C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

To ensure mRNA capping of C3P3-G1 transcripts, we pursued an approach that has benefited greatly from the vaccinia virus-T7RNAP expression system. This latter approach makes use of a recombinant vaccinia virus encoding the T7RNAP, while the target gene, under control of a T7 promoter, is either embedded in a plasmid or a sec- Figure 7 . C3P3-G1 expression system generates properly addressed proteins to cell compartments in comparison to standard CMV promoter-driven nuclear expression system. Fluorescent proteins with various trafficking signals were produced by the C3P3-G1 or standard CMV-promoter-driven nuclear expression system (pCMVScript) and were found appropriately addressed to the same corresponding cell compartments (A) green GFP fluorescent protein with SV40 eNLS signal featured by homogenous nuclear staining, (B) red RFP fused to the mitochondrial COX4 signal, which directs the fusion protein to the inner membrane of the mitochondria, was imaged as small aggregates spread in the cytoplasm, (c) red RFP fused to the transferrin receptor type 1 endosomal trafficking signal was visualized as multiple connected punctuates fluorescent patterns in cell cytoplasm. ond recombinant vaccinia virus (28, 29) . As the vaccinia virus is an intracellular parasite that replicates exclusively in the host-cell cytoplasm, the T7-transcripts are 5 -capped and 3 polyadenylated in host-cell cytoplasm by the vaccinia virus enzymes (30) . Although the amounts of mRNA made by this system are extremely high, comprising ∼30% of the total steady state RNA in the cytoplasm, only moderate amounts of T7-transcripts are properly 5 -capped (30) . A plausible explanation for these findings is provided by the crowded environment of the cytoplasm, which contains a dense network of cytoskeletal filaments, therefore impeding free diffusion of large macromolecules such as proteins, DNA and mRNA (61) . Due to the lack of coupling between the T7RNAP and vaccinia virus capping enzyme, the T7-transcripts are poorly 5 -processed by the vaccinia virus capping enzyme. This assumption was central for the development of the C3P3-G1 enzyme, which is preferably the fusion between the African Swine Fever Virus capping enzyme and a mutant bacteriophage K1E RNA polymerase.