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C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle

  • Volker Kinast
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    Faculty of Medicine, Department for Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
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  • Agnieszka Plociennikowska
    Affiliations
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany

    Division Virus-Associated Carcinogenesis, German Cancer Research Center, Heidelberg, Germany
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  • Anggakusuma
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    Vector Development department, research at uniQure, Paasheuvelweg 25A, 1105 BP, Amsterdam, The Netherlands
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  • Thilo Bracht
    Affiliations
    Medizinisches Proteom-Center, Ruhr University Bochum, Bochum, Germany
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  • Daniel Todt
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    Faculty of Medicine, Department for Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
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  • Richard J.P. Brown
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany
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  • Tujana Boldanova
    Affiliations
    Department of Biomedicine, University of Basel and Division of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland
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  • Yudi Zhang
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
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  • Yannick Brüggemann
    Affiliations
    Faculty of Medicine, Department for Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
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  • Martina Friesland
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
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  • Michael Engelmann
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    Faculty of Medicine, Department for Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
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  • Gabrielle Vieyres
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
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  • Ruth Broering
    Affiliations
    Department of Gastroenterology and Hepatology, University Hospital Essen, University Duisburg-Essen, Essen, Germany
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  • Florian W.R. Vondran
    Affiliations
    ReMediES, Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany

    German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany
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  • Markus H. Heim
    Affiliations
    Department of Biomedicine, University of Basel and Division of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland
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  • Barbara Sitek
    Affiliations
    Medizinisches Proteom-Center, Ruhr University Bochum, Bochum, Germany
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  • Ralf Bartenschlager
    Affiliations
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany

    Division Virus-Associated Carcinogenesis, German Cancer Research Center, Heidelberg, Germany

    German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany
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  • Thomas Pietschmann
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany
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  • Eike Steinmann
    Correspondence
    Corresponding author. Address: Department of Molecular and Medical Virology, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany. Tel.: +49 234 32 23189, fax: +49 234 32 14352
    Affiliations
    Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany

    Faculty of Medicine, Department for Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
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Open AccessPublished:April 12, 2020DOI:https://doi.org/10.1016/j.jhep.2020.03.047

      Highlights

      • C19orf66 is upregulated in vivo upon HCV infection and IFN therapy.
      • Expression of C19orf66 restricts HCV infection.
      • C19orf66 alters the morphology of the viral replication organelle.
      • The zinc finger motif of C19orf66 is crucial for its restriction capacity.

      Background & Aims

      HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive.

      Methods

      Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis.

      Results

      Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication.

      Conclusion

      C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication.

      Lay summary

      Interferon-stimulated genes are thought to be important to for antiviral immune responses to HCV. Herein, we analysed C19orf66, an interferon-stimulated gene, which appears to inhibit HCV replication. It prevents the HCV-induced elevation of phosphatidylinositol-4-phosphate and alters the morphology of HCV's replication organelle.

      Graphical abstract

      Keywords

      See Editorial, pages 496–498

      Introduction

      The World Health Organization estimates 71 million people are chronically infected with HCV, emphasizing that HCV still represents a global health problem with significant socioeconomic burden.
      World Health Organization
      Global Hepatitis Report 2017.
      Despite the success of direct-acting antivirals (DAAs), a combination of poor access to these treatments in developing countries, emerging DAA resistance and the high number of new infections (~1.7 million in 2015) continue to impede global eradication efforts.
      HCV is an enveloped, positive-strand RNA virus, classified as a member of the family Flaviviridae. This pathogen is transmitted via blood-blood contact and, in up to 85% of cases, the infection cannot be cleared by the host immune system and becomes chronic, frequently leading to liver cirrhosis and hepatocellular carcinoma.
      • Hoofnagle J.H.
      Hepatitis C: the clinical spectrum of disease.
      ,
      • Thrift A.P.
      • El-Serag H.B.
      • Kanwal F.
      Global epidemiology and burden of HCV infection and HCV-related disease.
      Innate immunity is key during the entire natural course of the disease, highlighted by the presence of viral immune evasion strategies that attenuate the innate immune response.
      • Heim M.H.
      Innate immunity and HCV.
      In addition, transcriptomic analysis of liver biopsies from HCV-infected patients revealed the induction of hundreds of interferon (IFN)-stimulated genes (ISGs),
      • Boldanova T.
      • Suslov A.
      • Heim M.H.
      • Necsulea A.
      Transcriptional response to hepatitis C virus infection and interferon-alpha treatment in the human liver.
      suggesting a pivotal role of ISGs in the intrinsic antiviral immune response against HCV. However, only a small number of HCV-triggered ISGs (e.g. CH25H
      • Anggakusuma
      • Romero-Brey I.
      • Berger C.
      • Colpitts C.C.
      • Boldanova T.
      • Engelmann M.
      • et al.
      Interferon-inducible cholesterol-25-hydroxylase restricts hepatitis C virus replication through blockage of membranous web formation.
      ) have been characterized with respect to their antiviral activity and their mode of action.
      In contrast, C19orf66 (also called SHFL) represents one of many under-investigated, potentially antiviral, genes which might play a role in the context of HCV infection. In 2011, the so far uncharacterized C19orf66 was detected in a screening approach, where Schoggins and colleagues aimed to identify novel ISGs.
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      The C19orf66 gene encodes a protein of 291 amino acids, which has no homology with any known protein family and no predicted enzymatic activity. Initial studies with immortalized cell lines suggested that C19orf66 expression is induced in an IFN-dependent manner and that the protein exhibits an antiviral activity against dengue virus.
      • Balinsky C.A.
      • Schmeisser H.
      • Wells A.I.
      • Ganesan S.
      • Jin T.
      • Singh K.
      • et al.
      IRAV (FLJ11286), an interferon-stimulated gene with antiviral activity against dengue virus, interacts with MOV10.
      ,
      • Suzuki Y.
      • Chin W.-X.
      • Han Q.E.
      • Ichiyama K.
      • Lee C.H.
      • Eyo Z.W.
      • et al.
      Characterization of RyDEN (C19orf66) as an interferon-stimulated cellular inhibitor against dengue virus replication.
      Recently, C19orf66 was reported to be a HIV-1 restriction factor by inhibiting programmed-1 ribosomal frameshifting (-1PRF).
      • Wang X.
      • Xuan Y.
      • Han Y.
      • Ding X.
      • Ye K.
      • Yang F.
      • et al.
      Regulation of HIV-1 Gag-Pol expression by shiftless, an inhibitor of programmed -1 ribosomal frameshifting.
      In addition, C19orf66 restricts Kaposi's sarcoma-associated herpesvirus (KSHV) while the C19orf66 RNA is able to escape the cleavage of KSHV.
      • Rodriguez W.
      • Srivastav K.
      • Muller M.
      • Jung J.U.
      C19ORF66 broadly escapes virus-induced endonuclease cleavage and restricts Kaposi's sarcoma-associated herpesvirus.
      These findings imply that C19orf66 is a multifunctional ISG with potential antiviral activities against several viruses. Despite C19orf66 garnering increasing attention, there remain several unknowns: i) the level of C19orf66 expression in primary cells; ii) how C19orf66 is regulated in vivo in response to IFN and HCV infection; iii) its potential antiviral activity and mode of action against HCV.
      In this study, we evaluated the expression of C19orf66 in vivo in response to HCV infection and IFN therapy. We validated these findings via ex vivo experiments with primary human hepatocytes (PHHs). Further, we generated C19orf66 knockout and C19orf66-expressing cells and used them to determine the antiviral activity of C19orf66 against different HCV strains representing multiple viral genotypes. In addition, we used a variety of molecular tools, determined the C19orf66-specific proteome and dissected the mode of action of this particular restriction factor. Collectively, our data suggest that C19orf66 contributes to the innate immune response against HCV in the liver.

