Influenza A Virus Polymerase Reconstitution

HEK 293T cells were transfected with plasmids expressing the subunits of the influenza A/WSN/33 (H1N1) virus RNA polymerase (pcDNA3-PB1, pcDNA3-PB2, and pcDNA3-PA), the viral nucleoprotein (pcDNA3-NP), and a viral RNA template based on segment 5 (NP-encoding genome segment) (14 (link), 23 (link)). As a negative control, a PB1 active-site mutant (PB1a) was used in which the active-site SDD motif was mutated to SAA (14 (link)). Enisamium iodide was added to the cell culture medium 15 min after transfection. Twenty-four hours after transfection, cells were washed in PBS and the total RNA was extracted using TRIzol and isopropanol precipitation (15 (link)). 5S rRNA and IAV vRNA and mRNA steady-state levels were subsequently analyzed by radioactive primer extension and 6% denaturing PAGE as described previously (15 (link), 24 (link)). Phosphorimaging was performed on an FLA7000 Typhoon scanner (GE Healthcare). GFP experiments were performed through transfection of pcDNA3-eGFP, and GFP expression was measured using a SpectraMax plate reader.

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te Velthuis A.J., Zubkova T.G., Shaw M., Mehle A., Boltz D., Gmeinwieser N., Stammer H., Milde J., Müller L, & Margitich V. (2021). Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity. Antimicrobial Agents and Chemotherapy, 65(4), e02605-20.

Publication 2021

FLA7000 Typhoon scanner

293t cells 5s rrna Cell culture Cells Culture medium Enisamium iodide Genome H1n1 virus Influenza Isopropanol Mrna Plasmids Primer Radioactive Rna polymerase Subunits Transfection Trizol Typhoon Viral rna

Corresponding Organization : University of Cambridge

Other organizations : Research Institute of Influenza, Ministry of Health of the Russian Federation, Icahn School of Medicine at Mount Sinai, University of Wisconsin–Madison, IIT Research Institute, Pharmalog (Germany)

Top 5 similar protocols

Variable analysis

independent variables

Plasmids expressing the subunits of the influenza A/WSN/33 (H1N1) virus RNA polymerase (pcDNA3-PB1, pcDNA3-PB2, and pcDNA3-PA)
Plasmid expressing the viral nucleoprotein (pcDNA3-NP)
Viral RNA template based on segment 5 (NP-encoding genome segment)
PB1 active-site mutant (PB1a) in which the active-site SDD motif was mutated to SAA
Enisamium iodide added to the cell culture medium

dependent variables

IAV vRNA and mRNA steady-state levels
GFP expression

control variables

HEK 293T cells used for transfection

positive control

Plasmids expressing the subunits of the influenza A/WSN/33 (H1N1) virus RNA polymerase (pcDNA3-PB1, pcDNA3-PB2, and pcDNA3-PA)
Plasmid expressing the viral nucleoprotein (pcDNA3-NP)
Viral RNA template based on segment 5 (NP-encoding genome segment)

negative control

PB1 active-site mutant (PB1a) in which the active-site SDD motif was mutated to SAA

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