Chikungunya and Venezuelan Equine Encephalitis Virus Constructs

All oligonucleotide sequences used are listed in S3 Table. All constructs were verified by sequence analysis. IRES-eGFP was amplified from pL-eGFP, a plasmid with a T7 promoter in which the foreign gene is under control of an encephalomyocarditis virus internal ribosomal entry site and inserted in plasmid pCAGGS/MCSII [104 (link)] using XhoI and BglII restriction sites using standard cloning techniques to create pCAGGS-IRES-GFP. The nucleotide sequences of CHIKV nsPs and protein domains were amplified from pCHIKV-LS3 [21 (link)] and pCHIKrepLS3-nCPE, a replicon derivative from pCHIKV-LS3 mutated in nsP2 (P718S, K649D and R650H), and inserted into pCAGGS-IRES-GFP using EcoRI or SacI and XhoI. Firefly luciferase was amplified from pGL3-MKP-1-Luc [105 (link)] and inserted into pCAGGS-IRES-GFP using EcoRI and XhoI.
pCAGGS-VEEV-nsP2-NTD-Hel was created by amplifying the nsP2-NTD-Hel sequence from cDNA prepared from the VEEV TC-83 stock. The PCR oligonucleotides introduced a start codon, N-terminal HA-tag, a stop codon and SacI and XhoI restriction sites to use for cloning.
pCAGGS-CHIKV-nsP2-NTD-Hel and pCAGGS-VEEV-nsP2-NTD-Hel Walker A and B mutants were created by cloning the CHIKV-nsP2-NTD-Hel or VEEV-nsP2-NTD-Hel PCR product into pCR2.1-TOPO (Thermo Fisher Scientific) for site-directed mutagenesis to introduce the Walker A and B-inactivating mutations prior to transferring the mutated sequences to pCAGGS-IRES-GFP. Renilla luciferase was expressed from phRL-TK or pRL-TK (both Promega).
pCAGGS-FLAG-eEF2 was created by cloning the human synthetic FLAG-eEF2 (IDT) sequence into pCAGGS-MCSII [104 (link)]. pCMV-FLAG-Ub expressed FLAG-tagged ubiquitin [106 (link)]. pCDNA3-FLAG-UbcH10 was a kind gift from prof. Akira Nakagawara (Chiba Cancer Centre, Japan) [107 (link)].