      Materials and methods

       Liver biopsies and informed consent

      Liver biopsies from patients with CHC (n = 25) and HCV negative liver biopsies (n = 26) were obtained in the outpatient clinic of the Division of Gastroenterology and Hepatology, University Hospital Basel (Table S1). METAVIR classification was used to assess grade and stage of CHC. All patients gave written informed consent in accord with local ethical committees.

       HCV infection assays

      Huh-7.5 cells were seeded at a density of 1 × 104/well in a 96-well plate or 3 × 104/well in a 24-well plate 24 h before inoculation. Cells were inoculated for 4 h at 37°C with the respective virus particles and viral inoculum was replaced by fresh culture medium afterwards. PHHs were infected with HCV Jc1 at 37°C for 6 h. Afterwards, the inoculum was removed, cells were washed 3 times with PBS, and 1 ml of fresh Hepatocyte Basal Medium from (HBM) (Lonza) was added.
      For further details regarding the materials and methods used, please refer to the CTAT table and supplementary information.

      Results

       C19orf66 is upregulated in vivo in the liver of patients with CHC and upon pegIFNα/ribavirin therapy

      To determine the in vivo regulation of C19orf66 in the context of HCV infection, we compared mRNA levels in liver biopsies from patients with CHC (n = 25) with those from patients with non-HCV-related chronic liver disease (n = 26). We detected significantly higher C19orf66 mRNA levels in liver biopsies from patients with CHC than in biopsies from the control group (Fig. 1A). Similar results were observed for Mx1 (Fig. 1B, Fig. S1A). Elevated C19orf66 expression levels did not correlate with viral loads, METAVIR scores or HCV genotype (Fig. S1B–E).
      Figure thumbnail gr1
      Fig. 1Induction of C19orf66 expression in CHC liver biopsies and upon pegIFNα/ribavirin treatment.
      Comparison of C19orf66 (A) and Mx1 (B) relative mRNA levels in liver biopsies from patients with non-HCV chronic liver diseases (n = 26) or CHC (n = 25). (unpaired 2-tailed Student's t test). Comparison of C19orf66 (C) and Mx1 (D) expression of pre-treatment and post-treatment biopsies at indicated time points after pegIFNα/ribavirin treatment. Raw data were obtained from Boldanova et al. (2017). (E) Comparison of virus production in mock-, HCV-infected (MOI 10) and 2′CMA [10 μM]-treated PHHs 48 hpi. Comparison of C19orf66 (F) and Mx1 (G) relative mRNA levels in mock-, HCV-infected (MOI 10) and 2′CMA [10 μM] -treated PHHs, 48 hpi. Comparison of C19orf66 (H) and Mx1 (I) relative mRNA levels in PHHs upon treatment with IFNα (1,000 IU/ml), IFNγ (1,000 IU/ml), and IFNλ1 (1,000 ng/ml). (E–I) Shown are means ± SD from 4 independent donors. (one-way ANOVA adjusted with Dunnett multiple comparison test). (A, B, F–I) mRNA levels were normalized to GAPDH as determined by RT-qPCR.
      2′CMA, 2′C-methyl-adenosine; CHC, chronic hepatitis C; MOI, multiplicity of infection; pegIFNα, pegylated interferon alfa-2; PHHs, primary human hepatocytes; RT-qPCR, quantitative reverse transcription PCR.
      For more than 25 years, pegylated interferon alfa-2 (pegIFNα) was an integral part of anti-HCV therapy, activating endogenous innate immunity to help combat this viral infection. Consequently, we analyzed samples from HCV-infected patients treated with pegIFNα/ribavirin by transcriptomics as reported previously.
      • Boldanova T.
      • Suslov A.
      • Heim M.H.
      • Necsulea A.
      Transcriptional response to hepatitis C virus infection and interferon-alpha treatment in the human liver.
      C19orf66 was significantly upregulated in pegIFNα/ribavirin treated patients at 4 h and 16 h post treatment (Fig. 1C), whereas Mx1 was significantly upregulated at all tested time points (Fig. 1D).

       C19orf66 is upregulated ex vivo in PHHs upon HCV challenge and IFN treatment

      To further validate our in vivo findings, we evaluated C19orf66 expression in PHHs upon HCV infection. PHHs were infected with cell culture-derived Jc1 virus and mock-treated or treated with the HCV replication inhibitor 2´C-methyl-adenosine (2´CMA) as a control. C19orf66 mRNA expression levels were significantly increased in HCV-infected PHHs, but not in 2´CMA-treated cells (Fig. 1E, F). The control ISG Mx1 was expressed at a higher level (Fig. 1G), consistent with the in vivo data (Fig. 1B). Additional infection assays with both replication-competent HCV and replication-deficient (UV-inactivated) HCV and PHHs, as well as publicly available transcriptomic gene expression data of HCV-infected PHHs (GSE132548) and hepatocyte-like cells (GSE132606), confirmed our observations that expression of C19orf66 is induced by HCV replication (Fig. S2A–C).
      To confirm C19orf66 is upregulated upon pegIFNα/ribavirin treatment, we next treated PHHs with IFNα, IFNγ and IFNλ1 for 4, 8 and 24 h. C19orf66 mRNA expression was strongly induced and significantly higher during the first 8 h of IFNα treatment. The response to IFNγ and IFNλ treatment was modest in comparison to IFNα (Fig. 1H). Like C19orf66 mRNA levels, Mx1 mRNA levels were more strongly induced by IFNα treatment (Fig. 1I). However, direct comparison may be affected by the different doses of interferon used in the case of type III IFN (IFNλ). Additionally, gene expression analysis of IFN-treated peripheral blood mononuclear cell-derived macrophages, as well as publicly available transcriptomic gene expression data of IFN-treated PHHs and IFN regulatory factor (IRF)1-expressing Huh-7.5 cells, supported that C19orf66 is induced in an IFN- and IRF1-dependent manner (Fig. S2D–E).
      Taken together, these data indicate that C19orf66 expression is induced in vivo in the human liver and ex vivo in PHHs in the context of HCV infection and IFN treatment.

