Author: Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.
Title: The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites Document date: 2016_2_10
ID: 1kuggdzj_50
Snippet: Cell culture, viral infection and transport inhibitor drugs HEK293T, A549, Huh7.5, and Huh7 cells were maintained in DMEM (Sigma) containing 10% FBS (Sigma). For HCV infection, Huh7.5 cells were seeded at a density of 2.5 × 10 5 cells/well in 6-well tissue culture plates, and 24 hours after plating, they were infected with 3 RNA genome equivalents of a serially passaged JFH-1 strain of HCV. For HAV infection, Huh7 cells were grown to 70% conflue.....
Document: Cell culture, viral infection and transport inhibitor drugs HEK293T, A549, Huh7.5, and Huh7 cells were maintained in DMEM (Sigma) containing 10% FBS (Sigma). For HCV infection, Huh7.5 cells were seeded at a density of 2.5 × 10 5 cells/well in 6-well tissue culture plates, and 24 hours after plating, they were infected with 3 RNA genome equivalents of a serially passaged JFH-1 strain of HCV. For HAV infection, Huh7 cells were grown to 70% confluence and infected with HAV/p16 virus at an MOI of 0.1. Cells were analyzed by immunofluorescence 3 weeks after infection. Huh7 cells containing the JFH-1 strain subgenomic replicon (encoding NS3 through to the C-terminus of the HCV polyprotein; received from the lab of Ralf Bartenschlager) were maintained in DMEM containing 10% FBS and 500 mg/mL G418. For the expression of constructs in HCV infected cells, cells were infected followed by transfection (as described below) at the indicated time after infection. For analysis of nuclear transport inhibition, uninfected and HCV-infected cells harbouring RIG-I expression constructs were treated with 40 μM importazole or 25 μM ivermectin for 3 hours.
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