Generation and Validation of CVB3 Mutants

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.