FIG.1.

Design of plasmids. (A) Adaptation and improvement of pol I cloning vectors. The original pol I vectors pHH21 (19, 29) and pHL1261 (11) were modified in several steps. The terminal 3′ nucleotide of the pol I transcript was changed (U→A), which facilitates cloning and precise pol I transcription with a terminal adenine (e.g., nairovirus and hantavirus genome segments), rather than a uracil residue (e.g., influenza virus and phleboviruses) (30, 46). A stop codon cassette to prevent false-positive background signals (13) and a spacer region between the two BsmBI cloning sites for a more efficient and controllable cleavage were also inserted. (B) Construction of chimeric CCHF virus reporter plasmids. Two PCR fragments, one containing the murine (m) pol I promoter and the CCHF virus S vRNA 5′ NCR (primers RF10 and RF186B [Fig. 2]) and the other containing the pol I terminator and the CCHF virus S vRNA 3′ NCR (primers RF183C and RF220 [Fig. 2]), were ligated with the large ApaI-NotI fragment obtained from plasmid pRF207 containing the components for bacterial amplification and selection. This resulted in plasmid pRF238 (pol I [m] CCHF virus S NCR), containing both CCHF virus S segment NCRs separated by a BbsI-BamHI-BbsI cassette. To construct CCHF virus minigenome plasmids, PCR-amplified reporter gene cassettes were inserted between the BbsI sites (shaded boxes) in the pol I-driven expression plasmid pRF238. The special feature (cleavage outside of the recognition site) of the restriction enzyme BbsI was used for exact insertion of the reporter gene sequence between the two NCRs of the CCHF virus S segment. Oligonucleotide sequences are in bold. Start and stop codons are underlined. Restriction enzyme cleavage sites are shown with a line between the sequences. Restriction enzyme recognition sites are shaded. Reporter gene ORFs are in italics. (C) Schematic diagram of the different CCHF virus S CAT and GFP minigenomes used for reverse genetics studies. P_polI(human) and P_{polI(murine),} human and murine pol I promoters, respectively; T_polI, murine pol I terminator sequence. vRNA minigenome constructs pRF243 and pRF249 contain CAT reporter genes in antisense orientation, whereas in pRF244 and pRF250 the CCHF virus N ORF is replaced by a GFP gene. cRNA transcription cassettes in sense orientation were inserted in constructs pRF283 and pRF284. The CAT and GFP genes are flanked by the NCR of the CCHF virus S segment, 168 nt representing the 5′ vRNA (or 3′ cRNA NCR) part and 55 nt representing the 3′ vRNA (or 5′ cRNA NCR) part.