       Knockout of endogenous C19orf66 attenuates IFN-induced suppression of HCV replication

      To dissect a potential antiviral effect of endogenous C19orf66 against HCV, we generated 3 independent CRISPR/Cas9-mediated knockout (KO) cell lines of C19orf66. To validate the loss of C19orf66, we treated the cell pools with IFNα and monitored the levels of C19orf66 protein by immunoblotting, demonstrating the lack of a C19orf66-specific band in the designated C19orf66 KO Huh-7.5 cells (Fig. 2A, Fig. S3A). Next, we evaluated if the missing C19orf66 expression could restore HCV replication in IFN-treated cells by pulse-treating the cells with IFNα 1 day prior to infection with the Jc1 Renilla-reporter virus or transfection with the HCV subgenomic replicon. Increased luciferase counts could be observed in the C19orf66 KO Huh-7.5 cell lines, indicating that endogenous IFN-induced C19orf66 in Huh-7.5 cells restricts HCV infection and replication (Fig. 2B, Fig. S3B, Fig. S3C).
      Figure thumbnail gr2
      Fig. 2Knockout of endogenous C19orf66 facilitates HCV replication and C19orf66 relocalizes upon HCV infection.
      (A) Western blot of non-targeted (nt) and C19orf66 KO Huh-7.5 after IFNα (10 IU/ml) treatment. (B) HCV JcR2a replication levels in IFNα treated nt- and C19orf66 KO Huh-7.5 cells 48 hpi. (mean ± SD.; n = 3; unpaired 2-tailed Student's t test; ∗p <0.05) (C) Western blot of Huh-7.5 cells stably expressing an empty vector (empty vector Huh-7.5) or 3xFLAG-C19orf66 (C19orf66 Huh-7.5). (D) Comparison of C19orf66 RPKM values of empty vector- and C19orf66 Huh-7.5 cells and publicly available data of PHHs, which were mock-treated, treated with IFNα (6 h post treatment) or challenged with HCV (72 hpi) (GSE132548). For PHHs, shown are means from 3 independent donors. RPKM values were determined via RNA-seq. (E) Scatter dot blot of RNA-seq expression analysis of empty vector- and C19orf66 Huh-7.5 cells. The dotted lines indicate a 2-fold difference of the RPKM values between empty vector- and C19orf66 Huh-7.5 cells. (F) Volcano blot representing whole cell proteome analysis of C19orf66 Huh-7.5 cells normalized to empty vector Huh-7.5 cells. White areas show the significant altered proteins (q-value <0.05, log2 fold change <−2 or q-value <0.05, log2 fold change >2) Shown are means of 6 experimental replicates. (G) Subcellular localization of C19orf66 in C19orf66 expressing Huh-7.5 cells upon mock or HCV infection. Staining: FLAG-tagged C19orf66 (green), HCV-NS5A (red), nuclear DNA (blue). Scale bar, 10 μm. (H) Colocalization coefficient of C19orf66 and lipid droplets in C19orf66 expressing Huh-7.5 cells after mock or HCV infection. Colocalization was determined by Pearson's R value with Costes threshold regression. 30 ROI were analyzed (n = 3). (I) Subcellular localization of C19orf66 and HCV proteins in C19orf66 expressing Huh-7.5 cells upon mock or HCV infection, 48 hpi. Staining: FLAG-tagged C19orf66 (green), HCV-Core, NS3 or NS5A (all red), nuclear DNA (blue). Scale bar, 10 μm. (J) Colocalization coefficients of C19orf66 and HCV proteins in C19orf66 expressing Huh-7.5 cells after mock or HCV infection. Colocalization was determined by Pearson's R value with Costes threshold regression. 30 ROI were analyzed (n = 3).
      FC, fold change; FDR, false discovery rate; IFN, interferon; KO, knockout; MOI, multiplicity of infection; pegIFNα, pegylated interferon alfa-2; PHH, primary human hepatocyte; RLU, relative light units; RNA-Seq, RNA sequencing; ROI, regions of interest. (This figure appears in color on the web.)

       Ectopically expressed C19orf66 restricts HCV irrespective of genotype

      To further evaluate the antiviral effect of C19orf66, we generated Huh-7.5 cells expressing empty vector or 3xFLAG-C19orf66 (C19orf66 Huh-7.5). After validating the expression of C19orf66 protein by immunoblotting (Fig. 2C), the transcriptomes of the empty vector and C19orf66 Huh-7.5 cells revealed comparable levels of C19orf66 mRNA in the C19orf66 Huh-7.5 cells relative to IFNα-treated or HCV-challenged PHHs (Fig. 2D). In addition, we observed, that ectopic expression of C19orf66 did not affect the expression of other gene subsets (Fig. 2E). By utilizing whole cell proteomic analysis of empty vector and C19orf66 Huh-7.5 cells, we detected similar expression levels of all identified (3,243 protein groups) proteins, except a significant higher abundance of C19orf66 in the C19orf66 Huh-7.5 cells (Fig. 2F). In summary, the ectopic expression of C19orf66 did not affect the expression levels of other genes and their related proteins.
      Next, we explored the subcellular localization of C19orf66 upon HCV infection of human hepatoma cells. In uninfected cells, the C19orf66 signal was homogenously distributed in the cytosol. Importantly, in HCV-infected cells, we observed clustering of the C19orf66 signal in punctae (Fig. 2G) and increased colocalization with lipid droplets, which are host organelles integral for the HCV life cycle (Fig. 2H, Fig. S3D). Furthermore, we detected partial colocalization of C19orf66 with HCV-Core, NS3 and NS5A (Fig. 2I, J). Collectively, these results demonstrate the re-localization of C19orf66 in HCV-infected cells to the cellular compartments where the HCV replicase complex is located.
      To further investigate the antiviral capacity of C19orf66 against HCV, we performed infection assays with the HCV genotype 2a/2a chimera Jc1 wild-type (WT) and the genotype 1a TNcc strain. We detected a reduced HCV antigen signal (Fig. 3A) as well as significantly decreased de novo progeny virus for both genotypes in the HCV-infected C19orf66 Huh-7.5 cells (Fig. 3B).
      Figure thumbnail gr3
      Fig. 3Expression of C19orf66 restricts HCV infection.
      (A) Detection of HCV proteins in empty vector and C19orf66 Huh-7.5 cells after mock, Jc1 WT (MOI 1) or HCV TNcc (MOI 0.2) infection, 48 hpi. Staining: HCV (red), nuclear DNA (blue). Scale bar, 10 μm. (B) HCV production in empty vector and C19orf66 expressing Huh-7.5 cells after mock, Jc1 WT or HCV TNcc infection. Newly released infectious virus particles were quantified by TCID50 assay. (C) Schematic of the JFH1 based Renilla-reporter chimeras. (D) HCV infection levels in empty vector- and C19orf66 expressing Huh-7.5 cells upon infection with HCV chimeras. Infection levels were determined by Renilla luciferase activity assay 24 hpi. (B,D) Means ± SD, n = 3. (One-way ANOVA adjusted with Dunnett multiple comparison test [for HCV Jc1 WT] and unpaired 2-tailed Student's t test [TNcc and JcR2a]).
      MOI, multiplicity of infection; RLU, relative light units; WT, wild-type. (This figure appears in color on the web.)
      To test the effect of C19orf66 on different HCV genotypes, we performed infection assays using JFH1-based Renilla-reporter chimeras, in which the structural proteins, Core and NS2 correspond to genotypes 1 to 7 (Fig. 3C). In agreement with infection data from non-reporter strains, a 10-fold decrease in luciferase activity for all 7 tested chimeras in the presence of stably expressed C19orf66 was observed (Fig. 3D). Thus, C19orf66 demonstrated potent anti-HCV activity against infection with all tested HCV intergenotypic chimeras.

