Producing Sindbis Virus Encoding ATP Sensor

The coding sequence of the ATP sensor ATeam1.03YEMK (Imamura et al., 2009 (link)) was subcloned into the viral vector pSinRep5. Sindbis virus was produced as previously described (Piquet et al., 2018 (link)). Recombinant pSinRep5 and helper plasmid pDH26S (Invitrogen) were transcribed in vitro into capped RNA using the Megascript SP6 kit (Ambion). Baby hamster kidney-21 cells (BHK-21, clone 13, Mesocricetus auratus, hamster, Syrian golden), negative for mycoplasma contamination and purchased from ATCC (CCL-10, RRID:CVCL_1915, lot number 1545545), were only used for viral production. BHK-21 cells were electroporated with sensor-containing RNA and helper RNA (2.10⁷ cells, 950 µF, 230 V) and incubated for 24 h at 37 °C in 5 % CO₂ in Dulbecco’s modified Eagle medium supplemented with 5 % fetal calf serum before collecting cell supernatant containing the viruses. The virus titer (10⁸ infectious particles/ml) was determined after counting fluorescent baby hamster kidney cells infected using serial dilution of the stock virus.