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1.
Figure 3

Figure 3. From: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

Replication of WNV genomes containing DV2 5’NCR substitution mutations. A. Nucleotide sequence alignment of WNV and DV2 5’NCRs. Conserved nucleotide segments are boxed. There is about 50% sequence identity. The translation start codon AUG is underlined. B. WNV genomes bearing substitutions of the 5’NCR by that of the DV2 genome are depicted, and the results of an IFA to detect virus replication in transfected Vero cells are shown. The experimental details are same as in Figure 2 legend.

Li Yu, et al. Virology. ;374(1):170-185.
2.

Figure 5. From: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

A. Growth of viable WN/D2 chimeric viruses in Vero and C6/36 cells. Plaque titers were determined for pools of viruses derived from transfected Vero cells. These viruses were used to infect confluent monolayers of Vero and C6/36 cells at a multiplicity of infection of 0.01. Viruses in the supernatant of infected cell monolayers were plaque titered on Vero cells on the days shown post-infection. Wt WNV, open triangles; WN/DV5’TR99nt virus, closed circles; WN/DV3’USL virus, open diamonds; WN/DN5’TR387nt virus, data points indicated by "x". B. Plaque size differences among chimeric mutant viruses as compared to wt WNV. Vero cells were infected with stock viruses as described under Materials and Methods, and plaques were stained with crystal violet at day 5 post-infection, except for plaques formed by WNV/DV5’TR387nt virus, which were stained at day 8 post-infection.

Li Yu, et al. Virology. ;374(1):170-185.
3.
Figure 6

Figure 6. From: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

Predicted RNA Secondary structures resulting from 5’ and 3’end interactions. The potential 5' and 3'end interactions were predicted using both RNAdraw (Matzura and Wennborg, 1996) and MFOLD v3.2 (Zuker et al, 1991) algorithms. a. Wt DV2 RNA; b. WNV RNA; c. WN/DV3’USL RNA; d. WN/DV5’99nt RNA; e. WN/DV5’TR387nt RNA. WN/DV5'99nt RNA contained an AAT to CAA mutation of nts 100–102, which was introduced in association with generating the chimera. The 5’/3’ CS base-pair interactions are indicated by ovals. Additional hydrogen bonding due to an interaction of the 5' and 3' UAR nt sequences (in the DV2 genome, 5’ nts 82–96 and 3’ nts 10642–10656) are boxed. The ATG codon is labeled with an asterisk. Arrows in 6a indicate 5’ and 3’ directions.

Li Yu, et al. Virology. ;374(1):170-185.
4.
Figure 2

Figure 2. From: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

Replication of WNV viruses containing DV2 3’NCR substitution mutations. The left panel shows schematic representations of the wt and mutant WNV genomes tested. The WNV ORF is represented as a stippled rectangle. WNV 5' and 3'NCR nt sequences are represented by open rectangles, and the DV2 5' and 3'NCR nt sequences are represented by closed rectangles. Nts comprising the 3'SL are indicated by vertically oriented rectangles. Nucleotide numbers in parenthesis indicate nts derived from the DV2 genome that were appended to the WNV genome at the site in the WNV genome indicated by the nt number to the left of the parenthesis. The right panel exhibits the results of IFA on the day indicated. Vero cells were transfected with RNAs of Wt WNV, WNV/DV3’SL, WNV/DV3’NCR, WNV/DV3’UCS, and WN/DV3’USL, as indicated on the left. At 5-day intervals up to 20 days post-transfection, the cells transferred to a coverslip were fixed with acetone, treated with mouse anti-WNV anti-serum, and stained with fluorescein isothiocyanate-conjugated goat-anti-mouse immunoglobulin G.

Li Yu, et al. Virology. ;374(1):170-185.
5.
Figure 4

Figure 4. From: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

Replication of WNV genomes containing heterologous or homologous DV2 5’ and 3’CS elements. A. WNV/DV2 chimeric genome RNAs containing the DV2 5’NCR and nts 100–159 of the 5'ORF, which includes the 5' cyclization sequence (CS), minus (WN/DV5’TR159nt RNA) or plus the entire DV2 3'NCR (WN/DV5’TR159nt3’NCR RNA) or the DV2 3'NCR upstream from the WNV 3'SL (WN/DV5’TR159nt3’USL RNA) are depicted on the left. These RNAs were transfected into vero cells, and WN virus replication was monitored by an IFA conducted at 5-day intervals up to day 20 post-tranfection, on an aliquot of cells that had been transferred to a coverslip. Negative results on day 20 post-transfection of the RNAs is indicated on the right. B. WNV/DV2 chimeric genomes analogous to those depicted in part A were constructed and are depicted on the left, except that in each case the entire ORF of the DV2 capsid gene segment (nts 100–387) was substituted for the ORF of the WNV capsid. Results of the IFA (described in part A and in the legend to Fig 2) on day 20 post-transfection of the indicated RNAs are shown on the right.

Li Yu, et al. Virology. ;374(1):170-185.
6.

Figure 1. Nt sequence requirements for in vitro RdRP activity using purified WNV and DV2 NS5. From: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

In vitro RdRP assays were performed as previously described (Nomaguchi et al., 2004; You and Padmanabhan, 1999). Radiolabeled RNA product molecules of in vitro RdRp reactions were separated by gel electrophoresis and visualized by autoradiography also as previously described (Ackermann and Padmanabhan, 2001). A. Transactivation of RNA synthesis off the WN3'NCR631nt RNA using heterologous or chimeric 5'TRs. RdRP reactions were carried out in the presence (+) or absence (−) of WN3'NCR631nt RNA. Lanes 1 and 2, reactions contained an RNA bearing the 5'terminal 230 nts of the dengue-2 strain NGC genome (DV5'TR230nt). Lanes 3 and 4, reactions contained a chimeric RNA representing the 5'NCR (nts 1–95) of the WNV genome covalently linked to nts 96–230 of the 5'terminus of the DV ORF (WN5'NCR-DV5'ORF). Lanes 5 and 6, reactions contained a chimeric RNA representing the DV 5'NCR nts 1–95 linked to the WN nts 96–230 (DV5'NCR-WN5'ORF). Lanes 7 and 8, reactions contained WN5'TR230nt RNA. Lane 9, reaction contained only WN3'NCR631nt RNA, as control. B and C: In vitro RdRP assays catalzyed by the WNV NS5 (B) or the DV2 NS5 (C) were done using homologous, heterologous, and chimeric WNV/DV2 minigenome RNAs as templates. WNV1051nt RNA contained the 5'terminal 281 nts of the WNV genome fused to the 3'terminal 770 nts, consisting of the 3'terminal 139 nts of the WN ORF plus the entire 631-nt 3'NCR. WNV818nt RNA contained WNV 5' nts 1–161 fused to the 3'terminal 656 nts of the WNV genome, consisting of the 3'terminal 25 nts of the ORF plus the 631-nt 3'NCR. DV2719nt RNA contained the 5'terminal 227 nts of the DENV2 genome fused to the 3'terminal 41 nt of the ORF plus the entire 451-nt 3'NCR. The construction of chimeric RNAs and conditions for PCR followed by in vitro transcription are described under Materials and Methods. The conditions for standard RdRP assays were previously described (Nomaguchi et al., 2004; You and Padmanabhan, 1999). The locations in the gel of 3'elongation products are indicated by solid arrows. The locations of de novo RNA products are indicated by open arrows.

Li Yu, et al. Virology. ;374(1):170-185.

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