Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

Abstract

Background: Dengue virus (DENV) is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined.

Results: In this study, we have used a targeted small interfering RNA (siRNA) library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study.

Conclusions: Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti-viral strategies and treatment of DENV infection.

Figure 1

Development of an image-based DENV detection assay for siRNA screening in Huh7 cells. (A) Detection of DENV infection in Huh 7 cells using immunofluorescence assay with antibody specific for the viral E protein and the cell nuclei are stained with DAPI. (B) Dosage-dependent inhibition of DENV infection is observed in Huh7 cells that are reverse-transfected with different concentrations of siRNA against PTB.

Figure 2

Identification of human genes that is important in endocytosis, vesicle trafficking and signaling as well as cytoskeleton rearrangement on DENV infection using siRNA screening platform. Huh7 cells were reverse-transfected with the panel of 119 siRNAs and, after 2 days, the cells were infected with DENV at an MOI of 1. After 48 hrs post-infection, the DENV infected cells were processed for immunofluorescence staining, auto-image capturing and data analysis. The data is expressed as the percentage of antigen-positive cells and the results are shown from three independent sets of experiments. To establish a baseline of infection and transfection efficiency, cells are treansfected with a control set (top left) of siRNAs and/or transfection reagent, which include transfection lipids alone or together with "non-targeting" siRNA pool, RISC-free siRNA, siGLO (fluorescently labeled) RISC free non-specific siRNA or siRNAs targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Cyclo B duplex and lamin A/C. Together with these controls permitted monitoring of transfection efficiency and cytotoxicity. Accession numbers and a brief description of the role of each gene are provided in Additional File 1.

Figure 3

Confirmation of siRNA suppression of host protein expression and reduction of DENV infection. Gene specific siRNA against (A) CLTC, (B) AP2B1, (C) DNM2, (D) ARRB1, (E) ATP6V0A1 and (F) ARPC1B were reverse-transfected into Huh7 cells at different concentrations (0 nM to 50 nM) and subjected to DENV infection. Dosage dependent inhibition of DENV infection can be observed for these selected genes. At the same time, Western blots were performed after treatment with siRNAs to ensure the knockdown of the specific protein expression. Dosage-dependent reduction of protein expression is also observed for the indicated genes corresponding to the concentrations of the transfected siRNA. The blots are also re-probed with β-actin-specific antibody which served as a gel-loading control (lower panels). (G) Deconvolution of siRNA smartpools that reduced infectious entry of DENV. The experiments shown were repeated with the deconvoluted siRNA sequences (4 individual siRNA) from the Smartpool. The data were displayed for 3 independent experiments.

Figure 4

Bio-imaging analysis of the interaction of clathrin molecules with DENV. DENV were stained green with anti-DENV E protein antibody conjugated to FITC, host clathrin stained red with anti-clathrin antibody conjugated to Texas Red (TR) and host nuclei stained blue with DAPI. (A) Attachment of DENV on the cell surface can be observed at 0 min p.i (arrow) with few co-localizations between DENV and clathrin molecules (arrows) (B) Obvious co-localization is observed between internalized DENV and clathrin by 5 minutes p.i (arrow). (C) Strong co-localization signals are observed between DENV2 and clathrin by 10 minutes p.i (arrows).

Figure 5

Clathrin-mediated endocytosis of DENV into Huh7 cells. Huh7 cells treated with (A) chlorpromazine shows marked reduction in the infectious entry of DENV, whereas (B) filipin does not significantly inhibit virus entry. The solvent (MET, methanol) control for filipin treatment is also included. Minimal cytotoxicity was observed for the concentration range of chlorpromazine and filipin used in this assay. The average of three independent experiments is shown. (C) Inhibition of DENV entry into Huh7 cells expressing EPS15 dominant-negative mutant protein. The infectious entry of DENV is significantly inhibited in Huh7 cells transfected with GFP-EPSΔ95/295 when compared to mock-transfected or pEGFP transfected cells. The number of viral E antigen-positive cells in relation to the total cell population is expressed as a percentage of viral antigen-positive cells. The average of three independent experiments is shown. (D) DENV (stained with TR failed to infect GFP-EΔ95/295 expressing cell. In contrast, internalized DENV particles (arrows) are observed within cells expressing the negative control plasmid (EGFP-C2) expressing GFP.

Figure 6

siRNA knockdown of clathrin heavy chain inhibits all DENV serotypes infection. Huh7 cells were reverse-transfected with different concentrations of clathrin heavy chain (CLTC) specific siRNA pool have resulted in dosage-dependent reduction of all DENV serotypes infection. The average of three independent experiments is shown.

Figure 7

Endosomal trafficking and low pH dependency is required to mediate infectious entry of DENV into cells. (A) Immunoflourescence analysis reveals co-localization of DENV particles with early endocytic vesicles. Anti-EEA1 was used to stain the early endosomes at 15 min p.i. while DENV particles were stained by anti-DENV E protein antibody conjugated with FITC. (B) By 25 min p.i., majority of the DENV particles are found in association with late endocytic vesicles as shown by confocal imaging. Lysotracker (specific stain for late endosomes) was used to stain the late endosomes within Huh7 cells. Perinuclear distribution pattern of the DENV-associated late endsomes is observed too. (C) Infectious entry of DENV is strongly inhibited in Huh7 cells transfected with dominant negative mutant of Rab5 (DNM) as compared to cells transfected with wild-type Rab5 or mock-transfected cells. (D) Bafilomycin A1 pretreatment of Huh7 cells significantly reduce DENV infection in a dosage-dependent manner. Minimal cytotoxicity is observed for the concentration range of bafilomycin A1 used in this assay. The solvent (DMSO) control was also included in this set of experiment. The average of three independent experiments is shown.

Figure 8

Involvement of the cytoskeleton networks in the entry process of DENV virus. (A) Cytochalasin D or (B) nocodazole-pretreated Huh7 cells is shown to inhibit the entry of DENV in a dosage-dependent manner. The percentage of viral antigen-positive cells is plotted against time. Minimal cytotoxicity was observed for the concentration range of the drugs used in this assay. The solvent (DMSO) control was also included in this set of experiment. The average of three independent experiments is shown.