Integrative Functional Genomics of Hepatitis C Virus Infection Identifies Host Dependencies in Complete Viral Replication Cycle

Abstract

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

“…The expression and binding of these proteins may not be simultaneous but instead may be dependent on cell cycle phases and the stages of the virus life cycle and disease (27). HuR is an RNA binding protein that has previously been shown to bind to the HCV 3= UTR and has also appeared in some whole-genome siRNA knockdown studies in the context of HCV (28,29,44,45). Our study using both HCV replicon and JFH1 infectious cell culture systems clearly demonstrates that depletion of HuR leads to a significant reduction in the HCV RNA and protein levels.…”

Engrafted human stem cell–derived hepatocytes establish an infectious HCV murine model

“…We compared this list, updated with all other host factors described in the literature (a total of 323 genes) (35), with a list of 374 genes shown by microarray as significantly induced (i.e., >4-fold) during one…”

Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

“…To verify whether Pim kinases were required for entry, we performed viral entry assays using HCV-like particles (HCV-LP). We performed viral entry assays using HCV-LP as described previously (21,22). In this system, HCV-LP can undergo singlecycle infection without producing virions.…”

“…Single-round infectious HCV (HCVsc) was generated from a replicon trans-packaging system as previously described (21,22). Briefly, Huh7.5 cells were cotransfected with pHH/SGRLuc plasmid, which carries a bicistronic HCV subgenomic (NS3-5) replicon firefly luciferase reporter with a Pol I promoter/terminator, and an HCV core-NS2 expression plasmid by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.…”