Viral NS1 Proteins and Host Protein Synthesis

To evaluate the effects of the different viral NS1 proteins on host protein synthesis, human 293T cells (24-well plate format, 2.5 × 10⁵ cells/well, triplicates) were transiently co-transfected, using LPF2000, with 1 μg/well of the indicated pCAGGS-HA COOH NS1 expression plasmids or an empty plasmid as control, together with 25 ng/well of pCAGGS plasmid expressing Gaussia luciferase (Gluc) (Capul and de la Torre, 2008 (link)). At 24 h post-transfection, Gluc activity was quantified from tissue culture supernatants using a Biolux Gaussia luciferase reagent (New England Bio-Labs) and a Lumicount luminometer (PacKard). The mean values and standard deviations (SD) were calculated using Microsoft Excel software.

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Nogales A., Aydillo T., Ávila-Pérez G., Escalera A., Chiem K., Cadagan R., DeDiego M.L., Li F., García-Sastre A, & Martínez-Sobrido L. (2019). Functional Characterization and Direct Comparison of Influenza A, B, C, and D NS1 Proteins in vitro and in vivo. Frontiers in Microbiology, 10, 2862.

Publication 2019

Biolux Gaussia luciferase reagent Lumicount luminometer

293t cells Cells Human Luciferase Plasmid Protein synthesis Tissue Transfection Viral proteins

Corresponding Organization : University of Rochester

Other organizations : Icahn School of Medicine at Mount Sinai, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, South Dakota State University

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Variable analysis

independent variables

Indicated pCAGGS-HA COOH NS1 expression plasmids

dependent variables

Gaussia luciferase (Gluc) activity

control variables

Human 293T cells (24-well plate format, 2.5 × 10^5 cells/well, triplicates)
LPF2000 transfection method
25 ng/well of pCAGGS plasmid expressing Gaussia luciferase (Gluc)
Empty plasmid as control

controls

Positive control: Cells co-transfected with Gluc expression plasmid
Negative control: Cells co-transfected with empty plasmid

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