       C19orf66 targets HCV RNA replication

      Since the entry process was not significantly affected by C19orf66 expression (Fig. 4A), we next made use of subgenomic replicons, bypassing virus entry and focusing on viral RNA translation and replication. To this end, we transfected subgenomic reporter replicons of Con1 (genotype 1b) or JFH1 (genotype 2a) (Fig. S4A) and detected significantly decreased luciferase activity for both genotypes with almost completely abolished reporter activity for Con1 (Fig. 4B, Fig. S4B).
      Figure thumbnail gr4
      Fig. 4C19orf66 targets an intracellular step of the HCV life cycle.
      (A) Effect of ectopically expressed C19orf66 on the entry of HCV pseudotyped particles. Shown are means ± SD, n = 2. (One-way ANOVA adjusted with Dunnett multiple comparison test) (B–C) Effect of ectopically expressed C19orf66 against (B) the subgenomic replicons of JFH1 NS3-3′, Con1/ET, JFH1/Con1 intergenotypic chimeras and (C) replication deficient GND mutants of JFH1 NS3-3′and Con1/ET (Schematics are depicted in ). HCV RNA replication was determined by Firefly luciferase activity assays at the indicated time points. Shown are means ± SD, n = 3. (One-way ANOVA adjusted with Dunnett multiple comparison test) (D) Schematic of the bicistronic RNA translation reporter construct. Firefly luciferase is translated in a cap-dependent manner and used for normalization, whereas the Renilla luciferase is translated in an HCV IRES-dependent manner. (E) Effect of C19orf66 against the cap-dependent and HCV IRES-dependent translation. 8 h post transfection, cap-dependent translation was determined by Firefly luciferase activity assay and HCV IRES-dependent translation was determined by Renilla luciferase activity assay. Shown are means ± SD, n = 3. (unpaired 2-tailed Student's t test).
      IRES, internal ribosome entry site; RLU, relative light units.
      To explore whether the magnitude of restriction by C19orf66 is dependent on the replication fitness, we transfected intergenotypic JFH1-based replicon chimeras containing replication-suppressing mutations in the 5´NTR (5′Con), the 3′ X tail (xCon) or both of them of Con1 (5′xCon) (Fig. S4A). Here, we detected reduced luciferase activity of all 3 tested replicon chimeras compared with the JFH1 replicon in the empty vector cells (Fig. 4B). Furthermore, we observed decreased luciferase activity in the presence of C19orf66 for all 3 tested replicon chimeras, but especially for the 5´xCon chimera, indicating that the magnitude of replication inhibition by C19orf66 correlates with the replication fitness.
      To further dissect the antiviral mechanism of C19orf66, we evaluated whether C19orf66 affects HCV internal ribosome entry site (IRES)-dependent translation. To this end, we used replication-deficient GND mutants of JFH1 and Con1 replicons to exclusively study the impact of C19orf66 on HCV RNA translation (Fig. S4A). These reporters exhibited no altered luciferase activity in the C19orf66 Huh-7.5 cells, compared with empty vector cells (Fig. 4C). We devised an additional approach with a bi-cistronic reporter construct, which enabled the simultaneous analysis of both, cap- and HCV IRES-dependent translation (Fig. 4D). In line with previously obtained results, the presence of C19orf66 did not suppress HCV IRES-dependent RNA translation, nor cap-dependent translation (Fig. 4E). Taken together, these data provide evidence that C19orf66 targets an HCV life cycle step, which is independent of translation, but reduces HCV RNA replication of different genotypes.

       The Zinc-finger motif of C19orf66 is crucial for its restriction capacity and ability to accumulate during viral infection and stress

