Hepatitis C Virus-Mediated Enhancement of MicroRNA miR-373 Impairs the JAK/STAT Signaling Pathway

ABSTRACTHepatitis C virus (HCV) is a serious global health problem and establishes chronic infection in a significant number of infected humans worldwide. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients, and the mechanism is not fully understood. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, including IFN signaling. To gain more insights into the role of cellular miRNAs in possible countermeasures of HCV for suppression of the host antiviral response, a miRNA array was performed by using primary human hepatocytes infected within vitrocell culture-grown HCV. A group of miRNAs were modulated in HCV-infected primary human hepatocytes. We focused on miR-373, as this miRNA was significantly upregulated in HCV-infected primary human hepatocytes. Here, we analyzed the function of miR-373 in the context of HCV infection. HCV infection upregulates miR-373 expression in hepatocytes and HCV-infected liver biopsy specimens. Furthermore, we discovered that miR-373 directly targets Janus kinase 1 (JAK1) and IFN-regulating factor 9 (IRF9), important factors in the IFN signaling pathway. The upregulation of miR-373 by HCV also inhibited STAT1 phosphorylation, which is involved in ISG factor 3 (ISGF3) complex formation and ISG expression. The knockdown of miR-373 in hepatocytes enhanced JAK1 and IRF9 expression and reduced HCV RNA replication. Taken together, our results demonstrated that miR-373 is upregulated during HCV infection and negatively regulated the type I IFN signaling pathway by suppressing JAK1 and IRF9. Our results offer a potential therapeutic approach for antiviral intervention.IMPORTANCEChronic HCV infection is one of the major causes of end-stage liver disease worldwide. Although the recent introduction of direct-acting antiviral (DAA) therapy is extremely encouraging, some infected individuals do not respond to this therapy. Furthermore, these drugs target HCV nonstructural proteins, and with selective pressure, the virus may develop a resistant strain. Therefore, understanding the impairment of IFN signals will help in designing additional therapeutic modalities. In this study, we provide evidence of HCV-mediated upregulation of miR-373 and show that miR-373 impairs IFN signaling by targeting JAK1/IRF9 molecules. The knockdown of miR-373 inhibited HCV replication by upregulating interferon-stimulating gene expression. Together, these results provided new mechanistic insights into the role of miR-373 in HCV infection and suggest a new potential target against HCV infection.

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Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

PLoS ONE ◽

10.1371/journal.pone.0121734 ◽

2015 ◽

Vol 10 (3) ◽

pp. e0121734 ◽

Cited By ~ 7

Author(s):

Jacques Friborg ◽

Petra Ross-Macdonald ◽

Jian Cao ◽

Ryan Willard ◽

Baiqing Lin ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Human Hepatocytes ◽

Type I ◽

Hepatitis C Virus Infection ◽

Type Iii ◽

Antiviral Responses ◽

Primary Human Hepatocytes ◽

Type Iii Ifn

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Toll-Like Receptor 3 Mediates Establishment of an Antiviral State against Hepatitis C Virus in Hepatoma Cells

Journal of Virology ◽

10.1128/jvi.01125-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 9824-9834 ◽

Cited By ~ 148

Author(s):

Nan Wang ◽

Yuqiong Liang ◽

Santhana Devaraj ◽

Jie Wang ◽

Stanley M. Lemon ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatitis Virus ◽

Hepatoma Cells ◽

Human Hepatocytes ◽

Hcv Infection ◽

Type I Interferons ◽

Type I ◽

Toll Like Receptor ◽

Virus Infections

ABSTRACT Toll-like receptor-3 (TLR3) senses double-stranded RNA, initiating signaling that activates NF-κB and interferon regulatory factor 3 (IRF-3), thereby inducing the synthesis of proinflammatory cytokines, type I interferons, and numerous interferon-stimulated genes (ISGs). This pathway has not been extensively investigated in human hepatocytes, and its role in sensing and protecting against hepatitis virus infections is uncertain. We show here that primary human hepatocytes express TLR3 and robustly upregulate ISGs upon poly(I·C) stimulation. We also show that TLR3 senses hepatitis C virus (HCV) infection when expressed in permissive hepatoma cells, acting independently of retinoic acid-inducible gene I and inducing IRF-3 activation and the synthesis of ISGs that restrict virus replication. In turn, HCV infection reduces the abundance of TRIF, an essential TLR3 adaptor, and impairs poly(I·C)-induced signaling. The induction and disruption of TLR3 signaling by HCV may be important factors in determining the outcome of infection and the ability of HCV to establish persistent infections.

