Journal: Viruses

Article Title: The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing

doi: 10.3390/v13030367

Figure Lengend Snippet: RIOK3 is involved in the signal transduction of the type I IFN response. ( A ) HEK293 cells and three RIOK3 KO cell lines were screened for RIOK3 protein expression by IP-Western blot. ( B ) HEK293 cells and RIOK3 KO cells were infected with MP-12 (MOI 1.0). The viral titer in the 24 hpi supernatant was quantified by plaque assay. ( C , D ) RT-qPCR targeting IFNB was performed on RNA from HEK293 cells and RIOK3 KO cells infected with MP12 ( C ) or treated with poly (I:C) ( D ) or 3p-hpRNA ( E ) for 18 hours. ( F ) RT-qPCR targeting IFNB was performed on RIOK3 KO cells transfected with a GFP (mock) or RIOK3 expression plasmid and treated with poly (I:C) 18 hours later. ( G ) Effect of RIOK3 KO on IFNB promoter activation. HEK293 cells and RIOK3 KO cells were co-transfected with pGL3-IFNB firefly reporter and phRL-CMV renilla control. Cells were stimulated with poly (I:C) or infected with MP-12 and the dual luciferase signals were measured after 18 or 48 h, respectively. In panels B–F, data are presented as mean +/− SEM from three biological replicates. Western blots in panels A is representative of duplicate experiments. Student’s t-test: ** p < 0.01, * p < 0.05.

Article Snippet: Since HEK293 cells lack TLR3 expression for stimulation of innate immunity via poly (I:C), which stimulates both the MDA5 and the RIG-I pathways, and 3p-hpRNA, which stimulates only the RIG-I pathway, we transfected 1 μg/mL poly (I:C) (Tocris/BioTechne, Minneapolis, MN, USA) or 3p-hpRNA (Invivogen, San Diego, USA) into HEK293 cells using Lipofectamine 2000 according to the manufacturer’s instructions (Thermo Fisher Scientific).

Techniques: Transduction, Expressing, Western Blot, Infection, Plaque Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Activation Assay, Luciferase

Journal: Viruses

Article Title: The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing

doi: 10.3390/v13030367

Figure Lengend Snippet: Activation of cytosolic innate immune RNA sensors, but not DNA sensors induces RIOK3 X2 variant alternative splicing, and RIOK3 splicing is vital for IFN expression. HEK293 cells were transfected with poly (I:C) ( A ), or infected with either the RNA virus Tacaribe (TCRV) ( B ), or the DNA virus adenovirus (ADV) ( C ). Total RNA was harvested 24 h post transcription or infection and RT-qPCR was used to quantify the relative fraction of X2 variant and full-length canonically spliced RIOK3 species. Data are presented as mean +/− SEM based on duplicate experiments. Student’s t -test: * p < 0.05. ( D ) HEK293 cells were transfected with 1 μg/mL 3p-hpRNA. RT-PCR targeted the region spanning RIOK3 exons 5–10, followed by agarose gel electrophoresis. Splicing isoforms are indicated. ( E ) Morpholino oligonucleotide targeting the canonical exon 8 splice donor site of RIOK3 pre-mRNA was transfected into HEK293 cells in increasing concentration (2–10 μM) for 18 h. RNA was processed via RT-PCR and run on agarose gel. ( F ) RT-qPCR was performed to measure the expression of IFNB mRNA after 18 h MO (8 μM) transfection and subsequent stimulation by either poly (I:C) or 3p-hpRNA. Data is presented as mean +/− SEM from triplicate experiments. Student’s t -test: ** p < 0.01, * p < 0.05.

Article Snippet: Since HEK293 cells lack TLR3 expression for stimulation of innate immunity via poly (I:C), which stimulates both the MDA5 and the RIG-I pathways, and 3p-hpRNA, which stimulates only the RIG-I pathway, we transfected 1 μg/mL poly (I:C) (Tocris/BioTechne, Minneapolis, MN, USA) or 3p-hpRNA (Invivogen, San Diego, USA) into HEK293 cells using Lipofectamine 2000 according to the manufacturer’s instructions (Thermo Fisher Scientific).

