Figure 1.
Strategies to interrogate host dependencies involved in the complete HCV replication cycle.
Flow diagram of various virologic studies for identification of host factors involved in various steps of the HCV replication cycle. Various virologic assays and the number of validated genes at each stage are indicated. Forty-six genes were not validated by virologic assays of cells treated with OTP SMARTpool siRNAs (either negative results or inconsistent border-line positive results) and therefore were considered as false-positives.
Figure 2.
Identification of HCV entry factors.
A) The effects of siRNA-mediated gene silencing on infection of firefly luciferase-encoded pseudotyped viruses bearing HCV, VSV or MLV envelopes in Huh7.5.1 cells. Inhibition in the heatmap is shown as blue (strong inhibition) to yellow (little or no inhibition), comparing with non-targeting control (NT). B) Firefly luciferase assay that reflects HCVpp (various genotypes), VSV-Gpp or MLVpp entry of Huh7.5.1 cells treated with siRNAs targeting various indicated viral entry factors. Data shown in A and B are subsets of those in Table S4. C) Image illustration and quantitative analyses of HCV core staining in Huh7.5.1 cells depleted of various viral entry factors by siRNAs and subsequently infected with HCV at an M.O.I. of 0.5 for 48 h. Percentages of core positive cells were quantified and normalized to siNT control (set as 1). Green: HCV core, blue: nuclei. Magnification 20×. Scale bars represent 100 µm. D) RT-PCR quantification of intracellular HCV RNA levels in Huh7.5.1 cells treated with indicated siRNAs prior to infection with HCV. Cells were harvested at 48 h post-infection, and total cellular RNA was then extracted. All values were normalized as relative to siNT (non-targeting control siRNA), and represent the mean ± SD, n = 5 (B) or 3 (C, D). D) The asterisks indicate statistically significant differences (*p<0.05; **p<0.01; Student's t test).
Figure 3.
Identification of host factors required for HCV RNA replication or IRES-mediated translation.
A) The effects of siRNAs against various host factors on HCV RNA replication (using JFH1-RLuc subgenomic replicon) and IRES-mediated translation (using pHCV-CLX-CMV RNA containing HCV IRES that harbors a firefly luciferase reporter gene). Values relative to siNT in the heatmap are depicted in a continuum of blue (reduced infection - more than 50% decrease of the replicon activity after siRNA-mediated silencing; proviral) to red (enhanced infection - more than 50% increase of the replicon activity after siRNA-mediated silencing; antiviral). Confirmed host factors in each assay are shown in green (proviral) or red (antiviral). B, C) HCV subgenomic replicon or IRES assays of Huh7.5.1 cells depleted of various indicated host factors with siRNAs. Data shown in B and C are subsets of those in Tables S5 and S6. D, E) Quantification of intracellular HCV RNA levels at 48 h post-infection in Huh7.5.1 cells (D) or PHHs (E) pre-treated with various indicated siRNAs. Knockdown efficiencies of various siRNAs in both cell lines were also determined. F) HCV subgenomic replicon assay of Huh7.5.1 cells transfected with GNB2L1, UBE2J1 or CHKA individual siRNAs. G, H) Effects of PIAS1 or USP11 individual siRNAs on HCV RNA replication or IRES-mediated translation, revealed by HCV subgenomic replicon assay or IRES assay, respectively. B–H) All values were normalized to siNT (as 1), and represent the mean ± SD, n = 5 (B, C, F–H) or 3 (D, E). The asterisks indicate statistically significant differences (*p<0.05; **p<0.01); NS, not significant.
Figure 4.
Identification of host factors important in HCV assembly or secretion.
