Sali hf

CVB3 mutants were generated by site-directed mutagenesis using the mCherry-CVB3 fluorescent infectious clone mentioned above^{49 (link)}. For each mutant, non-overlapping primers containing the desired mutation in one of the primers were used to introduce the mutation with Q5 polymerase, followed by DpnI (Thermo Scientific) treatment, phosphorylation, ligation, and transformation of chemically competent bacteria (NZY5α Competent Cells, NZY Tech). Successful mutagenesis was verified by Sanger sequencing. Subsequently, plasmids were linearized with SalI-HF (ThermoFisher), and 600 ng of plasmid were transfected into 5 × 10⁴ HEK293T cells, together with 600 ng of a plasmid encoding the T7 polymerase (Addgene 65974) using Lipofectamine 2000 (ThermoFisher) according to manufacturer’s instructions of use. Cells were then incubated until cytopathic effect (CPE) was observed and passage 0 virus was collected. Infectious virus titer was determined using an Incucyte system (Sartorius) to quantify the red fluorescence from the mCherry reporter signal as detailed above. If needed, mutants with low titers were amplified for an additional passage. The capsid region of the mutant virus populations was reverse transcribed, PCR-amplified, and sequenced to ensure no compensatory mutations or reversions arose during replication.

The pRib-EVD68/mCherry wild-type and mutant plasmids were linearized by cleavage with SalI-HF (Thermo Fisher Scientific) and purified using the mini spin columns (Epoch Life Science). Viral subgenomic replicon RNA was generated with TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and purified using the RNeasy mini spin columns (Qiagen). For replicon assays, U-87 MG or HaCaT cells grown in 12-well plates were transfected with T7-amplified RNA using the TransIT mRNA transfection kit (Mirus Bio). At 12 hours post transfection, the reporter mCherry fluorescence was determined by flow cytometry using BD LSR Fortessa (BD Biosciences) and the following optical configuration: 561 nm laser, 670/30 nm bandpass filter. Acquired data were analyzed with the FlowJo software. The level of RNA replication was expressed as a transfection efficiency-normalized percentage of cells with the mCherry signal above the threshold determined using the viral polymerase-lacking mutant Δ3D^pol.
To test the effect of PI4KB inhibition on virus replication, a PI4KB-specific inhibitor (compound 10 from [10 (link)] kindly provided by Radim Nencka) was added to the medium at a final concentration of 1 μM 30 min prior transfection of the viral subgenomic replicon RNA.