Techniques: Activation Assay, Variant Assay, Expressing, Transfection, Infection, Virus, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Concentration Assay

Journal: iScience

Article Title: Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses

doi: 10.1016/j.isci.2021.102477

Figure Lengend Snippet: SARS-CoV-2 infection does not inhibit type I IFN expression To determine if SARS-CoV-2 can modulate IFNβ gene expression and downstream stimulation of ISGs, Calu-3 cells were infected with SARS-CoV-2 for varying times, following which cells were mock transfected or transfected with poly(I:C). Mock-infected and mock-transfected cells served as controls. Transcript levels were quantified using qPCR. Protein expression was observed and quantified using immunoblot analysis. (A) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) for 0, 24, 48, and 72 h. Cells were fixed and stained to visualize the nucleus and SARS-CoV-2 nucleocapsid (N) protein. Scale bar indicates 300 μm. (B) SARS-CoV-2 genome (UpE) levels in Calu-3 cells infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h and transfected with 100 ng of poly(I:C) or mock transfected for 6 h (n = 6). Primers for the UpE region were designed to quantify SARS-CoV-2 genome levels (see methods). 1/dCT values are represented after normalizing Ct values for SARS-CoV-2 genome levels at 18 hpi with Ct values observed at 0 hpi (immediately after removal of virus inoculum). Gel (below): UpE qPCR amplicons were visualized on an agarose gel. (C) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 6). (D) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 6). (E) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 12 h. Twelve hpi, cells were either transfected with 100 ng of poly(I:C) or mock transfected for 6 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 6). (F) IFIT1, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for 24 h (n = 3). (G) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (H) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 0.1 or 1) or mock infected for 24 h. Twenty-four hpi, cells were transfected with 10 ng of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (I) IFNβ transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFNβ transcript levels were normalized to GAPDH transcript levels (n = 3). (J) IFIT1 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IFIT1 transcript levels were normalized to GAPDH transcript levels (n = 3). (K) IRF7 transcript levels in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with varying concentrations of poly(I:C) or mock transfected for 12 h. IRF7 transcript levels were normalized to GAPDH transcript levels (n = 3). (L) pTBK1-S172, TBK1, pIRF3-S396, IRF3, SARS-CoV-2 N, and ACTB protein expression in Calu-3 cells that were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h. Twenty-four hpi, cells were either transfected with 1,000 ng of poly(I:C) or mock transfected for an additional 24 h (n = 3). (M) Calu-3 cells were infected with SARS-CoV-2 (MOI 1) or mock infected for 24 h, followed by transfection with 1,000 ng of rhodamine-labeled poly(I:C) or mock transfection for 3 h. Cells were fixed and stained to visualize SARS-CoV-2 nucleocapsid (N) protein and rhodamine-labeled poly(I:C). SARS-CoV-2 N and poly(I:C)-rhodamine containing cells are indicated by arrows. Cells that only contained detectable levels of poly(I:C)-rhodamine are indicated by arrow heads. Scale bar indicates 150 μm. Data are represented as mean ± SD, n = 3 or 6, p∗∗<0.01, ∗∗∗<0.001, and ∗∗∗∗<0.0001 (Student's t test and Tukey's multiple comparisons test). pTBK1-S172 and pIRF3-S396 protein expression levels are expressed as ratios of pTBK1-S172/TBK1 and pIRF3-S396/IRF3 levels, respectively. Blots were quantified using Image Studio (Li-COR) (n = 3). Ct, cycle threshold. See also Figure S3 .

Article Snippet: Poly(I:C)-Rhodamine , InvivoGen , Cat#tlrl-piwr.

Techniques: Infection, Expressing, Transfection, Western Blot, Staining, Virus, Agarose Gel Electrophoresis, Labeling