FIG 3.

AUF1 p45 increases the accessibility of U residues predominantly in the bottom part of the DENV 3′SL. (A) (Left) Secondary structure of the 3′-terminal stem-loop (3′SL) of the DENV genome. Nucleotide position numbering starts at the 3′ end of the RNA. Nucleotides that were exposed in the presence of AUF1 p45 are highlighted in red. (Right) RNA structure probing of the DENV 3′SL with CMCT (modifies U and G residues in single-stranded RNA) in the presence of the indicated concentrations of AUF1 p45 (lanes 8 to 10). Control reaction mixtures in CMCT buffer were included (lanes 5 to 7). Products of the primer extension reaction, carried out with a radiolabeled primer, were analyzed by 8% denaturing PAGE along with a sequencing ladder (lanes 1 to 4). Note that due to the stop of the reverse transcriptase upon encountering alkylated Watson-Crick positions, the resulting product is one base shorter. Two regions of the gel are enlarged on the right. (B) Quantification of cDNA products of the primer extension analysis from panel A. (C) RNA structure probing of the DENV 3′SL with CMCT in the presence of the indicated concentrations of AUF1 p45 and hnRNPH1. Two regions of the gel where we observed changes of the secondary structure in the presence of AUF1 p45 are shown (see panel A).