Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants
Fig 1
Characterization of NS5-Mtase mutants.
A) Effects of MTase mutations on N7-MTase activities. B) Effects of MTase mutations on 2’-O-MTase activities. Recombinant wild-type (abbreviated as WT) and mutant (abbreviated as Mut). The mutant MTase of each DENV serotype contained double mutations within the K-D-K-E active site (with the underlined residues mutated to Alanine). MTases of all four DENV serotypes were assayed for GpppA-RNA→m7GpppA-RNA and m7GpppA-RNA→m7GpppAm-RNA conversions to indicate N7- and 2′-O-methylation activities, respectively. Relative methylation activities were indicated below each panel with WT activity set as 100%. C) Immunofluorescence analysis (IFA). BHK-21 cells were electroporated with equal amounts of in vitro transcribed WT and Mut genome-length RNAs of DENV-1 to -4. The mutant genome-length RNAs contained double mutations as described above. At indicated days post-transfection, intracellular E proteins were examined by IFA using mouse antibody 4G2 against DENV E protein and goat anti-mouse IgG conjugated with FITC as primary and secondary antibodies, respectively. D) Plaque morphology. WT and Mut viruses recovered from genome-length RNA-transfected cells were analyzed by standard CPE-based plaque assays using BHK cells. Plaques were developed on day 4 (DENV-2) or day 5 (DENV-1, -3 and -4) post-infection. E) Immunostaining. BHK-21 cells were infected with DENV-3 WT or Mut viruses harvested from genome-length RNA-transfected cells. On day 4 post-infection, cells were assayed by immunostaining using mouse antibody 4G2 against DENV E protein and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) as primary and secondary antibodies, respectively. F) Growth kinetics. BHK-21 and C3/36 cells were infected with WT and mutant viruses at an MOI of 0.01. Viral titers were measured at indicated time points using standard plaque assays (DENV-1, -2 and -4) or immunostaining (DENV-3). Average results and SD of three experiments are presented. Dash line indicates the limitation of detection (10 PFU/ml).