Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone

Schwarz, Megan C.; Sourisseau, Marion; Espino, Michael M.; Gray, Essanna S.; Chambers, Matthew T.; Tortorella, Domenico; Evans, Matthew J.

During cloning of the ZIKV RT-PCR products (scheme illustrated in Fig. 1C ), we were unable to generate plasmids carrying an intact NS1 through NS3 coding sequence. Our first attempts at cloning this region of the ZIKV genome resulted in plasmids with large deletions, which we deduced to likely be a result of homologous recombination events by noting short stretches of sequence similarity at either side of the deleted segment. In an attempt to circumvent this issue, we screened a panel of bacterial strains with documented inefficiencies at homologous recombination. Indeed, using New England BioLabs' Turbo competent cells allowed us to propagate a plasmid bearing the entire ZIKV cDNA. However, all such plasmids continued to lack an intact viral open reading frame due to the presence of either nonsense or frameshift mutations within the NS1 coding sequence. At this stage, we hypothesized that translation of viral polyprotein sequence from this region led to toxicity in bacteria. To test this hypothesis, we inserted a synthetic intron in NS1, such that the coding sequence would be disrupted in bacteria but splicing in mammalian cells would restore the viral RNA ( Fig. 1B and C) . Indeed, this plasmid could be stably propagated in bacteria, thus completing the generation of a plasmid containing the entire ZIKV cDNA. As a negative control for subsequent replication studies, we also generated a version of this plasmid encoding an inactivating GDD to GNN mutation in the viral NS5 RNA-dependent RNA polymerase (RDRP) catalytic active site [Pol(Ϫ)].