      To identify the interactome of C19orf66, we performed co-immunoprecipitation of the 3xFLAG peptide in empty vector and C19orf66-expressing Huh-7.5 cells in the presence or absence of HCV followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. In addition to C19orf66, the stress granule (SG)-associated ribonucleoproteins RO60,
      • Gallois-Montbrun S.
      • Kramer B.
      • Swanson C.M.
      • Byers H.
      • Lynham S.
      • Ward M.
      • et al.
      Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules.
      RBPMS
      • Farazi T.A.
      • Leonhardt C.S.
      • Mukherjee N.
      • Mihailovic A.
      • Li S.
      • Max K.E.A.
      • et al.
      Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.
      and CELF1
      • Fujimura K.
      • Kano F.
      • Murata M.
      Dual localization of the RNA binding protein CUGBP-1 to stress granule and perinucleolar compartment.
      were exclusively identified in C19orf66 samples, but not in empty vector samples (Fig. 5A). Furthermore, SG- and P-body-associated components also dominated the proteins, which were significantly enriched in C19orf66 samples (Fig. S5).
      Figure thumbnail gr5
      Fig. 5The Zinc-finger motif of C19orf66 is important for the restriction capacity against HCV and its re-localization upon stress.
      (A) LFQ intensities of proteins exclusively identified in FLAG-immunoprecipitations of C19orf66 Huh-7.5 cells, but not in immunoprecipitations of empty vector Huh-7.5 cells. Experiments were performed in the presence or absence of HCV. Shown are values of 5 independent specimens. (B) Western blot of Huh-7.5 cells stably expressing an empty vector, C19orf66-WT or a mutated variant of C19orf66 (C) Effect of different C19orf66 variants against subgenomic replicons of JFH1 NS3-3′ and Con1/ET. HCV RNA replication was determined by Firefly luciferase activity assays. Shown are means ± SD, n = 3. (One-way ANOVA adjusted with Dunnett multiple comparison test). (D) Cellular localization of C19orf66-WT or C19orf66-Zincmut and HCV proteins, 48 hpi. Staining: FLAG-tag (green), HCV-Core, HCV-NS3 or NS5A (all red), nuclear DNA (blue). White lines in the merged images were selected for the intensity line profiles shown in the lower panels. Scale bar, 10 μm. (E) Localization of C19orf66-WT and C19orf66-Zincmut upon mock or Arsenite treatment (5 μM, 30 min) or pI:C transfection (2 μg/ml, 16 h). Staining: FLAG-tag (green), nuclear DNA (blue). White arrows point to C19orf66 punctae. Scale bar, 50 μm. (F) Quantification of C19orf66 punctae positive cells upon mock or Arsenite treatment (5 μM, 30 min) or pI:C transfection (2 μg/ml, 16 h). Shown are means ± SD, n = 3. (One-way ANOVA adjusted with Sidak’s multiple comparison test). (G) Colocalization of C19orf66-WT and C19orf66-Zincmut with RBPMS upon mock or Arsenite treatment (5 μM, 30 min). Staining: FLAG-tag (green), RBPMS (red) nuclear DNA (blue). White lines in the merged images were selected for the intensity line profiles shown in the right panels. Scale bar, 10 μm.
      LFQ, label-free quantification; RLU, relative light units; WT, wild-type. (This figure appears in color on the web.)
      To determine the C19orf66 protein domains that are crucial for its antiviral activity, we generated Huh-7.5 cells, which ectopically express mutated versions of C19orf66 (Fig. 5B, Fig. S6A). Intriguingly, we observed no inhibition of replication for C19orf66-Zincmut (Fig. 5C). In addition, we detected no prominent re-localization of C19orf66-Zincmut upon HCV infection and hence less colocalization with viral proteins, compared with C19orf66 WT (Fig. 5D, Fig. S6B–E). Considering, that especially SG-associated proteins co-immunoprecipitated with C19orf66 (Fig. 5A, Fig. S5) the questions arose whether other forms of stress elicit the formation of C19orf66-WT but not C19orf66-Zincmut accumulations. Both, arsenite, which induces oxidative stress, and pI:C, which mimics double-stranded RNA, induced the re-localization of C19orf66-WT, but not of C19orf66-Zincmut, into punctae (Fig. 5E–G). Importantly, the SG-associated protein RBPMS not only co-immunoprecipitated with C19orf66 (Fig. 5A), but also colocalized with it during arsenite treatment (Fig. 5G).
      Taken together, the ability of C19orf66 to form punctae in response to stress may be crucial for its capacity to restrict HCV, as suggested by the C19orf66-Zincmut, which has an impaired restriction ability and fails to form punctae during stress. C19orf66 was reported to restrict HIV-1 via inhibition of -1PRF, but ribosomal frameshifting is not reported to be crucial for HCV replication. Therefore, we analyzed the restriction capacity of the splice variants including C19orf66-209, which failed to restrict HIV-1 via inhibition of -1PRF
      • Wang X.
      • Xuan Y.
      • Han Y.
      • Ding X.
      • Ye K.
      • Yang F.
      • et al.
      Regulation of HIV-1 Gag-Pol expression by shiftless, an inhibitor of programmed -1 ribosomal frameshifting.
      (Fig. S7A, B). Importantly, ectopically expressed C19orf66-209 significantly restricted HCV replication in Huh-7.5 cells (Fig. S7C, D), suggesting that HCV inhibition by C19orf66 was independent of -1PRF.

       C19orf66 alters formation of the HCV-induced membranous web

      HCV is known to remodel endoplasmic reticulum-derived membranes by the induction of exvaginations that appear as single-, double-, and multi-membrane vesicles (SMVs, DMVs and MMVs, respectively). These accumulate within the cytosol, forming a structure designated the membranous web (MW), which is the site of HCV replication.
      • Romero-Brey I.
      • Merz A.
      • Chiramel A.
      • Lee J.-Y.
      • Chlanda P.
      • Haselman U.
      • et al.
      Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
      It is thought that remodeled membranes serve as scaffold for the assembly of the viral replicase machinery. Therefore, we explored whether C19orf66 interferes with the formation of a proper HCV-induced MW. Huh7-Lunet cells stably expressing the T7 RNA polymerase and C19orf66-WT, C19orf66-Zincmut or the empty vector were transfected with constructs encoding the HCV replicase of the JFH1 or the Con1 strain (Fig. 6A). This expression-based system does not rely on HCV replication, thus enabling the analysis of a potential influence of C19orf66 on MW formation. Similar levels of NS5A protein were detected in cells expressing or not expressing C19orf66-WT and C19orf66-Zincmut (Fig. 6B). HCV-induced MWs were quantified using correlative light and electron microscopy, where we observed an altered morphology with apparently smaller, more homogenous and clustered vesicles as well as an altered composition of DMVs and MMVs in the presence of stably expressed C19orf66-WT, but not C19orf66-Zincmut (Fig. 6C–F, Fig. S8A,B). In addition, the diameter of DMVs was reduced in cells expressing C19orf66-WT (Fig. 6G) and we observed a slight increase in the numbers of DMVs per μm2 in these cells relative to the empty vector control cells (Fig. 6H). Collectively, these data suggest that C19orf66 affects the formation and/or integrity of the HCV-induced MW.
      Figure thumbnail gr6
      Fig. 6Aberrant formation of the HCV-induced membranous web by C19orf66.
      (A) Schematic illustration of the experimental setup. (B) Abundance of NS5A was determined by western blotting in Huh7-Lunet/T7 cells transfected with non-tagged constructs encoding HCV NS3–NS5B of JFH1 or Con1 WT. (C and D) Fluorescence images of C19orf66 expressing Huh7-Lunet/T7 cells grown on gridded dishes overlapped with the matching bright field image (left). Higher magnification fluorescence image of the boxed cell (middle). Merged electron microscopy and fluorescence image (right). (E and F) Representative electron micrographs of empty vector Huh7-Lunet/T7 cells expressing NS3-5B proteins of HCV JFH1 (E) or HCV Con1 WT (F). Lower panels show enlarged image of the membranous web enriched area highlighted with a white square in the upper panels. (G) Quantification of DMV diameters in empty vector, C19orf66-WT and C19orf66-Zincmut expressing Huh7-Lunet/T7 cells. (H) Quantification of DMVs per square micrometer in empty vector, C19orf66-WT and C19orf66-Zincmut expressing Huh7-Lunet/T7 cells. (G and H) The surface of 7 cell profiles was measured, and the size and number of DMVs within every cell profile was determined. Shown are the results from 2 independent experiments. (One-way ANOVA adjusted with Dunnett multiple comparison test).
      DMV, double-membrane vesicle; WT, wild-type. (This figure appears in color on the web.)

