(A) Terminal RNA structures of the DENV4 genome. The UAR, DAR and CS elements are highlighted in yellow, green and red, respectively. Pseudoknotted interactions are labeled. The UFS stem region is …
The UAR, DAR and CS elements are shown in yellow, green and red, respectively. The sequence of the unwound UFS duplex is shown in blue.
The ORF sequences of 48 flavivirus species were obtained from Genbank and aligned by the Clustal W method. The evolutionary relationships of flaviviruses were inferred by using the Maximum …
The SHAPE reactivity results are labeled in the structure models of different flaviviruses. Highly reactive nucleotides (SHAPE reactivity > 0.85) are labeled in red, whereas nucleotides with …
Source data for Figure 2.
SHAPE analysis of DENV3 5′-300 nt RNA molecules containing UFS mutations. the SHAPE result of WT 5′-300 nt RNA is shown in parallel. SHAPE diagrams of 5′ 25–255 nt are shown, and the regions …
(A) The organization of the DENV4 replicon constructs. In p4-cHPstop-SP-IRES-Rluc-Rep, the translation of viral nonstructural proteins is controlled by the EMCV IRES, and artificial stop codons (red …
Source data for 3B, 3D and Figure 3—figure supplement 1.
(A) Demonstration of mutants targeting the AUG start codon. Mutation sites are shown in purple. These mutants were designed without affecting either genome cyclization or the terminal topology of …
SHAPE analysis was performed for DENV4 5′-300 nt RNA containing the UFS mutations M1A, M1C, M3A or M3C. The SHAPE result for WT 5′ end RNA is shown in parallel. SHAPE diagrams of 5′ 20–200 nt are …
Source data for Figure 4.
(A) DENV4 UFS M5 series mutants. Mutations are shown in purple. (B–D) vRNA replication profiles of different UFS mutants in transfected BHK-21 cells. WT and NS5-inactive GVD mutant vRNA, measured in …
Source data for 5B and 5C.
Source data for 5D
Source data for 5F
Source data for 5G, 5H and 5I.
Indirect immunofluorescence assay of WT and UFS-mutated ZIKV vRNA-transfected BHK-21 cells were performed. The secondary structures of mutants were demonstrated and mutation sites are shown in purple.
(A) Simplified diagrams of DENV4 virus 5′ end RNA constructs used for RdRp binding and/or in vitro RdRp activity assays. The region corresponding to the UFS element is shown in blue, and the red …
(A) JEV UFS mutants. Mutations are shown in purple. (B) SDS-PAGE of purified recombinant JEV NS5Pol. (C) The binding of JEV NS5Pol to 5′-320 nt RNA molecules containing UFS mutations was analyzed by …
Lane 1: dsRNA prepared by the annealing of the positive and negative strand of DENV4 5′-160 nt RNA. Lane 2–4: products of RdRp assays using different 5′-160 nt RNAs as templates. Lane 2: WT, Lane 3: …
(A) Demonstration of UFS mutants. The mutations are indicated in purple. The M2C mutant contained two UA-to-GC base pair substitutions, whereas the M4C mutant contained four substitutions in the UFS …
Source data for Figure 7 and Figure 7—figure supplement 1.
(A) Demonstration of mutants targeting the internal loop between SLB and UFS. Mutation sites are shown in purple. (B) Relative replication efficiency of UFS mutants shown above at 72 hr …
The binding of NS5Pol to 5′-300 nt WT, M2A and M2C RNA was analyzed by EMSA assay. The left first lane in each group contained no NS5Pol. The NS5Pol concentrations in the reactions were …
(A) Schematic diagram of experimental design. As 5′-300 nt RNA is incubated with 3′ UTR RNA, a 5′-3′ RNA bimolecular complex is formed due to the interactions that promote genome cyclization. …
3′ UTR RNA (20 ng/μl) was incubated with different amount (16, 40, 80 and 160 ng/μl) of the corresponding 5′-300 nt RNA mutants in 20 μl reactions. The formation of RNA complexes was then analyzed …
Reactions containing a series of concentrations of different 5′-300 nt RNAs and fixed concentration (approximately 36.9 nM) of 3′ UTR RNA were resolved in native PAGE gels. The gels were analyzed by …
(A) SHAPE analysis was performed for DENV4 5′-300 nt WT and M2C RNA in the presence of various amounts of 3′ UTR RNA. For convenience, only the results for nucleotides 60–150, which include critical …
Source data for Figure 9.
(A) Schematic diagram showing the conformational changes of the DENV4 genome. Two major conformations of vRNA, the linear and circular form, exist in equilibrium. The regions involved in terminal …
A proposed mechanistic model of flavivirus vRNA replication. (i) After the viral genome is released into the cytoplasm, first, translation occurs on the circularized genome to generate a sufficient …
Sequences involved in terminal interactions are shown in red. The 5′ RNA structures that are immediately downstream of SLA elements and consistently involved in genome cyclization are indicated by …
The demonstrated sequence was based on DENV4. Sequences involved in genome cyclization are highlighted in yellow. The gray regions are not included in the demonstration of Figure 12. The mfold …
The demonstrated sequence was based on WESSV. Sequences involved in genome cyclization are highlighted in yellow. The gray regions are not included in the demonstration of Figure 12. The mfold …
The demonstrated sequence was based on TBEV. Sequences involved in genome cyclization are highlighted in yellow. The gray regions are not included in the demonstration of Figure 12. The mfold …
The demonstrated sequence was based on RBV. Sequences involved in genome cyclization are highlighted in yellow. The gray regions are not included in the demonstration of Figure 12. The mfold …
Mfold prediction for the terminal RNA structures of different flaviviruses.
Mfold prediction for the 5′ end RNA structures of yokose virus clade of NKV group.
Mfold prediction for analysis of the influence of DENV4 UFS mutations on the overall genome terminal RNA structures.
Structure models of DENV4 5′ WT, M1A, M1C, M3A and M3C RNA generated by RNAstructure software using SHAPE constraints.