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.
Highlights
Hepatitis C virus (HCV) is a hepatotropic member of the Flaviridae family and a primary etiologic agent of chronic hepatitis that can progress to cirrhosis and hepatocellular carcinoma (HCC) [1]
To functionally interrogate HCV host dependencies uncovered from the genome-wide small interfering RNA (siRNA) screen [4] and study how these cellular functions are relevant for the HCV replication cycle, we selected 205 host factors of interest based on their known molecular functions and potential interactions with HCV
F) HCV subgenomic replicon assay of Huh7.5.1 cells transfected with GNB2L1, UBE2J1 or CHKA individual siRNAs
Summary
Hepatitis C virus (HCV) is a hepatotropic member of the Flaviridae family and a primary etiologic agent of chronic hepatitis that can progress to cirrhosis and hepatocellular carcinoma (HCC) [1]. Standard therapy for hepatitis C was a combination of peginterferon and ribavirin, curing only about half of the patients with substantial side effects [2]. The recent development of direct-acting antivirals (DAAs) significantly improves treatment response in patients infected with HCV genotype 1 [2]. The newer regimens are still suboptimal and problematic concerning adverse effects, viral resistance, drug-drug interactions and variable efficacies among HCV genotypes [2]. HCV exploits host factors extensively for infection and propagation [3,4,5,6]. Identification of these host dependencies may provide potential antiviral targets, and critical insights into mechanisms of HCV-mediated pathogenesis and chronic liver disease
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