RIG-I, MDA5 and TLR3 Synergistically Play an Important Role in Restriction of Dengue Virus Infection

Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus

Over-expression of RIG-I and MDA5 in wild type HUH-7 cells.

<p>(A) RIG-I and MDA5 constructs were over-expressed in HUH-7 cells and lysates harvested 48 hours post infection. Immunoblotting for antibodies stated on the left shows a decrease in viral antigen in cells double transfected with both plasmids. Real-time RT-PCR analysis of gene expression in mock and RIG-I and/or MDA5 transfected cells. Total RNA was isolated, used for cDNA preparation and subjected to real-time RT-PCR analysis. Rows below figure show negative strand viral RNA. (B) IFN-β production was assayed using ELISA. Symbols indicate significantly different from vector transfected and infected cells (*P<0.05).</p

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

<p>(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.</p

Endoplasmic reticulum (ER) stress-induced expression of XBP1 mRNA and calreticulin protein expression.

<p>(A, upper panel) XBP1 splicing in DV1-infected cells. Total RNA samples were prepared and RT-PCR analysis was performed. sXBP1, spliced form of XBP1; uXBP1, unspliced form of XBP1. (A, lower panel) Analysis of calreticulin expression in DV1-infected cells. Whole cell lysate from DV1-infected cells were subjected to Western blot analysis and probed for the calreticulin and visualized by enhanced chemiluminescence. The nitrocellulose membranes were then reprobed for β-actin (loading control). Note increase in calreticulin expression in DV1-infected shRIG-I cells. (B, upper panel) <i>In situ</i> DNA fragmentation analysis (TUNEL) of mock and DV1-infected cells was carried out by flow cytometry. (B, lower panel) Flow cytometric analysis of DNA content by PI staining: DV1-infected HUH-7 and shRIG-I cells were analyzed after 72 hpi. The PI uptake rate was determined by flow cytometry and analyzed using WinMDI 2.7 software. Percentage of cells in sub-G1 (M1 region), indicative of cell death, in each sample is shown. DV1-infected shRIG-I cells showed significant increase in sub-G1 content.</p

siRNA silencing of TLR3 in HUH7 and shRIG-I cell lines.

<p>siTLR3 was transfected in HUH7 and shRIG-I cells and total RNA was tested for DV1 negative strand RNA by semi-quantitative RT-PCR. Rows below figure shows quantitative analysis of DV1 negative strand RNA, TLR3 and IFN-β production in infected HUH7 and shRIG-I cells. Symbols indicate significantly different from vector transfected and infected cells (**P<0.01).</p

DV1 replication and propagation in HUH-7 and shRIG-I cells.

<p>(A) Flow cytometric analysis showed enhanced DV1 infection in shRIG-I cells as compared to HUH-7 cells. Cells were stained with anti-Dengue E or IBV S (an isotype control) antibodies and analyzed by flow cytometry. (B) Tissue Culture Infectious Dose 50 (TCID50) assay. Virus titers in 50% tissue culture infectious doses (TCID₅₀)/ml were determined according to Reed and Muench <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000926#pntd.0000926-Reed1" target="_blank">[45]</a>. Symbols indicate significantly different from infected HUH-7 (**P<0.01). (C) Qualitative RT-PCR detection of IFN-related gene expression. Cells grown in 6-well plates were infected with DV1 for up to 72 h. Total RNA was extracted, used for cDNA synthesis and subjected to RT-PCR analysis. GAPDH was used as a control for equal RNA templates. (D) Real-time RT-PCR analysis of gene 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. IFN-β production was assayed using ELISA (lower panel). Symbols indicate significantly different from infected HUH-7 cells (**P<0.01). (E) Total cellular protein was extracted and analyzed by immunoblot. IRF3 dimerization was assessed by native PAGE with anti-IRF3 antibody as a probe. Tubulin protein level was assessed by SDS PAGE to ensure equal loading of cell lysate. Arrows indicate IRF3 dimer and monomer. Row below figure shows ratio of dimer: monomer analysed by densitometry.</p