Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Entirely plasmid-based reverse genetics system for rotaviruses

doi: 10.1073/pnas.1618424114

Figure Lengend Snippet: IFN-β promoter activation by the NSP1 mutant. (A) Expression of NSP1 protein from a recombinant virus. MA104 cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 0.1 pfu per cell. As a control, cells were transfected with NSP1 expression plasmid (pCAG-NSP1). Viral protein expression in cells was assessed by immunoblotting using anti-NSP1 antibody after 48 h of incubation. An actin-specific antibody was used as a loading control. Molecular masses were determined by coelectrophoresis of prestained protein markers. (B) IFN-β promoter activation assay. The 293T cells were transfected with a firefly luciferase (Fluc) reporter plasmid under the control of the IFN-β promoter (pIFN-β–Fluc) and a pSV40-Rluc reporter plasmid that expresses Renilla luciferase (Rluc) under the control of the SV40 promoter (Promega). Twelve hours after transfection, cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 10 pfu per cell. Cells were harvested 10 h postinfection, and luciferase activities were determined. Relative luminescence units (Fluc/Rluc) are shown. *P < 0.05, **P < 0.01 (Tukey’s multiple comparison test).

Article Snippet: The 293T cells were transfected with a firefly luciferase (Fluc) reporter plasmid under the control of the IFN-β promoter (pIFN-β–Fluc) and a pSV40-Rluc reporter plasmid that expresses Renilla luciferase (Rluc) under the control of the SV40 promoter (Promega).

Techniques: Activation Assay, Mutagenesis, Expressing, Recombinant, Infection, Transfection, Plasmid Preparation, Western Blot, Incubation, Luciferase

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV phosphorylated IRF-3 and weakly activated the IFN-β promoter in MARC-145 at 24 h p.i., but their activities were rapidly inhibited in the following infection. (A) MARC-145 cells were infected with PRRSV at an MOI of 0.05. After 6 h, 18 h, 30 h respectively, the cells were co-transfected with p-284 Luc and phRL-TK, and 18 h later, cells were harvested for dual luciferase reporter assay system in MicroBeta-1450. Cells were transfected with Poly(I:C) for 9 h as positive controls. (B) MARC-145 cells were infected with PRRSV at an MOI of 0.05, and harvested at 24, 36, 48 h p.i. for the immunoblots of IRF-3 and pIRF-3. The results shown were from one of three independent experiments with similar observations, and each including three or two replicates. Control (Con): Sham virus mentioned in part of Section were added into MARC-145 cells. *: p < 0.05 compared to the control.

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Infection, Transfection, Luciferase, Reporter Assay, Western Blot, Control, Virus

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV nsp1 inhibited the activations of IFN-β promoter and pIRF-3-dependent synthetic promoter (p55C1B Luc) induced by Poly(I:C). (A) The mRNA of nsp1 and N in MARC-145 cells transfected with pcDNA3.1-nsp1 (nsp1), pcDNA3.1-N (N) or pcDNA3.1-His (Con) were analyzed by RT-PCR. Lanes 1–3 were amplified by PRRSV N primers, while lanes 4–6 were amplified by PRRSV nsp1 primers. (B) Western blot analyzed nsp1 and N protein by anti-His primary antibody in MARC-145 transfected with pcDNA3.1-nsp1 (nsp1) or pcDNA3.1-N (N). The control cells were transfected with pcDNA 3.1-His (Con). Since the nsp1 should be cleaved into nsp1α and nsp1β subunits, the arrow-pointed band was only the nsp1β subunits. (C) MARC-145 cells were co-transfected with p-284 Luc, phRL-TK and different expression plasmid, and 24 h later, cells were either mock-treated (Con) or transfected with Poly(I:C) for 6 h. Cells were analyzed by dual luciferase reporter assay. (D) MARC-145 cells were co-transfected with p55C1B Luc, phRL-TK and different expression plasmid. Twenty four hours later, cells were either mock-treated (Con) or transfected with Poly(I:C) for 6 h. Cells were analyzed by dual luciferase reporter assay. LCPS was the luminescence counts per second detected by luminescence counting on the Microbeta-1450. Bars showed relative luciferase activity (Relative LCPS) normalized to phRL-TK activity (relative LCPS = LCPS of firefly- luciferase/LCPS of Renilla- luciferase). Data represented means of three replicates, and experiments were repeated three times. Vector (pcDNA3.1-His) showed in figure was the control plasmid. *: p < 0.05 compared to the cells transfected with pcDNA3.1-His (Con).

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Control, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV nsp1 inhibited activities of IFN-β promoter and pIRF-3 dependent promoters (p55C1B Luc) induced by RIG-N, VISA, TRIF or TBK1, but did not inhibit the activities of IFN-β promoter and pIRF-3 dependent promoter induced by IRF-3(5D). MARC-145 cells were co-transfected with p-284 Luc (A) or p55C1B Luc (B), phRL-TK and different expression plasmids. Thirty hours later, cells were harvested for dual luciferase reporter assay. Con: cells transfected with pcDNA3.1-His. Data represented means of three replicates, and experiments were repeated three times. *: p < 0.05 compared to cells transfected with pcDNA3.1-His (Con).

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Transfection, Expressing, Luciferase, Reporter Assay