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Replication of Subgenomic Hepatitis C Virus Replicons in Mouse Fibroblasts Is Facilitated by Deletion of Interferon Regulatory Factor 3 and Expression of Liver-Specific MicroRNA 122

Journal of Virology ◽

10.1128/jvi.00559-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9170-9180 ◽

Cited By ~ 54

Author(s):

Liang-Tzung Lin ◽

Ryan S. Noyce ◽

Tram N. Q. Pham ◽

Joyce A. Wilson ◽

Gary R. Sisson ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mouse Models ◽

Type I Interferon ◽

Hcv Infection ◽

Host Factors ◽

Hcv Rna ◽

Type I ◽

Interferon Signaling ◽

Mouse Cells

ABSTRACT Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3−/− IRF-9−/− MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation.

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Dual Effects of Let-7b in the Early Stage of Hepatitis C Virus Infection

Journal of Virology ◽

10.1128/jvi.01800-20 ◽

2020 ◽

Author(s):

Yung-Ju Yeh ◽

Ching-Ping Tseng ◽

Sheng-Da Hsu ◽

His-Yuan Huang ◽

Michael M. C. Lai ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Early Stage ◽

Hcv Infection ◽

Hcv Rna ◽

Type I ◽

Seed Region ◽

Type I Ifn ◽

Stat Signaling ◽

Rna Accumulation

MicroRNA let-7b expression is induced by infection of hepatitis C virus (HCV) and is involved in the regulation of HCV replication by directly targeting the HCV genome. The current study demonstrated that let-7b directly targets negative regulators of type I interferon (IFN) signaling thereby limiting HCV replication in the early stage of HCV infection. Let-7b-regulated genes which are involved in host cellular responses to HCV infection were unveiled by microarray profiling and bioinformatic analyses followed by various molecular and cellular assays using Huh7 cells expressing wild type or the seed region-mutated let-7b. Let-7b targeted the cytokine signaling 1 (SOCS1) protein, a negative regulator of JAK/STAT signaling, which then enhanced STAT1-Y701 phosphorylation leading to increased expression of the downstream interferon stimulated genes (ISGs). Let-7b augmented RIG-I signaling, but not MDA5, to phosphorylate and nuclear translocate IRF3 leading to increased expression of IFN-β. Let-7b directly targeted the ATG12 and IKKα transcripts and reduced the interaction of the ATG5-ATG12 conjugate and RIG-I leading to increased expression of IFN, which may further stimulate JAK/STAT signaling. Let-7b induced by HCV infection elicits dual effects on IFN expression and signaling, along with targeting the coding sequences of NS5B and 5'-UTR of HCV genome, limited HCV RNA accumulation in the early stage of HCV infection. Controlling let-7b expression is thereby crucial in the intervention of HCV infection. Importance: HCV is a leading cause of liver disease, with an estimated 71 million people infected worldwide. During HCV infection, type I IFN signaling displays potent anti-viral and immuno-modulatory effects. Host factors, including microRNAs, play a role in up-regulating IFN signaling to limit HCV replication. Let-7b is a liver abundant miRNA that is induced by HCV infection and targets the HCV genome to suppress HCV RNA accumulation. In this study, we demonstrated that let-7b, as a positive regulator of type I IFN signaling, plays dual roles in against HCV replication by increasing the expression of IFN and ISRE-driven ISGs in the early stage of HCV infection. This study sheds new insight into understanding the role of let-7b in combatting HCV infection. Clarifying IFN signaling regulated by miRNA during the early phase of HCV infection may help researchers understand the initial defense mechanisms to other RNA viruses.

