FIG. 11.

(A) Northern hybridization analysis of negative-strand WN RNAs in cells transfected by wt and lethal mutant RNAs. Total cellular RNAs were isolated from mock-transfected cells (lane 4) and from cells transfected with wt WN RNA (lane 3) or WNmutA1, WNmutA2, WNmutC2, and WN/DN-SL RNAs (lanes 5 to 8, respectively) 40 h p.e. RNAs were then electrophoresed on a denaturing 1.2% agarose gel for 4 h at 120V, transferred to a membrane, and hybridized to an in vitro-synthesized ³²P-labeled ∼4.7-kb positive-sense ssDNA probe representing nt 1894 to 6777 of the WN virus genome. Full-length in vitro-synthesized WN negative-strand RNA (lane 1) and WN positive-strand RNA isolated from infectious virus (lane 2) served as controls to establish the specificity of the hybridization for WN virus negative-strand RNA. (B) After electrophoresis and prior to transblotting, the agarose gel was stained with ethidium bromide and photographed on a UV light box to visualize 18S and 28S rRNAs in preparations of total cellular RNA (lanes 3 to 8).