an IRF-3-dependent synthetic promoter, by medium poly (I-C) (hatched bars) or SeV infection (solid bars) in HEK293-TLR3 cells with expression of SARS-CoV PLpro-TM (TM) or a control vector (Vect). B and C. Activation of IFN-β (B) and p55C1B (C) promoters by ectopic expression of various signaling molecules within TLR3 and RIG-I/MDA5 pathways at or above the level of IRF-3 in HEK293 cells with (solid bars) or without (empty bars) expression of SARS-CoV PLpro-TM. N-RIG and N-MDA5 denote the CARD domain of RIG-I and MDA5, respectively.

A. HeLa PLpro-4 cells were manipulated to repress or induce PLpro-TM expression for 3 days, and were subsequently mock-treated, or incubated with 50 μg/ml poly (I-C) in culture medium for 6 h, or challenged with SeV for 16 h. Cell lysates were separated on native PAGE followed by immunoblot analysis to detect IRF-3 monomer and dimer forms (top panel, empty arrow indicates IRF-3 dimer), or subjected to SDS-PAGE and immunoblot analysis of IRF-3, ISG56, PLpro, SeV and Actin (lower panels). B. HeLa PLpro-4 cells were cultured to repress or induce PLpro-TM expression and subsequently mock-infected or infected with SeV for 16 h. Where indicated, cells were incubated with 0.05 μg/ml of okadaic acid (OA) for the last 4 h of SeV infection/mock infection. Cells were then harvested for immunoblot analysis of IRF-3, p396-IRF3, PLpro (anti-V5), and SeV. * denotes a nonspecific band. C. Lanes 1 through 4, HeLa cells were mock-infected or infected with SeV for 16h. Where indicated, OA was included in the last 4-h duration of infection/mock infection. Lanes 5 through 8, Cell lysates of HeLa cells infected with SeV were mock-treated, treated with CIP, or OA, or CIP in the presence of OA, as indicated in the Experimental Procedures. IRF-3 and p396-IRF3 were detected by immunoblot analysis. * denotes a nonspecific band. D. Immunofluorescence staining of IRF-3 subcellular localization in HeLa PLpro-4 cells repressed (upper panels) or induced (lower panels) for PLpro-TM expression, and mock-infected (left) or infected (right) with SenV for 16h. Numbers in SeV-infected cells were the averages of the percentage of cells that had IRF-3 nuclear translocation and were counted from ten random fields (40 X magnification). E. HeLa PLpro-4 cells were cultured to repress or induce PLpro-TM expression and subsequently mock-infected or infected with SeV. Cell lysates were subjected to immunoprecipitation with a rabbit anti-CBP antibody (upper panels) or with a rabbit anti-IRF-3 antiserum (lower panels). The immunoprecipitates were separated on SDS-PAGE, followed by immunoblot analysis of IRF-3 (using an mAb anti-IRF-3) or CBP (using a rabbit anti-CBP antibody).

MA104 cells were mock-infected (lane 1), infected with SeV for 10 h (lane 2), infected with SARS-CoV (MOI=10) for 18 and 28 h (lane 3 and 4), respectively, or infected with SARS-CoV for 18 h then superinfected with SeV for an additional 10 h (lane 5). Expression of IRF-3, RIG-I, MxA, ISG56, SARS-CoV NC and nsp3, and SeV proteins in cell lysates was determined by immunoblot analysis (upper panels). A nonspecific band detected by the rabbit anti-MxA antiserum (bottom panel) indicates equal loading. Data shown are representative of three independently conducted experiments. In lower panels, the cell lysates were subjected to immunoprecipitation with a rabbit anti-IRF3 antiserum, followed by immunoblot analysis of SARS-CoV nsp3, and IRF-3 (using an mAb anti-IRF3 antibody). Data shown are representative of two independently conducted experiments.

A. Huh7 DN-18 cells with tet-regulated, conditional expression of a dominant negative form of IRF-3 (DN IRF-3) were cultured in the presence or absence of tet for 3 days, to repress or induce DN IRF-3 expression, respectively, and were subsequently mock-infected, or challenged with SeV for 12 h. Expression of IRF-3, ISG56, SeV and actin proteins were detected in cell lysates by immunoblot analysis. The endogous IRF-3 (FL) and the DN IRF-3 (DN) bands were marked as filled and empty circles, respectively. The asterisk (*) in ISG56 panel denotes a nonspecific band. B. Huh7 DN-18 cells repressed or induced for DN IRF-3 expression were infected with SARS-CoV at an MOI of 0.01 at 37 °C for 1h. The virus inoculum was then removed and cells were washed extensively and refed with complete medium. Cell-free culture supernatants were collected at the indicated times postinfection and stored at −80 °C until they were subjected to assessment of infectious viral titers using a standard TCID₅₀ assay in Vero E6 cells, as described in Experimental Procedures. The experiments were terminated at day 3 postinfection as CPE was observed in ~30% and ~70% of infected cells without and with DN IRF-3 expression, respectively. The growth curve of SARS-CoV shown was representative of three independently conducted experiments.