RNA Helicase DDX3 Interacts with the Capsid Protein of Hepatitis E Virus and Plays a Vital Role in the Viral Replication

Figure 1. GO Enrichment of pORF2-proximal proteins. The selection of pORF2 interactors is indicated by color-coded nodes and grouped according to their intracellular localization (GO CC; (A)) and functions (GO BP; (B)). Selected hits and corresponding relevant categories are indicated. BioID interactions are depicted in red, and reported interactions between hits (collected from http://iid.ophid.utoronto.ca/ (accessed on 10 December 2020); using experimental evidence as a threshold; sources detailed in Supplementary Table S2) are shown by dashed blue edges.

Figure 2. DDX3 co-precipitates the capsid protein, independent of RNA association. (A) Co-IP of DDX3 precipitates the capsid protein. HEK293T cells were co-transfected with FLAG-DDX3 and Myc-BioID2-pORF2 and harvested for Co-IP with the FLAG-tag antibody at 36 h post-transfection (hpt). The IP precipitate and input were subjected to Western blotting (WB). A Myc-BioID2 empty vector (EV) was used as a negative control. Original Western blot images can be found in Figure S1 and S2. (B) The co-precipitation of DDX3 and the capsid protein is independent of RNA. HEK293T cells were co-transfected with HA-DDX3 and Myc-BioID2-pORF2. The cells were harvested 24 hpt. The cell lysate was treated with RNase A at 333 μg/mL for 20 min at room temperature, followed by Co-IP and WB. Original Western blot images can be found in Figures S3 and S4. (C) IFA and confocal microscopy show the co-localization of pORF2 (red) and DDX3 (green). HepG2/C3A cells were co-transfected with Myc-BioID2-pORF2 and FLAG-DDX3 for 42 h. The cells were fixed and probed with a rabbit anti-Myc-tag antibody and a mouse anti-FLAG antibody. The bars in the lower right corners of the images denote 10 μm. The Pearson’s CC is indicated…

Figure 3. The C-terminal domain of DDX3 interacts with the capsid protein. (A) A model of the interaction between DDX3 and the capsid protein. The capsid protein is in a homodimeric form and the chains are colored yellow and blue, and DDX3 is colored orange. “N” stands for the N-terminus and “C” for the C-terminus; these are labeled for each protein. (B) A schematic illustration of the two DDX3 domains and the truncations. The numbers above the lines indicate amino acid residues of DDX3. (C) The capsid protein co-precipitates the C-terminal domain of DDX3. The HEK293T cells were transfected with plasmids of DDX3-D1, D2, and pORF2. Co-IP with the antibody against pORF2 was conducted, followed by Western blotting with an antibody against the HA-tag for DDX3. Cell lysate input was included in the Western blotting as a control. Doublet bands were detected for HA-DDX3-D1, and its expected size is indicated by an arrow. Original Western blot image can be found in Figure S5.

Figure 4. Depletion of DDX3 leads to the inhibition of IFN-β expression. (A) Depletion of DDX3 in GP2-293 cells. The cells were transfected with pSIREN-RetroQ-ZsGreen-shDDX3 and pVSV-G plasmids for 3 days and were then harvested for WB. (B) DDX3 silencing has a minimal effect on GP2-293 cell viability. The cells were co-transfected with pSIREN-RetroQ-ZsGreen-shDDX3 and pVSV-G plasmids for 3 days and were then harvested for the cell viability assay. Three replicates were used for each sample. NS: no significant difference. (C) DDX3 depletion leads to a significant reduction in poly(I:C)-induced interferon-β expression. DDX3-depleted GP2-293 cells were transfected with the capsid protein or the YFP plasmid for 24 h and then transfected with poly(I:C) overnight. The cells were harvested for RNA extraction and RT-qPCR for IFN-β expression. Three replicates were used for each sample. The transcript of the housekeeping gene RPL32 was also determined for normalization. Relative levels in comparison with the YFP and shRNA control are shown. *** denotes p < 0.001, ** for p < 0.01 and * for p < 0.05. Original Western blot image can be found in Figure S6.

Figure 5. Depletion of DDX3 results in a significant reduction in HEV replication. (A) Western blot detection of DDX3 in Huh7.5.1 cells after the DDX3 depletion. The cells were transduced with a retrovirus containing DDX3-shRNA (shDDX3) or control shRNA (shCONT) for 3 days and were harvested for WB detection of DDX3. (B) Depletion of DDX3 has a minimal effect on the cell growth. The Huh7.5.1 cells were transduced with a retrovirus containing shDDX3 for 3 days before the cell viability was tested. Three replicates were used in each group. NS: no significant difference. (C) DDX3 depletion reduces the luciferase yield from the HEV replicon. Huh7.5.1 cells with or without DDX3 silencing were electroporated with the RNA of the p6/luc replicon. The daily culture supernatant samples were assayed for luciferase activity. (D) WB of DDX3 trans-compensation in DDX3-depleted Huh7.5.1 cells. The cells were transduced with a retrovirus encoding shDDX3 for 2 days and then transfected with FLAG-DDX3 or EV (empty vector) for another 2 days. (E) Ectopic expression of DDX3 restores most HEV replication. The Huh7.5.1 cells with DDX3 reconstitution were electroporated with the RNA of the HEV replicon p6/luc.…

Figure 6. The ATPase motif of DDX3 is extremely important in HEV replication. (A) Schematic illustration of DDX3 and its mutants. The numbers above the lines indicate the amino acid residues of DDX3. (B) WB detection of the protein expression of the DDX3 and mutants in DDX3-silenced Huh7.5.1 cells. The Huh7.5.1 cells were transduced with a retrovirus encoding shDDX3 for 2 days and then transfected with DDX3, YFP vector, or mutant DDX3 (K230E and S382L) plasmids for another 2 days. (C) Restoration of HEV replication in cells trans-compensated with the DDX3 wild-type and the helicase mutant. Two days after the reconstitution with the wild-type or mutant DDX3, the Huh7.5.1 cells were electroporated with the RNA of the HEV replicon p6/luc. The luciferase activity in the samples of the culture supernatant was determined on day 5. Relative luciferase activity is shown. NS: no significant difference; ***: p < 0.001. Original Western blot image can be found in Figure S9.