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.

We have synthesized a series of novel coplanar chromophores in which heteroarenes, namely, thiophene, benzothiophene, and carbazole, were fused to neighboring phenylene ring(s) through intramolecular annulation via sp(3)-hybridized carbon atoms bearing two p-tolyl groups as peripheral substituents. The molecular configurations of the pi-conjugated backbones were determined by X-ray crystallographic analysis; the heteroarene-fused molecular frameworks of these novel molecules exhibit nearly coplanar conformations. [structure: see text]

Cellular microRNAs (miRNAs) have been shown to regulate hepatitis C virus (HCV) replication, yet a systematic interrogation of the repertoire of miRNAs impacting HCV life cycle is lacking. Here we apply integrative functional genomics strategies to elucidate global HCV–miRNA interactions. Through genome-wide miRNA mimic and hairpin inhibitor phenotypic screens, and miRNA–mRNA transcriptomics analyses, we identify three proviral and nine antiviral miRNAs that interact with HCV. These miRNAs are functionally linked to particular steps of HCV life cycle and related viral host dependencies. Further mechanistic studies demonstrate that miR-25, let-7, and miR-130 families repress essential HCV co-factors, thus restricting viral infection at multiple stages. HCV subverts the antiviral actions of these miRNAs by dampening their expression in cell culture models and HCV-infected human livers. This comprehensive HCV–miRNA interaction map provides fundamental insights into HCV-mediated pathogenesis and unveils molecular pathways linking RNA biology to viral infections.