Citation
- Authors: Ma A. et al.
- Year: 2021
- Journal: Genomics 113 2400-2412
- Applications: in vitro / DNA / jetOPTIMUS
- Cell types:
- Name: Grass carp ovary cell line (CO)
- Name: HEK-293
Description: Human embryonic kidney Fibroblast
Known as: HEK293, 293
Method
Apoptosis assay:
HEK-293 cells were transfected with the DNA plasmid pEGFP-C1 and pEGFP-C1-Lj-MAVS (5 μg or 10 μg) for 24 h using jetOPTIMUS® according to the manufacturer's instructions in 6-well chamber slides. All plasmids were extracted using the Pure LinkTM Hipure Plasmid Filter Midiprep kit. After 24 h, apoptosis-related proteins were detected via Western blot analysis.
Luciferase activity assay:
HEK-293 cells were seeded in 96-well plates and incubated for 16 h, after which the cells were transfected with plasmids (pEGFP-C1 and pEGFP-C1-Lj-MAVS or pcDNA3.1 and pcDNA3.1-Lj-RIG-I) using jetOPTIMUS®. After 24 h, each well was co-transfected with 0.2 ng luciferase reporter plasmid and 25 ng Renilla luciferase internal control vector. At 24 h post-transfection, IFN-β at 25 ng/mL was added to one well to stimulate the cells, which were then washed with PBS and lysed to measure luciferase activity using the Dual Luciferase Reporter Assay System.
Antiviral activities of RRP4:
The CO cells were transfected with 0.5 μg DNA plasmid pEGFP-N1-Lj-RRP4 or pEGFP-N1 with 1 μL jetOPTIMUS® according to the manufacturer's protocol.
After 36 h, the cells were cultured in medium containing G418 to enrich the transfected cells. When a minimum of 90% of cells were RRP4- and EGFP-positive, they were collected, and total protein was extracted for WB to detect the effect of stabilization. Subsequently, for antiviral assays, the stably transfected RRP4 and EGFP cells were seeded into a 96-well plate at approximately 80% confluence, 12 h before GCRV infection.
Abstract
Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are well-known viral RNA sensors in the cytoplasm. RIG-I-mediated antiviral signals are activated by interacting with the adapter protein mitochondrial antiviral signaling (MAVS), which triggers interferon (IFN) responses via a signaling cascade. Although the complete RIG-I receptor signaling pathway has been traced back to teleosts, definitive evidence of its presence in lampreys is lacking. Here, we identified 13 pivotal molecules in the RIG-I signaling pathway in lamprey, and demonstrated that the original RIG-I/MAVS signaling pathway was activated and mediated the expression of unique immunity factors such as RRP4, to inhibit viral proliferation after viral infection in vivo and in vitro. This study confirmed the conservation of the RIG-I pathway, and the uniqueness of the RRP4 effector molecule in lamprey, and further clarified the evolutionary process of the RIG-I antiviral signaling pathway, providing evidence on the origins of innate antiviral immunity in vertebrates.