       Expression of C19orf66 diminishes the HCV-induced elevation of PI(4)P

      The formation of the MW is driven by HCV non-structural proteins, most notably NS5A.
      • Romero-Brey I.
      • Berger C.
      • Kallis S.
      • Kolovou A.
      • Paul D.
      • Lohmann V.
      • et al.
      NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication.
      In addition, multiple host cell factors are involved in changing the lipid composition of DMVs, one of them being phosphatidylinositol 4-kinase IIIα (PI4KA), which converts phosphatidylinositol to phosphatidylinositol 4-phosphate [PI(4)P]. The interaction of NS5A with PI4KA is assumed to activate the kinase, leading to elevated levels of PI(4)P in HCV-infected cells.
      • Berger K.L.
      • Kelly S.M.
      • Jordan T.X.
      • Tartell M.A.
      • Randall G.
      Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.
      Importantly, knockdown of PI4KA dampens HCV-induced elevation of PI(4)P,
      • Wang H.
      • Perry J.W.
      • Lauring A.S.
      • Neddermann P.
      • de Francesco R.
      • Tai A.W.
      Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking.
      correlating with an aberrant morphology of MWs with decreased DMV size, DMV clustering and a rather homogenous vesicle appearance.
      • Reiss S.
      • Rebhan I.
      • Backes P.
      • Romero-Brey I.
      • Erfle H.
      • Matula P.
      • et al.
      Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.
      Since ectopic expression of C19orf66 caused a phenotype similar to PI4KA knockdown, we assumed that C19orf66 expression might reduce HCV-induced elevation of PI(4)P. To put this hypothesis to the test, we performed immunofluorescence studies with empty vector, C19orf66-WT and C19orf66-Zincmut Huh7-Lunet/T7 cells expressing the HCV NS3-5B viral replicase proteins of the JFH1 and Con1 strains. As reported earlier, an ~4-fold elevation of PI(4)P was found in control cells expressing HCV NS3-5B, whereas PI(4)P levels in HCV-negative cells were unaltered. In contrast, accumulation of PI(4)P was markedly reduced in C19orf66-WT-expressing cells (Fig. 7A–C; Fig. S9). Taken together, these data indicate that C19orf66 expression impairs HCV-induced elevation of PI(4)P, which interferes with the formation of a fully functional HCV-induced MW.
      Figure thumbnail gr7
      Fig. 7Expression of C19orf66 reduces HCV-induced elevation of PI(4)P.
      (A) HCV-induced accumulation of PI(4)P in empty vector, C19orf66-WT and C19orf66-Zincmut expressing Huh7-Lunet/T7 cells. Empty vector, C19orf66-WT and C19orf66-Zincmut expressing Huh7-Lunet/T7 cells were transfected with HCV NS3-3'constructs. Staining: PI(4)P (green), NS5A (red) nuclear DNA (blue). Representative images of mock-transfected cells and cells transfected with HCV variants of JFH1 or Con1 are shown. White lines in the merged images were selected for the intensity line profiles shown in . Scale bars, 10 μm. (B) Quantification of immunofluorescence signals of NS5A in HCV-positive cells, n = 3 (One-way ANOVA adjusted with Dunnett multiple comparison test). For each condition, z-stacks from 30 cells were analyzed and the results are displayed as integrated density in arbitrary units. (C) Quantification of immunofluorescence signals of PI(4)P in HCV-positive cells, n = 3 (One-way ANOVA adjusted with Dunnett multiple comparison test). For each condition, z-stacks from 30 cells were analyzed and the results are displayed as integrated density in arbitrary units.
      PI4P, phosphatidylinositol 4-phosphate; WT, wild-type. (This figure appears in color on the web.)

