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. 2011 Jan 4;5(1):e926. doi: 10.1371/journal.pntd.0000926

Figure 1. DV1 infection of HUH-7 and shRIG-I cells.

Figure 1

(A) Whole cell lysate from DV1-infected cells were subjected to Western blot analysis and probed for the antibodies indicated and visualized by enhanced chemiluminescence. The nitrocellulose membranes were then reprobed for β-actin (loading control). (B) Qualitative RT-PCR for MDA5 mRNA level in DV1-infected cells from different time points. Total RNA was isolated and subjected to RT-PCR analysis. GAPDH was used as a control for equal RNA templates. (C) Uninfected shRIG-I and HUH-7 cells were stimulated with synthetic polyI:C. Cell lysates were assessed after 24 h of stimulation by Western blot for MDA5. β-actin was used as a control for equal loading of cell lysates. (D) Real-time RT-PCR analysis of MDA5 expression in mock and DV1-infected cells. Total RNA was isolated, used for cDNA preparation and subjected to real-time RT-PCR analysis. Bar histograms show the average difference in gene expression between mock and DV1-infected cells based on at least two independent experiments. (E) siRNA silencing technique was used to silence RIG-I gene. In HUH-7 cells, and not in A549 cells, RIG-I silencing co-induced silencing of MDA5 gene. (F) A549 cells were transfected with siRNA for EGFP, RIG-I and/or MDA5. Whole cell lysate from mock and DV1-infected A549 cells were subjected to immunoblotting (IB) and RT-PCR analysis. Results show that DV1 infection is significantly higher in cell line deficient for both RIG-I and MDA5. (G) Whole cell lysate from HUH-7 and shRIG-I cells infected with either wild type or UV-treated DV1 virus were subjected to immunoblotting (IB) and RT-PCR analysis. DV1 was UV-inactivated by exposing the virus to a UV-lamp (wavelength, 254 nm) at a distance of 5 cm for 1 hour. Results show that UV-treated DV1 infection did not activate RIG-I and MDA5. (H) Specificity of monoclonal NS3 antibody. Myc-tagged DV1 NS1 and NS3 constructs were transfected into HUH-7 cells. Cell lysates were harvested 24 h later and used to test specificity of monoclonal NS3 antibody. DV1-infected HUH-7 cell lysate was used as a control. Anti-NS3 antibody was found to be specific for DV1 NS3 protein.