FIG. 6.

Complementation of full-length KUN virus RNA with a deletion of the SAM binding site in the NS5 gene (construct FLdSAM). Results of IF analysis with anti-E antibodies (A) and Northern blot analysis with the E-specific probe (B) for detection of replicating FLdSAM RNA are shown. Photos 1 to 3 in panel A and the corresponding repBHK lanes in panel B demonstrate replication of electroporated FLdSAM RNA in repBHK cells at 3, 5, and 7 days (3d, 5d, and 7d, respectively). BHK lanes in panel B show the absence of replication of FLdSAM RNA at 3, 5, and 7 days after transfection into normal BHK cells. Photo 4 in panel A shows results of IF analysis with anti-E antibodies of repBHK cells at 2 days after infection with complemented FLdSAM virus recovered at 7 days after transfection of repBHK cells with FLdSAM RNA. Photos 5 and 6 show results of dual-IF analysis with anti-E (fluorescein isothiocyanate [FITC] stain; photo 5) and anti-NS3 (Texas Red [TR] stain; photo 6) antibodies of normal BHK cells infected with complemented FLdSAM virus recovered at 7 days after transfection of repBHK cells with FLdSAM RNA and immunostained 2 days later. CF, culture fluid. The arrow in panel B indicates the position of RNA of about 11 kb, determined as described in the legend to Fig. 4. The Northern blot was exposed to X-ray film for 24 h, compared to 3 h for the blot in Fig. 5B.