      Discussion

      IFN was an integral part of anti-HCV therapy for more than 25 years, highlighting the sensitivity of HCV to the IFN-mediated immune response.
      • Heim M.H.
      Innate immunity and HCV.
      However, the mechanism(s) by which IFNs achieve an antiviral state within the cell are incompletely defined.
      • Schneider W.M.
      • Chevillotte M.D.
      • Rice C.M.
      Interferon-stimulated genes: a complex web of host defenses.
      In this study, we identified C19orf66 as an IFN-induced restriction factor in the human liver, which reduced HCV infection by interfering with the formation of the HCV-induced MW. Gene expression analysis revealed that C19orf66 was upregulated in the livers of patients with CHC compared to control liver biopsies (Fig. 1A) and in PHHs upon HCV infection and upon IFN therapy (Fig. 1F, H), thus classifying C19orf66 as an early induced ISG. In addition, multiple published studies, using microarrays to quantify the host transcriptomic response to RNA and DNA virus infections, identified C19orf66 dysregulation,
      • Wang J.
      • Nikrad M.P.
      • Phang T.
      • Gao B.
      • Alford T.
      • Ito Y.
      • et al.
      Innate immune response to influenza A virus in differentiated human alveolar type II cells.
      • Miyazaki D.
      • Haruki T.
      • Takeda S.
      • Sasaki S.-I.
      • Yakura K.
      • Terasaka Y.
      • et al.
      Herpes simplex virus type 1-induced transcriptional networks of corneal endothelial cells indicate antigen presentation function.
      • Kash J.C.
      • Mühlberger E.
      • Carter V.
      • Grosch M.
      • Perwitasari O.
      • Proll S.C.
      • et al.
      Global suppression of the host antiviral response by Ebola- and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence.
      underlining a pathogen-triggered induction of this gene.
      Importantly, we observed that C19orf66 suppressed HCV replication (Fig. 3, Fig. 4C), while knockout of the gene attenuated IFN-induced suppression of HCV replication (Fig. 2B). These data indicate C19orf66 contributes to the IFN-mediated control of HCV replication in the human liver.
      Transcriptomic analysis and whole cell proteomics revealed that the ectopic expression of C19orf66 did not induce the dysregulation of additional antiviral genes (Fig. 2D, E), suggesting a potential effector function of C19orf66 rather than being modulatory. The re-localization of C19orf66 to the HCV replication compartment and its aggregation (Fig. 2G–J) support the hypothesis that C19orf66 directly interferes with the HCV life cycle. Consistently, we observed a restriction of HCV subgenomic replicons, with the magnitude of restriction correlating with viral replication fitness (Fig. 4V).
      By utilizing co-immunoprecipitation followed by LC-MS/MS, we identified a SG and P-body protein-dominated interactome of C19orf66 (Fig. 5A). Accordingly, C19orf66 is reported to co-localize with markers of SGs and P-bodies upon dengue virus infection.
      • Balinsky C.A.
      • Schmeisser H.
      • Wells A.I.
      • Ganesan S.
      • Jin T.
      • Singh K.
      • et al.
      IRAV (FLJ11286), an interferon-stimulated gene with antiviral activity against dengue virus, interacts with MOV10.
      ,
      • Suzuki Y.
      • Chin W.-X.
      • Han Q.E.
      • Ichiyama K.
      • Lee C.H.
      • Eyo Z.W.
      • et al.
      Characterization of RyDEN (C19orf66) as an interferon-stimulated cellular inhibitor against dengue virus replication.
      SG and P-body components play an important role in turnover and degradation of RNA. They are known to be recruited to replication sites of different Flaviviridae, but also support viral replication, indicating a paradoxical relationship between viruses and these proteins.
      • Ruggieri A.
      • Dazert E.
      • Metz P.
      • Hofmann S.
      • Bergeest J.-P.
      • Mazur J.
      • et al.
      Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection.
      • Fernández-Carrillo C.
      • Pérez-Vilaró G.
      • Díez J.
      • Pérez-Del-Pulgar S.
      Hepatitis C virus plays with fire and yet avoids getting burned. A review for clinicians on processing bodies and stress granules.
      • Chahar H.S.
      • Chen S.
      • Manjunath N.
      P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication.
      Importantly, experiments with replication-deficient subgenomic replicons and a translation reporter system demonstrated that the C19orf66-mediated anti-HCV effect is not based on increased degradation of viral RNA or the inhibition of RNA translation (Fig. 4C–E). However, the ability of C19orf66 to accumulate in response to stress may be crucial for its antiviral capacity, as suggested by C19orf66-Zincmut, which has impaired restriction ability and fails to form punctae during stress (Fig. 5C–G).
      Recently, Wang and colleagues observed that C19orf66 is a host HIV-1 restriction factor that causes premature termination of translation.
      • Wang X.
      • Xuan Y.
      • Han Y.
      • Ding X.
      • Ye K.
      • Yang F.
      • et al.
      Regulation of HIV-1 Gag-Pol expression by shiftless, an inhibitor of programmed -1 ribosomal frameshifting.
      In contrast, the current knowledge about HCV virology in combination with our obtained data suggests that C19orf66 restricts HCV via a different mode of action: ribosomal frameshifting is not reported to be crucial for HCV replication and we did not observe an inhibitory effect on HCV IRES-dependent translation (Fig. 4C–E). In addition, the splice variant C19orf66-209 failed to restrict HIV-1 via inhibition of -1PRF, but restricted HCV replication (Fig. S7C, D), arguing for an additional mechanism by which C19orf66 targets viruses.
      A hallmark of a HCV infection is the formation of the MW, the presumed site of viral RNA replication.
      • Romero-Brey I.
      • Merz A.
      • Chiramel A.
      • Lee J.-Y.
      • Chlanda P.
      • Haselman U.
      • et al.
      Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
      ,
      • Egger D.
      • Wolk B.
      • Gosert R.
      • Bianchi L.
      • Blum H.E.
      • Moradpour D.
      • et al.
      Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex.
      Importantly, the formation of MWs can be induced by sole expression of the replicase proteins of HCV.
      • Romero-Brey I.
      • Merz A.
      • Chiramel A.
      • Lee J.-Y.
      • Chlanda P.
      • Haselman U.
      • et al.
      Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
      Thus, this experimental setting allows MW formation to be uncoupled from viral RNA replication. Using this technique, we observed an altered morphology of the MW with clustering of smaller and rather homogenous DMVs in cells expressing C19orf66, whereas in control cells DMVs are scattered throughout the cell and have a highly heterogenous morphology (Fig. 6E–H). This phenotype is reminiscent of earlier observations in cells deficient in the lipid kinase PI4KA.
      • Berger K.L.
      • Kelly S.M.
      • Jordan T.X.
      • Tartell M.A.
      • Randall G.
      Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.
      • Wang H.
      • Perry J.W.
      • Lauring A.S.
      • Neddermann P.
      • de Francesco R.
      • Tai A.W.
      Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking.
      • Reiss S.
      • Rebhan I.
      • Backes P.
      • Romero-Brey I.
      • Erfle H.
      • Matula P.
      • et al.
      Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.
      Consistently, we observed that C19orf66 expression impairs HCV-induced elevation of PI(4)P (Fig. 7A–C). These results suggest that C19orf66 might interfere with PI4KA activation by HCV. In summary, C19orf66 restricts HCV replication, by perturbing the formation of the viral replication organelle.

       Abbreviations

      -1PRF, programmed-1 ribosomal frameshifting; 2′CMA, 2′C-methyl-adenosine; CHC, chronic hepatitis C; DAA, direct-acting antivirals; DMV, double-membrane vesicle; FC, fold change; FDR, false discovery rate; HBM, hepatocyte basal medium; IFN, interferon; IRES, internal ribosome entry site; IRF, interferon regulatory factor; ISG, interferon-stimulated gene; KO, knockout; KSHV, Kaposi's sarcoma-associated herpesvirus; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LFQ, label-free quantification; MMV, multi-membrane vesicle; MOI, multiplicity of infection; MW, membranous web; pegIFNα, pegylated interferon alfa-2; PHH, primary human hepatocyte; PI4KA, phosphatidylinositol 4-kinase IIIα; PI(4)P, phosphatidylinositol 4-phosphate; RLU, relative light units; RNA-Seq, RNA sequencing; ROI, regions of interest; RT-qPCR, quantitative reverse transcription PCR; SG, stress granule; SMV, single-membrane vesicle; WT, wild-type.

      Financial support

      T.P. was supported by grants from the Helmholtz-Alberta Initiative For Infectious Disease Research (HAI-IDR). M.H. was supported by the Swiss National Science Foundation (SNF) grant 310030B_147089 . R.B. was supported by the Deutsche Forschungsgemeinschaft, Germany ( DFG) grant ( TRR179 , TP9 ).

      Authors' contributions

      V.K., A.P., A.K. T.Br., R.B. and E.S. designed research; V.K., A.P., A.K., T.Br., D.T., R.J.P.B., Y.Z., Y.B., M.F., M.E. and G.V. performed research; T.Br., T.Bo., Y.B., R.Br., F.W.R.V., M.H., B.S., R.B., T.P. and E.S. contributed reagents/analytic tools; V.K., A.P., A.K., T.Br., D.T., R.J.P.B., Y.Z., Y.B., G.V., R.B. and E.S. analyzed data; V.K., R.B. and E.S. wrote the original draft; V.K., A.P., A.K., T.B., D.T., R.J.P.B., T.Bo., Y.Z., Y.B., G.V., R.Br., F.W.R.V., M.H., B.S., R.B., T.P. and E.S. reviewed and edited the original draft.

      Conflict of interest

      T.P. has received consulting fees from Biotest AG and from Janssen Global Services, L.L.C.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgments

      We are grateful to Takaji Wakita and Jens Bukh for JFH1 and J6CF isolates, respectively, to Volker Lohmann for the intergenotypic JFH1-based replicon chimeras, to Charles Rice for Huh-7.5 cells and the 9E10 monoclonal antibody. Furthermore, we thank Yves Rouillé for the bicistronic reporter pIRF1b and Marc Schmidt-Supprian and Klaus Heger for the pLenti CRISPR v2 ccdB plasmid. Moreover, we thank all members of the Institute of Experimental Virology, TWINCORE, and the Department for Molecular and Medical Virology, Ruhr-University Bochum, for helpful support, suggestions and discussions. R.B. and A.P. gratefully acknowledge the expert technical assistance by Uta Haselmann, the EM core facility at Heidelberg University, headed by Stefan Hillmer, and the Light Microscopy Facility (LMF) at German Cancer Research Center, headed by Felix Bestvater.

