Plasmid-based generation of recombinant coxsackievirus B3 particles carrying capsid gene replacement replicons

Recombinant infectious coxsackievirus B3 (CVB3) particles were generated by packaging of modified viral genomes in which the capsid coding P1-region was replaced by an EGFP-luciferase reporter gene. Efficient packaging of the recombinant genome was achieved by a novel method based on cotransfection of a plasmid encoding the subgenomic viral replicon together with two alternative helper plasmids carrying expression cassettes of the CVB3 capsid proteins, and a T7 RNA polymerase expression plasmid. Transcription of a reporter gene and expression of capsid proteins were achieved in a single step, eliminating the need of a helper virus. Recombinant viral stocks were used to infect human embryonal cardiomyocytes (hCMC) and other cell types, and luciferase activity was measured at different timepoints after infection. Neither progeny virus nor wildtype CVB3 was produced upon infection of target cells, facilitating analyses of infected cells without viral spread. The presence of an IRES sequence upstream of the P1 open reading frame in the helper plasmids was indispensable for the generation of recombinant particles, as no packaging was observed using helper plasmids without this feature. Luciferase data obtained by transfection of reporter plasmids with and without upstream 5'-NTR sequences suggests that the CVB3 IRES facilitates translation in T7 RNA polymerase-dependent gene transcription, both in presence and absence of viral replication.