Model of AUF1 p45-mediated WNV genome cyclization. The genomic and structural organizations of the WNV RNA's 5′ and 3′ termini in the linear and circularized forms are shown. Conserved RNA elements that are required for WNV replication are highlighted by heavy lines: SLA (stem-loop A); SLB (stem–loop B); CS (conserved sequence); UAR (located upstream of the AUG translation initiation region); DAR (located downstream of the AUG translation initiation region); 3′ SL (3′ stem-loop). The RNA secondary structures of the UTRs and of the 5′-terminal region of the core (C) protein coding region are depicted according to the experimental data reported by Dong et al. (2008). The translational start codon AUG of the ORF is indicated; it is located within SLB. The residual part of the ORF encoding the viral structural (C, prM, and E) and nonstructural (NS) proteins is represented as a bar. (Upper panel) Linear form of the viral genome. (Lower panel) Circular conformation, mediated by complementary base pairing of sequences in the 5′- and 3′CS, UAR, DAR1, and DAR2 elements, respectively. Previously, we reported that AUF1 p45 stimulates the formation of the circular conformation of the WNV RNA (Friedrich et al. 2014). Here we show that the AUF1 p45-supported RNA cyclization and stimulation of viral RNA synthesis is most efficient when the protein is methylated (AUF1 p45aDMA). The cyclization of the WNV RNA is enhanced by the RNA chaperone activity of AUF1 p45 that comprises an RNA destabilizing as well as an RNA annealing activity. Although the RNA annealing activities of AUF1 p45 and AUF1 p45aDMA are comparable, the RNA destabilizing activity is more pronounced with the methylated AUF1 p45aDMA (indicated by a bold tick).
