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.
The ubiquitous ATP-dependent RNA helicase DDX3X is involved in many cellular functions, including innate immunity, and is a pivotal host factor for hepatitis C virus (HCV) infection. Recently, we showed that DDX3X specifically recognizes the HCV 3= untranslated region (
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.
Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCVinduced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.epatitis C virus (HCV), a member of the Hepacivirus genus in the Flaviviridae family, is an enveloped, single-stranded and positive-sense RNA virus that infects humans and other higher primates, with a selective tropism to the liver. The virus is estimated to infect 2.8% of the world's population (1), and has evolved into a major causative agent of end-stage liver diseases, including cirrhosis and hepatocellular carcinoma (HCC) (2). Chronic hepatitis C is also the leading indication for liver transplantation in the United States (3). To date, a protective vaccine is not available. Current therapeutic regimens applying direct-acting antivirals with or without ribavirin have made it possible to cure the majority of patients with HCV (4).HCV infection gains chronicity in ∼75-85% of patients, facilitated by various viral mechanisms to evade host immune responses and exploit the cellular machinery (5). The replication cycle of HCV in the host cell consists of multiple sequential steps, beginning with the lipo-viro-particle binding and entry, followed by viral RNA translation and replication, packaging and assembly of the virion, and finally secretion from host cells (6, 7). Each...
BACKGROUND & AIMS The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor beta (TGFB) signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6, and another inhibitory SMAD (SMAD7), promote HCV infection in human hepatoma cells and hepatocytes. METHODS We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with JFH1 HCVcc; we measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. HCV entry in HepG2/miR122/CD81 cells, which support entry and replication of HCV, were transfected with small-interfering (si)RNAs and gene expression profiles were analyzed. Uptake of labeled low-density lipoprotein (LDL) and cholesterol were measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 mRNAs. RESULTS siRNA knockdown of SMAD6 blocked the binding and infection of cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased infection by HCV. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells following SMAD6 knockdown. SMAD6 knockdown also reduced transcription of gene encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR (VLDLR), and the scavenger receptor class B member 1 (SCARB1 or SR-BI) in hepatocytes; SMAD6 knockdown also reduced cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. HCV infection of cells increased expression of SMAD6, which required the activity of nuclear factor-kB (NF-kB), but not TGFB. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. Conclusions In studies of hepatoma cell lines and human primary hepatocytes, we found that infection with HCV leads to activation of NF-κB, leading to increased expression of SMAD6 and SMAD7. Upregulation of SMAD6 and SMAD7 lead to increased expression of HSPGs, such as SDC1, as well as LDLR, VLDLR, and SR-BI, which promote HCV entry and propagation, as well as cell uptake of cholesterol and lipoprotein.
Cellular microRNAs (miRNAs) have been shown to modulate HCV infection via directly acting on the viral genome or indirectly through targeting the virus-associated host factors. Recently we generated a comprehensive map of HCV–miRNA interactions through genome-wide miRNA functional screens and transcriptomics analyses. Many previously unappreciated cellular miRNAs were identified to be involved in HCV infection, including miR-135a, a human cancer-related miRNA. In the present study, we investigated the role of miR-135a in regulating HCV life cycle and showed that it preferentially enhances viral genome replication. Bioinformatics-based integrative analyses and subsequent functional assays revealed three antiviral host factors, including receptor interacting serine/threonine kinase 2 (RIPK2), myeloid differentiation primary response 88 (MYD88), and C-X-C motif chemokine ligand 12 (CXCL12), as bona fide targets of miR-135a. These genes have been shown to inhibit HCV infection at the RNA replication stage. Our data demonstrated that repression of key host restriction factors mediated the proviral effect of miR-135a on HCV propagation. In addition, miR-135a hepatic abundance is upregulated by HCV infection in both cultured hepatocytes and human liver, likely mediating a more favorable environment for viral replication and possibly contributing to HCV-induced liver malignancy. These results provide novel insights into HCV–host interactions and unveil molecular pathways linking miRNA biology to HCV pathogenesis. Electronic supplementary material The online version of this article (10.1007/s12250-018-0055-9) contains supplementary material, which is available to authorized users.
Tissue-resident-memory T cells (TRM) populate the body’s barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation via T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN-γ signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN-γ pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases.
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