Engineered Chikungunya Virus with Altered 3'UTR

To facilitate mutations within the 3’UTR of CHIKV-Cbn [17 (link)] and CHIKV-LR [67 (link)] infectious clones, a unique SacI restriction site was introduced downstream of the stop codon of the viral structural proteins. To this end, the PCR product generated with primers sense 94 and antisense 92, and the product of a second PCR generated with primers sense 93 and antisense 95, were fused by overlapping PCR. The XhoI- NotI fragment of CHIKV-Cbn or CHIKV-LR was replaced by the XhoI- NotI fragment of the overlapping PCR products to generate CHIKV-Cbn SacI or CHIKV-LR SacI, respectively. A 3’UTR cloning cassette between unique SacI and NotI restriction sites allowed us to exchange the wild type sequences by mutant 3’UTRs, as described in S1 Table. Nucleotide sequences of primers used for PCRs are listed in S2 Table.
The chimeric viruses were obtained digesting the infectious clones with SacI and NotI and introducing the products in the alternate lineage. The DNAs of the recombinant constructs were linearized by digestion with NotI enzyme and used as templates for transcription by SP6 polymerase in the presence of GpppG cap structure analog, using the mMessage mMachine transcription kit (Thermo Fisher) according to manufacturer’s instructions. The RNAs were gel-quantified and used for transfection in cell culture.