Materials and Reagents

1. HEV Kernow-C1 p6/luc plasmid (genotype 3 subgenomic replicon expressing Gaussia luciferase; a kind gift from Suzanne Emerson, NIH) (Shukla et al., 2012)
   
2. MluI restriction endonuclease with accompanying 10x buffer D (Promega Corporation, catalog number: R6381 )
   
3. pT7-IRES-FFLuc-YFsfRNA plasmid (constructed in-house; IRES, internal ribosome entry site; FFLuc, firefly luciferase; YFsfRNA, yellow fever virus small flaviviral RNA) (Debing et al., 2014)
   
4. Primers #1 (5’-CATATGTCGACTAATACGACTCACTATAGGGATCCGCCCCTCTCCC-3’) and #2 (5’-AGTGGTTTTGTGTTTGTCATCC-3’) (custom order) (Integrated DNA Technologies) (dissolved to a final concentration of 10 µM in buffer TE)
   
5. Kapa HiFi HotStart ReadyMix master mix (Kapa Biosystems, catalog number: KK2601 )
   
6. SeaKem LE agarose for gel electrophoresis (Lonza, catalog number: 50001 )
   
7. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
   
8. T7 RiboMAX large scale RNA production system (Promega Corporation, catalog number: P1300 )
   
9. RNeasy mini kit (QIAGEN, catalog number: 74104 )
   
10. ScriptCap m7G capping system (CELLSCRIPT^TM, catalog number: C-SCCE0610 )
    
11. Huh7 human hepatoma cell line (Japanese Collection of Research Bioresources, catalog number: JCRB0403)
    
12. Dulbecco’s modified Eagle’s medium with high glucose (DMEM) (Life Technologies, catalog number: 41965-039 )
    
13. Fetal bovine serum (FBS) (not heat-inactivated) (Life Technologies, catalog number: 10270-106 )
    
14. Opti-MEM I reduced serum medium (Life Technologies, catalog number: 31985-062 )
    
15. Lipofectin transfection reagent (Life Technologies, catalog number: 18292-011 )
    
16. Dulbecco’s phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (Life Technologies, catalog number: 14190-094 )
    
17. Renilla luciferase assay system (Promega Corporation, catalog number: E2810 )
    
18. 5x passive lysis buffer (Promega Corporation, catalog number: E1941 ) and demineralized H₂O for dilution
    
19. Luciferase assay system (Promega Corporation, catalog number: E1501 )
    
20. CellTiter 96 AQueous MTS reagent powder [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega Corporation, catalog number: G1111 )
    
21. Phenazine methosulphate (PMS) (Sigma-Aldrich, catalog number: P9625 )
    
22. Minimum essential medium (MEM) (no glutamine, no phenol red) (Life Technologies, catalog number: 51200-046 )
    
23. Tris(hydroxymethyl)aminomethane powder (Trizma base) (Sigma-Aldrich, catalog number: T1503 ) (dissolved to 1 M and pH 8.0 in H₂O)
    
24. Ethylenediaminetetraacetic acid disodium salt solution (EDTA, 0.5 M) (Sigma-Aldrich, catalog number: E7889 )
    
25. MTS/PMS solution (see Recipes)
    
26. Buffer TE (see Recipes)
    

Equipment

1. Spectrophotometer to determine RNA concentrations (e.g. Thermo Fischer Scientific, NanoDrop, model: ND-1000 )
   
2. Falcon transparent 96-well plate (Corning Incorporated, catalog number: 353072 )
   
3. CulturPlate white 96-well plate (PerkinElmer, catalog number: 6005680 )
   
4. 37 °C and 35 °C 5% CO₂ cell culture incubators
   
5. Plate shaker
   
6. Saphire² microplate reader for both luminescence and absorbance (Tecan Trading AG)
   

Procedure