Dimerization of the U85CUG88 mutants is not affected by the introduced mutations. Schematic representation of: (A) The FIV genome, (B) the full-length unspliced gRNA up to 744 nt expressed from a T7 expression plasmid. The dashed arrow shows the 744-nt long, 5′ region of the unspliced FIV gRNA that was cloned for in vitro transcription. The table describes the different mutations introduced in the U85CUG88 motif of the gRNA for biochemical experiments. (C) The envelope (env) spliced mRNA up to 744 nt expressed from a T7 expression plasmid. The dashed arrow shows the 5′ 744-nt long region of spliced env mRNA that was cloned for in vitro transcription. (D) A representative gel image showing in vitro dimerization of WT gRNA (AD26) and U85CUG88 mutant RNAs in TBM buffer. M and D below the lanes of the gel indicate the monomer and dimer buffers, respectively, in which the dimerization experiments were performed. The positions of the monomeric and dimeric RNA species are indicated by the letters M and D, respectively, on the left side of the gel. (WT) Wild type. (E) The histograms show dimerization efficiencies of all mutant RNAs compared to the WT calculated by performing a densitometric analysis of the bands from three independent experiments (shown as open circles). Dimerization efficiency was calculated by measuring the intensity of the dimeric RNA band divided by the intensity of total RNA bands (i.e., sum of intensities of dimer and monomer bands).