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Hepatitis C Virus Infection Induces Hepatic Expression of NF-κB-Inducing Kinase and Lipogenesis by Downregulating miR-122

mBio ◽

10.1128/mbio.01617-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 2

Author(s):

Brianna Lowey ◽

Laura Hertz ◽

Stephan Chiu ◽

Kristin Valdez ◽

Qisheng Li ◽

...

Keyword(s):

Chronic Hepatitis ◽

Hepatitis C Virus ◽

Lipid Metabolism ◽

Hepatitis C ◽

Chronic Hepatitis C ◽

Human Hepatocytes ◽

Hcv Infection ◽

Pivotal Role ◽

Cellular Lipid

ABSTRACT Hepatitis C virus (HCV) harnesses host dependencies to infect human hepatocytes. We previously identified a pivotal role of IκB kinase α (IKK-α) in regulating cellular lipogenesis and HCV assembly. In this study, we defined and characterized NF-κB-inducing kinase (NIK) as an IKK-α upstream serine/threonine kinase in IKK-α-mediated proviral effects and the mechanism whereby HCV exploits this innate pathway to its advantage. We manipulated NIK expression in Huh7.5.1 cells through loss- and gain-of-function approaches and examined the effects on IKK-α activation, cellular lipid metabolism, and viral assembly. We demonstrated that NIK interacts with IKK-α to form a kinase complex in association with the stress granules, in which IKK-α is phosphorylated upon HCV infection. Depletion of NIK significantly diminished cytosolic lipid droplet content and impaired HCV particle production. NIK overexpression enhanced HCV assembly, and this process was abrogated in cells deprived of IKK-α, suggesting that NIK acts upstream of IKK-α. NIK abundance was increased in HCV-infected hepatocytes, liver tissues from Alb-uPA/Scid mice engrafted with human hepatocytes, and chronic hepatitis C patients. NIK mRNA contains an miR-122 seed sequence binding site in the 3′ untranslated region (UTR). miR-122 mimic and hairpin inhibitor directly affected NIK levels. In our hepatic models, miR-122 levels were significantly reduced by HCV infection. We demonstrated that HNF4A, a known transcriptional regulator of pri-miR-122, was downregulated by HCV infection. NIK represents a bona fide target of miR-122 whose transcription is downregulated by HCV through reduced HNF4A expression. This effect, together with the sequestering of miR-122 by HCV replication, results in “derepression” of NIK expression to deregulate lipid metabolism. IMPORTANCE Chronic hepatitis C virus (HCV) infection is a major global public health problem. Infection often leads to severe liver injury that may progress to cirrhosis, hepatocellular carcinoma, and death. HCV coopts cellular machineries for propagation and triggers pathological processes in the liver. We previously identified a pivotal role of IKK-α in regulating cellular lipid metabolism and HCV assembly. In this study, we characterized NIK as acting upstream of IKK-α and characterized how HCV exploits this innate pathway to its advantage. Through extensive mechanistic studies, we demonstrated that NIK is a direct target of miR-122, which is regulated at the transcription level by HNF4A, a hepatocyte-specific transcription factor. We show in HCV infection that NIK expression is increased while both HNF4A and miR-122 levels are decreased. NIK represents an important host dependency that links HCV assembly, hepatic lipogenesis, and miRNA biology.

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Hepatitis C Virus Infection Impairs IRF-7 Translocation and Alpha Interferon Synthesis in Immortalized Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.00900-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 10991-10998 ◽

Cited By ~ 31

Author(s):

Amit Raychoudhuri ◽

Shubham Shrivastava ◽

Robert Steele ◽

Srikanta Dash ◽

Tatsuo Kanda ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Signaling Pathway ◽

Nuclear Translocation ◽

Human Hepatocytes ◽

Hcv Infection ◽

Oligoadenylate Synthetase ◽

Downstream Signaling ◽

Inducible Protein ◽

Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces beta interferon (IFN-β) expression (T. Kanda, R. Steele, R. Ray, and R. B. Ray, J. Virol. 81:12375-12381, 2007). However, the regulation of the downstream signaling pathway for IFN-α production by HCV is not clearly understood. In this study, the regulation of the IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1 but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces IFN-stimulated response element activation, as evidenced by upregulation of 2′,5′-oligoadenylate synthetase 1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV-infected IHH, IFN-α expression initially increased (up to 24 h) and then decreased at later time points, and IFN-α-inducible protein 27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly(I-C) or IFN-α treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-α signaling pathway may, in part, be responsible for establishment of chronic HCV infection.

