Design and characterization of Coxsackievirus B3 with catalytically-inactive 2A protease (2A pro ).

(A) Schematic representation of the wild-type (wt) Coxsackievirus B3 (CVB3) genome and its engineered derivative viruses with inactivated 2A pro (2Amut). The 2A pro cleavage site was replaced by a Theiler’s murine encephalomyelitis virus (TMEV)-derived IRES sequence, a T2A peptide, or a sequence corresponding to the cleavage site of 3C/3 CD protease (3C pro ) (3 CD cs ). Specific cleavage sites of the viral proteases are indicated with blue (2A pro ) and green (3C pro ) triangles. (B) Growth kinetics of recombinant 2A pro wt viruses (3 CDcs-2Awt, T2A-2Awt and IRES-2Awt) in HeLa-R19 cells infected at a multiplicity of infection (MOI) of 5, as measured by TCID 50 assay (upper panel) or qPCR (lower panel). (C) Growth kinetics of the different 2Awt and 2Amut viruses in HeLa-R19 cells. Experiment was performed as in panel (B). (D) HeLa-R19 or HEK293-T cells were transfected with equal amounts of in vitro transcribed RNA of Renilla-luciferase (RLuc)-3 CDcs-2Awt or -2Amut, and luciferase levels were determined at the indicated times post infection. (E) Growth curves of 2Awt and 2Amut viruses obtained from infected HeLa-R19, BGM, Vero E6 and A549 cells. Data represent the mean ± SEM of one representative experiment (B-D) or three independent experiments (E). The illustration in (A) was created in BioRender. Schipper, J. (2025) https://BioRender.com/7bqvkpe . Statistical significance was assessed by multiple two-tailed unpaired t-tests with multiple comparisons corrections using the Bonferroni-Dunn method (* p < 0.05; ** p < 0.01; *** p < 0.001).