Figure 1.
Hydrophobic network and GTR sequence of JEV NS5 are highly conserved in the Flavivirus genus.
(A) Schematic depiction of the key components within the MTase-RdRP interface of NS5. RdRP interacting module (residues 112–128) and GTR sequence from MTase, and the middle, index, and ring finger subdomains of RdRP are shown in grey. Six hydrophobic residues are shown as boxes. The hydrophobic interactions among six residues are indicated by dashed lines. (B) Representative NS5 sequences from the Flavivirus genus were aligned using ClustalW software [http://embnet.vital-it.ch/software/ClustalW.html]. Specifically, both WNV and JEV belong to the JEV group, DENV1-4 belong to DENV group and YFV belongs to YFV group. The representative viruses mentioned above are all from Mosquito-borne viruses. TBEV comes from Tick-borne viruses and Modoc virus (MODV) from no known arthropod vector. Gray shading indicates the conservative residues of hydrophobic network and GTR sequence in MTase-RdRP interface of NS5.
Figure 2.
Functional analysis of hydrophobic network mutations in NS5 interface in a JEV infectious cDNA clone.
(A) Immunofluorescence analysis and plaque morphology of JEV genome-length viral RNA replication containing hydrophobic network mutations in transfected BHK-21 cells at the indicated time points. Monoclonal antibody against SLEV envelope protein and Texas Red-conjugated goat anti-mouse IgG were used as primary and secondary antibodies, respectively. The supernatants collected at 120 hpt were assayed for plaque morphology analysis by double-layer plaque assay as described in Materials and Methods. (B) The supernatants were collected at each time point post transfection, and subjected to monolayer plaque assay for measurement of virus production. The visible plaques were used to calculate titers of JEV WT and mutants.
Figure 3.
Functional analysis of NS5 GTR sequence mutations in JEV infectious cDNA clone.
(A) Immunofluorescence analysis and plaque morphology of JEV genome-length viral RNA replication containing NS5 GTR sequence mutations in transfected BHK-21 cells at the indicated time points. Monoclonal antibody against SLEV envelope protein and Texas Red-conjugated goat anti-mouse IgG were used as primary and secondary antibodies, respectively. The plaque morphology of GTR sequence mutants and WT virus was determined by the double-layer plaque assay using the supernatants collected at 120 hpt. (B) Virus production of the transfected cells at each time point post transfection was detected by monolayer plaque assay, and the visible plaques were used to calculate titers of JEV WT and GTR mutants.
Figure 4.
Functional analysis of NS5 interface and GTR sequence mutations in a DENV-2 infectious cDNA clone.
(A) Immunofluorescence analysis and plaque morphology of DENV-2 genome-length viral RNA replication containing representative hydrophobic network and GTR sequence mutations of NS5 in transfected BHK-21 cells. Monoclonal antibody 4G2 against DNEV-2 envelope and Alexa Fluor 488-conjugated goat anti-mouse IgG were used as primary and secondary antibodies, respectively. The supernatants collected at 120 hpt were assayed for double layer plaque assay for size difference analysis as described in Materials and Methods. (B) Virus production of the supernatants of the transfected cells at each time point post transfection was detected by monolayer plaque assay, and the visible plaques were used to calculate titers of DENV-2 WT and NS5 interface and GTR sequence mutants.
Figure 5.
Characteristics and sequencing analysis of recovered viruses from hydrophobic network and GTR sequence mutations of NS5 in JEV.
(A) Comparison of plaque morphology changes of JEV between representative NS5 mutations and their corresponding passaged viruses. JEV NS5 P113D and F467D viruses were serially passaged in BHK-21 cells, and the plaques at passage 0 (P0) and passage 5 (P5) were compared with that of WT. (B) Summary of sequencing results for all recovered viruses from P5. (C) Crystal structure of JEV NS5 showing the key residues in the MTase-RdRP interface and the recovered secondary compensatory mutations. A combination of cartoon and surface representations was used with MTase in cyan, RdRP palm in grey, thumb in light blue, index finger in green, middle finger in orange, ring finger in yellow, pinky finger in light red, N-terminal extension of RdRP in pink, priming loop in purple, and signature sequence SGDD in magenta. Six hydrophobic residues and the GTR sequence are shown as small spheres that are represented and then labeled at the top left corner. The recovered mutations are shown as large spheres with the same color of its original mutations.
Figure 6.
Compensatory mutations analysis for P113D and F467D.
(A–B) Functional analysis of adaptive mutations of P113D and F467D using a JEV infectious clone. IFA was performed on BHK-21 cells transfected with genome-length RNAs at the indicated time points. Plaque morphology of WT and mutant viruses was compared. (C) Virus production of the transfected cells at each time point post transfection was detected by monolayer plaque assay, and the visible plaques were used to calculate titers of viruses.
Figure 7.
Replicon analysis of P113D and F467D and their compensatory mutations.
(A) The structure of JEV-Rluc-Rep is depicted. JEV-Rluc-Rep was constructed by replacing the structural genes with the Renilla luciferase (Rluc) gene. (B) JEV-Rluc-Rep was used for direct measurement of the effects of P113D and F467D and their compensatory mutations on viral RNA replication. Equal amounts of WT and mutant JEV-Rluc-Rep RNAs were transfected into BHK-21 cells and assayed for luciferase activity at the indicated time points post transfection. Three independent transfections were performed and the representative data were presented.