FIG. 5.
Complementation of full-length KUN virus RNA with a GDD deletion in the NS5 gene (construct FLdGDD). Results of IF analysis with anti-E antibodies (A) and Northern blot analysis with the E-specific probe (B) for detection of replicating FLdGDD RNA are shown. Photos 1 and 2 in panel A and the corresponding repBHK lanes in panel B demonstrate replication of FLdGDD RNA in repBHK cells at 3 and 5 days (3d and 5d), respectively, after electroporation. Photo 3 in panel A and the BHK lanes in panel B indicate the absence of replication of FLdGDD RNA as late as 5 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 the complemented FLdGDD virus recovered at 5 days after transfection of repBHK cells with FLdGDD 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 for 2 days with complemented FLdGDD virus recovered at 5 days after transfection with FLdGDD RNA. CF, culture fluid. The control lane in panel B contains 10 ng of in vitro-transcribed FLdGDD RNA. The arrow 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 3 h.