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Hepatitis C Virus Infection Induces the Beta Interferon Signaling Pathway in Immortalized Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.01695-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12375-12381 ◽

Cited By ~ 33

Author(s):

Tatsuo Kanda ◽

Robert Steele ◽

Ranjit Ray ◽

Ratna B. Ray

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Signaling Pathway ◽

Human Hepatocytes ◽

Hcv Infection ◽

Oligoadenylate Synthetase ◽

Hcv Genotype ◽

Beta Interferon ◽

Genotype 1A

ABSTRACT Beta interferon (IFN-β) expression is triggered by double-stranded RNA, a common intermediate in the replication of many viruses including hepatitis C virus (HCV). The recent development of cell culture-grown HCV allowed us to analyze the IFN signaling pathway following virus infection. In this study, we have examined the IFN-β signaling pathway following infection of immortalized human hepatocytes (IHH) with HCV genotype 1a (clone H77) or 2a (clone JFH1). We observed that IHH possesses a functional Toll-like receptor 3 pathway. HCV infection in IHH enhanced IFN-β and IFN-stimulated gene 56 (ISG56) promoter activities; however, poly(I-C)-induced IFN-β and ISG56 expression levels were modestly inhibited upon HCV infection. IHH infected with HCV (genotype 1a or 2a) exhibited various levels of translocation of IRF-3 into the nucleus. The upregulation of endogenous IFN-β and 2′,5′-oligoadenylate synthetase 1 mRNA expression was also observed in HCV-infected IHH. Subsequent studies suggested that HCV infection in IHH enhanced STAT1 and ISG56 protein expression. A functional antiviral response of HCV-infected IHH was observed by the growth-inhibitory role in vesicular stomatitis virus. Together, our results suggested that HCV infection in IHH induces the IFN signaling pathway, which corroborates observations from natural HCV infection in humans.

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Hepatitis C virus infection and membranoproliferative glomerulonephritis in Japan.

Journal of the American Society of Nephrology ◽

10.1681/asn.v62220 ◽

1995 ◽

Vol 6 (2) ◽

pp. 220-223

Author(s):

H Yamabe ◽

R J Johnson ◽

D R Gretch ◽

K Fukushi ◽

H Osawa ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Membranoproliferative Glomerulonephritis ◽

Immunoglobulin A ◽

Hcv Infection ◽

Renal Diseases ◽

Hcv Rna ◽

Type I ◽

Hcv Antibody ◽

Polymerase Chain

The prevalence of hepatitis C virus (HCV) infection was determined in 146 adult patients with various types of glomerulonephritis and renal diseases monitored between 1990 and 1993. Serum HCV antibody (HCV Ab) was evaluated, and positive cases were tested for HCV RNA by polymerase chain reaction. HCV infection was present in 1 (1.7%) of 58 cases of immunoglobulin A nephropathy, 0 (0%) of 14 cases of lupus nephritis, 0 (0%) of 12 cases of minimal change nephrosis, and 0 (0%) of 28 cases of other renal diseases, which is similar to the 2% prevalence observed in healthy blood donors in Japan. In contrast, HCV Ab was observed in 2 (8.3%) of 24 cases of membranous nephropathy and 6 (60%) of 10 cases of membranoproliferative glomerulonephritis (MPGN) Type I. The prevalence of HCV infection in MPGN patients was significantly higher than the frequency of HCV infection observed in the other patients with renal diseases (P < 0.001). HCV RNA was present in all cases in which HCV Ab was present. The six patients with HCV-MPGN were similar to the four patients with idiopathic MPGN with respect to age, presence of nephrotic syndrome, and renal dysfunction, but had a higher incidence of liver dysfunction, cryoglobulinemia, rheumatoid factor, and hypocomplementemia (low C3). HCV infection is present in a large percentage of patients with MPGN in Japan and clinically may differ slightly from other cases of MPGN.

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