Construction of expression vectors for CHIKV replicase

CHIKV sequences were based on the isolate LR2006 OPY1 representing the East Central South African genotype. The sequence encoding for ns polyprotein was optimized according to human codon usage (hereafter this coding sequence is referred to as P1234) and obtained as synthetic DNA (Genscript, USA; other synthetic DNAs were also obtained from the same company). A construct, where only 120 codons from the 5′ end and 52 codons from the 3′ end of CHIKV ns ORF were to correspond to human codon usage, was made for comparison. It contains native codon usage in 93% of ns polyprotein encoding region and was designated P1234-NAT.

In order to express ns polyprotein, two different approaches were used. First, expression of mRNA for ns polyprotein was driven by the RNA polymerase of bacteriophage T7. To achieve efficient ns polyprotein translation from non-capped transcripts, the P1234 was placed under control of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) [52]. To increase the stability of mRNAs, the IRES-P1234 cassette was flanked by the 5′ and 3′ UTRs from mRNA encoding for human beta actin. 60 adenine residues were added to the 3′ end of the construct; this sequence was followed by the antisense strand ribozyme from hepatitis delta virus (HDV) and terminator for T7 RNA polymerase. The sequence containing all these elements was obtained as synthetic DNA and cloned into pUC57Kan plasmid; the final construct was designated T7-P1234 (Fig 1A). Second, expression of mRNA for ns polyprotein was triggered by the immediate early promoter of human cytomegalovirus (CMV). In this case, the P1234 and P1234-NAT were cloned into pMC-gtGTU2 vector (FIT Biotech Plc, Finland) that contains the CMV promoter, leader sequence from the thymidine kinase of herpes simplex virus (HSV TK) with an inserted synthetic intron and late polyadenylation signal from simian virus 40 (SV40). The constructs were designated CMV-P1234 (Fig 1A) and CMV-P1234-NAT. In addition, the cassette containing the CMV promoter, P1234 and SV40 polyadenylation signal was inserted into pMC.BESPX minicircle production vector [53] to generate MCC-P1234. In order to obtain polymerase-inactive forms of CHIKV replicase the catalytic Gly-Asp-Asp (GDD) motif in nsP4 was changed to Gly-Ala-Ala (GAA) using PCR-based site-directed mutagenesis. The resulting construct was designated P1234^GAA.

(A) Schematic presentation of T7- and CMV promoter-based expression constructs of CHIKV replicase. T7—promoter for T7 RNA polymerase; IRES—EMCV IRES; CMV—CMV promoter; LI—HSV TK leader region with an intron. Arrows below the drawings point to the position of the inactivating GDD to GAA mutation in the catalytic site of nsP4. (B) Schematic presentation of T7—and CMV promoter-based constructs expressing template RNAs. The 5′ and 3′ UTRs are from CHIKV; N77—region encoding for the 77 N-terminal amino acid residues of nsP1; SG—CHIKV SG promoter. The position of the human beta globin intron (hBG), removed by splicing, is marked. For both A and B, the antisense strand ribozyme of HDV (RZ), T7 transcription terminator and SV40 late polyadenylation region are shown. The drawings are not in scale. (C) U2OS cells were co-transfected with CMV-P1234 + CMV-Fluc-Gluc or CMV-P1234-NAT + CMV-Fluc-Gluc. Gluc activity in the medium was measured at 24 h post transfection. An average of three independent experiments is shown, error bars represent standard deviation. ** designates p<0.01 (Student’s paired t-test). (D) BSR cells were co-transfected with T7-P1234 + T7-Fluc-Gluc or CMV-P1234 + CMV-Fluc-Gluc. Aliquots of growth media were collected at the indicated time points and used for measurement of Gluc activity. Each point represents an average from three parallel experiments; error bars are too small to be shown.