Journal: Journal of Virology
Article Title: Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription
doi: 10.1128/JVI.00279-17
Figure Lengend Snippet: VHSV M blocks nascent cellular RNA synthesis. (A) EPC cells were left untreated, treated with 2 μg/ml α-amanitin or 1 μg/ml actinomycin D, or infected with VHSV-IVb (MOI = 1) for 24 h and then cultured in 100 μM 5-EU for 2 h. 5-EU was visualized with Alexa Fluor 594 via a click chemistry reaction. VHSV-transfected cells were visualized using a polyclonal anti-VHSV antibody, followed by goat anti-rabbit FITC secondary staining. (B) EPC cells were cotransfected with 0.4 μg GFP and 0.1 μg IVb M for 24 h and then labeled with 100 μM 5-EU for 2 h. 5-EU was visualized with Alexa Fluor 594 via a click chemistry reaction. (C) EPC cells were transfected with an SV40/luc, ITS1/luc, or U6/luc reporter construct with 0.25 μg of pCD-M per 1 × 106 cells for 24 h, followed by total RNA extraction and reverse transcription to cDNA. Luciferase mRNA levels were quantified by RT-qPCR and normalized to EPC β-actin mRNA levels. (D) The data in panel C are expressed as a percentage of the control values for each construct, demonstrating that relative levels of inhibition were similar for all.
Article Snippet: Primers for PCR analysis and cloning Luciferase driven by a human U6 promoter reporter construct (pGL3-U6-Luc) was generated by subcloning the human U6 promoter from the pLKO.1 puro vector (Addgene plasmid 10879) into the pGL3 luciferase reporter vector (Promega E1751) upstream of the luciferase gene using Gibson Assembly (NEB E5520).
Techniques: Infection, Cell Culture, Transfection, Staining, Labeling, Construct, RNA Extraction, Reverse Transcription, Luciferase, Quantitative RT-PCR, Control, Inhibition
