Figure 4.

Deletion of the 3′ NCR sequence inhibited the N protein-panhandle interaction in vivo. A, a cartoon showing the M1GFP plasmid in which GFP flanked by the 5′ NCR sequence of CCHFV S-segment vRNA was cloned between T7 promoter and HDV sequence. The mRNA expressed from T7 promoter will not form the panhandle structure due to the deletion of 3′ NCR sequence in the mRNA. B, BSRT7.5 cells were transfected with plasmids exactly as described in the legend to Fig. 3B, except the pPanGFP plasmid in Fig. 3B was replaced with M1GFP plasmid in B. Cells were examined by fluorescence microscopy to monitor GFP expression (upper six panels), as described in the legend to Fig. 3B. The experiment was repeated and the cells were stained with DAPI (lower six panels). C, cell lysates from B were tested by Western blot analysis for the expression of proteins from transfected plasmids as described in the legend to Fig. 3C. D, cells from B were examined by FACS analysis and the quantified GFP signal was plotted exactly as described in the legend to Fig. 3C. E, the cell lysates from B, containing equal amounts of GFP mRNA, were loaded on Ni-NTA beads and total RNA was purified from the material eluted from washed beads. GFP mRNA was detected by PCR as described in the legend to Fig. 3E. F, GFP mRNA levels in the cell lysates from B were quantified by real time PCR as described in the legend to Fig. 3F.