A) Effects of siRNA-mediated host factor silencing in HCVcc (core staining and viral RNA quantification), HCVpp and replicon assays shown in a heatmap format. The numerical data are also shown in Tables S2, S3, S4 and S5, respectively. Values relative to siNT in the heatmap are depicted in a continuum of blue (reduced infection) to red (enhanced infection). Gene symbols are displayed on the right. Green represents HPF and red, HAF. Part-1, core staining part-one; Part-2, core staining part-two; IC, intracellular HCV RNA; EC, extracellular HCV RNA. B) Image illustration and quantitative analyses of HCV core staining (part-one and part-two) in Huh7.5.1 cells depleted of indicated host factors. Percentages of core positive cells were quantified, and relative ratios normalized to siNT control (set as 1) were shown. Red: HCV core, blue: nuclei. Magnification 20×. Scale bars represent 100 µm. C, D) Effect of IDE or NDRG1 silencing on HCV RNA production or secretion of infectious HCV (D) in Huh7.5.1 cells. HCV RNA quantification and infectivity assay were performed at 48 h post-infection. E) Knockdown efficiencies of various siRNAs in Huh7.5.1 cells. Gene expression assay was conducted at 72 h after siRNA transfection. F, G) HCV subgenomic replicon assay (F) or quantification of HCVsc infection (G) in Huh7.5.1 cells upon IDE or NDRG1 silencing. SiRNA against PIK4CA or ApoE served as the positive or negative control for both assays, respectively. F, G) Data shown are subsets of those in Tables S5 and S7, respectively. B–G) All values were normalized to siNT (as 1), and represent the mean ± SD, n = 3 (B–E) or 5 (F, G). Asterisks indicate statistically significant differences (**p<0.01); NS, not significant.
Figure 5.
Identification of host factors involved in a yet-to-be-defined early stage of the HCV replication cycle.
A) Effects of siRNAs targeting various indicated host factors on HCVcc (core staining and viral RNA quantification), HCVpp, replicon and HCVsc assays are shown in a heatmap. All the numerical data are shown in Tables S2, S3, S4, S5 and S7. Values relative to siNT in the heatmap are depicted in a continuum of blue (inhibition) to yellow (no effect). Gene symbols are displayed on the right. Part-1, core staining part-one; Part-2, core staining part-two; IC, intracellular HCV RNA; EC, extracellular HCV RNA. B) Quantification of intracellular and extracellular HCV RNA levels of CD81-deficient Huh7.25 cells. Cells were treated with various indicated siRNAs for 72 h prior to transfection with full-length JFH-1 HCV RNA. After 48 h, cells and culture media were collected, and viral RNA was extracted and subsequently quantified by Q-RT PCR. C-G) Quantification of HCV core staining part-one and part-two (C), HCV RNA levels (D), HCVsc infection (E), HCV subgenomic replicon RNA (F) and HCVpp and VSV-Gpp entry (G) of Huh7.5.1 cells upon TRAPPC5 or UBE2M silencing. Data shown are subsets of those in Tables S2 (C), S3 (D), S7 (E), S5 (F) and S4 (G), respectively. H, I) Effects of various TRAPPC5 (H) or UBE2M (I) individual siRNAs on HCV infection, determined by measuring intracellular and extracellular HCV RNA levels of Huh7.5.1 cells at 48 h post-infection. B–I) All values were normalized to siNT (as 1), and represent the mean ± SD, n = 3 (B–D, H, I) or 5 (E–G). Asterisks indicate statistically significant differences (*p<0.05; **p<0.01); NS, not significant.
Figure 6.
Integrated map of host dependencies in the complete replication cycle of HCV.
Using the complete HCV replication cycle (from entry to secretion) as a framework, all verified HCV host dependencies from this study were placed based on their predominant subcellular localization and relevance to particular stages of the viral life cycle. In addition, multiple datasets from other HCV siRNA screens and existing publications were mined, explored and integrated into a comprehensive up-to-date dataset of HCV interacting host factors (see Tables S8 and S9). Computational mapping was performed to reconstitute the map that was further refined manually. HPFs (host proviral factors) are shown in red square, HAFs (host antiviral factors) are shown in green circle. Previously published HCV host dependencies (see Table S9) that were also identified in this study are shown in yellow, and other known HCV host factors that were not identified in this study are shown in orange (HPF in circle and HAF in square).