      Supplementary data

      References

        • World Health Organization
        Global Hepatitis Report 2017.
        2017
        • Hoofnagle J.H.
        Hepatitis C: the clinical spectrum of disease.
        Hepatology. 1997; 26: 15S-20S
        • Thrift A.P.
        • El-Serag H.B.
        • Kanwal F.
        Global epidemiology and burden of HCV infection and HCV-related disease.
        Nat Rev Gastroenterol Hepatol. 2017; 14: 122-132
        • Heim M.H.
        Innate immunity and HCV.
        J Hepatol. 2013; 58: 564-574
        • Boldanova T.
        • Suslov A.
        • Heim M.H.
        • Necsulea A.
        Transcriptional response to hepatitis C virus infection and interferon-alpha treatment in the human liver.
        EMBO Mol Med. 2017; 9: 816-834
        • Anggakusuma
        • Romero-Brey I.
        • Berger C.
        • Colpitts C.C.
        • Boldanova T.
        • Engelmann M.
        • et al.
        Interferon-inducible cholesterol-25-hydroxylase restricts hepatitis C virus replication through blockage of membranous web formation.
        Hepatology. 2015; 62: 702-714
        • Schoggins J.W.
        • Wilson S.J.
        • Panis M.
        • Murphy M.Y.
        • Jones C.T.
        • Bieniasz P.
        • et al.
        A diverse range of gene products are effectors of the type I interferon antiviral response.
        Nature. 2011; 472: 481-485
        • Balinsky C.A.
        • Schmeisser H.
        • Wells A.I.
        • Ganesan S.
        • Jin T.
        • Singh K.
        • et al.
        IRAV (FLJ11286), an interferon-stimulated gene with antiviral activity against dengue virus, interacts with MOV10.
        J Virol. 2017; 91: e01606-e01616
        • Suzuki Y.
        • Chin W.-X.
        • Han Q.E.
        • Ichiyama K.
        • Lee C.H.
        • Eyo Z.W.
        • et al.
        Characterization of RyDEN (C19orf66) as an interferon-stimulated cellular inhibitor against dengue virus replication.
        PLoS Pathog. 2016; 12: e1005357
        • Wang X.
        • Xuan Y.
        • Han Y.
        • Ding X.
        • Ye K.
        • Yang F.
        • et al.
        Regulation of HIV-1 Gag-Pol expression by shiftless, an inhibitor of programmed -1 ribosomal frameshifting.
        Cell. 2019; 176: 625-635.e14
        • Rodriguez W.
        • Srivastav K.
        • Muller M.
        • Jung J.U.
        C19ORF66 broadly escapes virus-induced endonuclease cleavage and restricts Kaposi's sarcoma-associated herpesvirus.
        J Virol. 2019; 93 (e00373-19)
        • Gallois-Montbrun S.
        • Kramer B.
        • Swanson C.M.
        • Byers H.
        • Lynham S.
        • Ward M.
        • et al.
        Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules.
        J Virol. 2007; 81: 2165
        • Farazi T.A.
        • Leonhardt C.S.
        • Mukherjee N.
        • Mihailovic A.
        • Li S.
        • Max K.E.A.
        • et al.
        Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.
        RNA. 2014; 20: 1090-1102
        • Fujimura K.
        • Kano F.
        • Murata M.
        Dual localization of the RNA binding protein CUGBP-1 to stress granule and perinucleolar compartment.
        Exp Cell Res. 2008; 314: 543-553
        • Romero-Brey I.
        • Merz A.
        • Chiramel A.
        • Lee J.-Y.
        • Chlanda P.
        • Haselman U.
        • et al.
        Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
        PLoS Pathog. 2012; 8: e1003056
        • Romero-Brey I.
        • Berger C.
        • Kallis S.
        • Kolovou A.
        • Paul D.
        • Lohmann V.
        • et al.
        NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication.
        mBio. 2015; 6: e00759
        • Berger K.L.
        • Kelly S.M.
        • Jordan T.X.
        • Tartell M.A.
        • Randall G.
        Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.
        J Virol. 2011; 85: 8870-8883
        • Wang H.
        • Perry J.W.
        • Lauring A.S.
        • Neddermann P.
        • de Francesco R.
        • Tai A.W.
        Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking.
        Gastroenterology. 2014; 146: 1373-1385.e1–11
        • Reiss S.
        • Rebhan I.
        • Backes P.
        • Romero-Brey I.
        • Erfle H.
        • Matula P.
        • et al.
        Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.
        Cell Host Microbe. 2011; 9: 32-45
        • Schneider W.M.
        • Chevillotte M.D.
        • Rice C.M.
        Interferon-stimulated genes: a complex web of host defenses.
        Annu Rev Immunol. 2014; 32: 513-545
        • Wang J.
        • Nikrad M.P.
        • Phang T.
        • Gao B.
        • Alford T.
        • Ito Y.
        • et al.
        Innate immune response to influenza A virus in differentiated human alveolar type II cells.
        Am J Respir Cell Mol Biol. 2011; 45: 582-591
        • Miyazaki D.
        • Haruki T.
        • Takeda S.
        • Sasaki S.-I.
        • Yakura K.
        • Terasaka Y.
        • et al.
        Herpes simplex virus type 1-induced transcriptional networks of corneal endothelial cells indicate antigen presentation function.
        Invest Ophthalmol Vis Sci. 2011; 52: 4282-4293
        • Kash J.C.
        • Mühlberger E.
        • Carter V.
        • Grosch M.
        • Perwitasari O.
        • Proll S.C.
        • et al.
        Global suppression of the host antiviral response by Ebola- and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence.
        J Virol. 2006; 80: 3009-3020
        • Ruggieri A.
        • Dazert E.
        • Metz P.
        • Hofmann S.
        • Bergeest J.-P.
        • Mazur J.
        • et al.
        Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection.
        Cell Host Microbe. 2012; 12: 71-85
        • Fernández-Carrillo C.
        • Pérez-Vilaró G.
        • Díez J.
        • Pérez-Del-Pulgar S.
        Hepatitis C virus plays with fire and yet avoids getting burned. A review for clinicians on processing bodies and stress granules.
        Liver Int. 2018; 38: 388-398
        • Chahar H.S.
        • Chen S.
        • Manjunath N.
        P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication.
        Virology. 2013; 436: 1-7
        • Egger D.
        • Wolk B.
        • Gosert R.
        • Bianchi L.
        • Blum H.E.
        • Moradpour D.
        • et al.
        Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex.
        J Virol. 2002; 76: 5974-5984