Fig. 4: Self-complementarity of the side loop is required for viral replication.
From: Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase
a The size exclusion chromatography of tRNA-SLADENV. The RNA elutes in two peaks, the early peak likely corresponding to a dimer (D, 91 kDa), and the second peak corresponding to the monomer (M, 45.5 kDa). Protein molecular weight standards are shown on top of the elution profile of tRNA-SLADENV. The size exclusion chromatography was performed twice with similar results. b Side loop in SLA mediates RNA-RNA interactions in solution. Interaction between tRNA-SLADENV and fluorescein-labeled RNAs (F-RNA) was examined by EMSA. See Supplementary Fig. 5 for the RNA sequences. The tRNA-SLADENV interacts with SLA, SLA(-), and the ssRNA complementary to the side loop sequence (Side-M). The tRNA-SLADENV does not bind 8-mer random sequence (negative control), the ssRNA complementary to the top stem (Top), or the self-complementary side-loop RNA (Side). Thus, side loop in SLA mediates RNA-RNA interactions. Binding experiments repeated twice with similar results. Source data are provided as a Source Data file. c Replication of DENV containing SLA mutations. DENV2 SLA was replaced with either ZIKV (SLA-ZIKV) or 9 A (SLA-9A) in the side-loop, and replication of WT and mutant viruses were monitored by immunofluorescence staining using monoclonal anti-NS1 antibody (green fluorescence color) and nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI, blue fluorescent color). Scale bar represents 50 μm. Replication experiments were repeated three times with similar results. d RNA-seq analysis of recovered SLA-9A virus. The numbers and percentage of each haplotypes were calculated from 8,489 total reads. The cutoff of inclusion is >4% abundance. Altered nucleotides in 8 haplotypes are shown in red. Potential intermolecular side-loop interactions resulting from the mutations are shown on the right.
