A genome-wide genetic screen for host factors required for hepatitis C virus propagation

Abstract

Hepatitis C virus (HCV) infection is a major cause of end-stage liver disease and a leading indication for liver transplantation. Current therapy fails in many instances and is associated with significant side effects. HCV encodes only a few proteins and depends heavily on host factors for propagation. Each of these host dependencies is a potential therapeutic target. To find host factors required by HCV, we completed a genome-wide small interfering RNA (siRNA) screen using an infectious HCV cell culture system. We applied a two-part screening protocol to allow identification of host factors involved in the complete viral lifecycle. The candidate genes found included known or previously identified factors, and also implicate many additional host cell proteins in HCV infection. To create a more comprehensive view of HCV and host cell interactions, we performed a bioinformatic meta-analysis that integrates our data with those of previous functional and proteomic studies. The identification of host factors participating in the complete HCV lifecycle will both advance our understanding of HCV pathogenesis and illuminate therapeutic targets.

Fig. 1.

The siRNA screen. (A) siRNAs against CD81 or ApoE inhibit HCV infection (green: HCV core). Percent infected (lower left corner) is relative to control throughout (Fig. S1A and B). NT, Nontargeting siRNA #2. Magnification, 20×. (B and C) The results of the HCVcc siRNA screen part one (B) and part two (C) are shown with the siRNA SMARTpools ranked in order of average Z-score, from lowest (decreased infection) to highest (increased infection). The position of known HCV-host factors and several newly identified genes that scored in the screen are indicated.

Fig. 2.

Network showing connections between HCV proteins, HCV-interacting human proteins and candidate proteins from the HCVcc siRNA screen. Network extension analysis of HCV-host protein interactions derived from the infection mapping project (I-MAP, 20) revealed extensive first-order (direct) interactions between host factors implicated in the I-MAP and those found in the HCVcc siRNA screen (enrichment P = 0.0001).

Fig. 3.

Network clusters derived from Fig. 2 showing interactions between HCV-host factors from the siRNA screen and host proteins interacting with HCV core, E1, E2, F, p7 or NS components (from the infection mapping project (I-MAP, 20) thereby placing the siRNA screen hits in the network neighborhood of the respective HCV proteins. For clarity, interactions between HCV proteins and their human interacting partners are not displayed, but are shown in Fig. 2.

Fig. 4.

Common features of host-Flaviviridae interactions. (A) Common network modules for HCV-dependency factors identified in siRNA screens for Flaviviridae. The network was generated using first-order (direct) interactions between siRNA hits from HCV, WNV, and dengue virus screens to identify common functional modules potentially used by Flaviviridae. Network clusters obtained were examined for enrichment (P < 0.05) in KEGG signaling pathways. (B and C) Common network modules for HCV host factors identified in the HCVcc and HCV replicon siRNA screens. (B) Network cluster generated using second order interactions for overlapping siRNA hits reveals a significant enrichment for the TGF-β-signaling pathway (P = 5 × 10⁻¹⁰). (C) First order interactions identify connections between HCV replicon screen hits and a number of TGF-β- and VEGF pathway-associated siRNA hits from the HCVcc screen. Other binary or triple-member connections include subunits (BCKDHA and BCKDHB) of the branched chain keto acid dehydrogenase E1 enzyme complex, and components (TRRAP and YEATS4) of the NuA4 histone acetyltransferase complex.