Shilin Li

MicroRNA-130a inhibits HCV replication by restoring the innate immune response.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and hepatocellular carcinoma. Currently pegylated interferon (IFN) combined with ribavirin remains the best therapeutic approach, although patients infected with HCV genotype I may benefit from adding protease inhibitors as ‘triple therapy’. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression and have recently been shown to play an important role in human innate immune response and as an antiviral in chimpanzees. We studied the effect of miR‐130a on the HCV replication. We found that miR‐130a significantly inhibits HCV replication in both HCV replicon and J6‐/JFH1‐infected cells. Over expression of miR‐130a upregulated the expression of type I IFN (IFN‐α/IFN ‐β), ISG15, USP18 and MxA, which are involved in innate immune response and decreased expression of miR‐122, a well‐defined miRNA promoting HCV production. In conclusion, miR‐130a inhibits HCV replication/production by restoring host innate immune responses and/or downregulating pro‐HCV miR‐122. miR‐130a might be a potential drug target by modulating host innate immune responses to combat HCV infection.

Activation of endogenous type I IFN signaling contributes to persistent HCV infection

HCV infection is a major world health problem, leading to both end‐stage liver disease and primary liver cancer. Great efforts have been made in developing new therapies for HCV infection; however, combination therapy with pegylated IFN‐α and ribavirin (pegIFN‐RBV) remains the first choice of treatment for chronic HCV infection in most countries. The treatment response to pegIFN‐RBV remains relatively low. Understanding the molecular mechanisms of persistent HCV infection and pegIFN‐RBV resistance will suggest ways of improving the current standard of care and offers new antiviral therapies for both HCV and other viral infections. Recent data suggest that increased expression of hepatic IFN‐stimulated genes (ISGs) before treatment is associated with treatment nonresponse in patients chronically infected with HCV. Although ISGs are generally antiviral in nature, in the case of HCV, the virus may exploit some of them to its benefit. This is not unique to HCV: Blockade of type I IFN signaling has been shown to control persistent LCMV infection. Thus, in certain viral infections, preactivation or overactivation of type I IFN signaling may contribute to viral persistence. In this review, we briefly summarize the findings from high‐throughput gene expression profiling from patients chronically infected with HCV, then focus on a novel ubiquitin‐like signaling pathway (ISG15/USP18) and its potential role in HCV persistence. Finally, the role of activation of endogenous type I IFN signaling in persistent HCV infection will be discussed in the context of recent studies indicating that blocking IFN signaling controls persistent LCMV infection. Copyright

Severe fever with thrombocytopenia syndrome virus inhibits exogenous Type I IFN signaling pathway through its NSs in vitro

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel bunyavirus (SFTS virus, SFTSV). At present there is still no specific antiviral treatment for SFTSV; To understand which cells support SFTSV life cycle and whether SFTSV infection activates host innate immunity, four different cell lines (Vero, Hela, Huh7.5.1, and Huh7.0) were infected with SFTSV. Intracellular/extracellular viral RNA and expression of IFNα, and IFNß were detected by real-time RT- PCR following infection. To confirm the role of non-structural protein (NSs) of SFTSV in exogenous IFNα-induced Jak/STAT signaling, p-STAT1 (Western Blot), ISRE activity (Luciferase assay) and ISG expression (real-time PCR) were examined following IFNα stimulation in the presence or absence of over-expression of NSs in Hela cells. Our study showed that all the four cell lines supported SFTSV life cycle and SFTSV activated host innate immunity to produce type I IFNs in Hela cells but not in Huh7.0, Huh7.5.1 or Vero cells. NSs inhibited exogenous IFNα-induced Jak/STAT signaling as shown by decreased p-STAT1 level, suppressed ISRE activity and down-regulated ISG expression. Suppression of the exogenous Type I IFN-induced Jak/STAT signaling by NSs might be one of the mechanisms of SFTSV to evade host immune surveillance.

MxA Is A Positive Regulator of Type I IFN Signaling in HCV Infection

Type I interferons (IFNs) are a family of primordial cytokines that respond to various pathogen infections including Hepatitis C virus (HCV). Type I IFNs signal through Jak/STAT pathway leading to the production of a few hundred interferon stimulated genes (ISGs). The aim of this study was to explore the role of one of these ISGs, MxA in HCV infection and type I IFN production. Plasmid encoding MxA was cloned into PcDNA3.1‐3×tag vector and MxA expression was confirmed both at mRNA (RT‐PCR) and protein (Western blot, WB) levels. IFNα and IFNβ productions were quantified by RT‐PCR from cell lysate and by ELISA kit from culture medium following MxA over‐expression in Huh7.5.1 cells. The activation status of Jak/STAT signaling pathway was examined at three levels: p‐STAT1 (WB), interferon sensitive response element (ISRE) activity (dual luciferase reporter gene assay), and levels of ISG expression (RT‐qPCR). J6/JFH1 HCV culture system was used to study the role of MxA in HCV replication. Our findings indicated that MxA over‐expression inhibited HCV replication and potentiated the IFNα‐mediated anti‐HCV activity; MxA stimulated the production of IFNα, IFNβ, and enhanced IFNα‐induced activation of Jak‐STAT signaling pathway. We concluded that MxA is a positive regulator of type I IFN signaling in HCV infection.

ISG12a inhibits HCV replication and potentiates the anti-HCV activity of IFN-α through activation of the Jak/STAT signaling pathway independent of autophagy and apoptosis

Interferon stimulated (sensitive) genes (ISGs) are the effector molecules downstream of type I/III interferon (IFN) signaling pathways in host innate immunity. ISG12a can be induced by IFN-α. Although ISG12a has been reported to inhibit the replication of HCV, the exact mechanism remains to be determined. In this study, we investigated the possible mechanisms of ISG12a anti- HCV property by exploring the production of type I IFN and the activation of Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway, apoptosis and autophagy in Huh7.5.1 cells transiently transfected with ISG12a over-expression plasmid. Interestingly, we found that ISG12a inhibited HCV replication in both Con1b replicon and the HCV JFH1-based cell culture system and potentiated the anti-HCV activity of IFN-α. ISG12a promoted the production of IFN α/β and activated the type I IFN signaling pathway as shown by increased p-STAT1 level, higher Interferon sensitive response element (ISRE) activity and up-regulated ISG levels. However, ISG12a over-expression did not affect cell autophagy and apoptosis. Data from our current study collectively indicated that ISG12a inhibited HCV replication and potentiated the anti-HCV activity of IFN-α possibly through induced production of type I IFNs and activation of Jak/STAT signaling pathway independent of autophagy and cell apoptosis.

Vitamin D Potentiates the Inhibitory Effect of MicroRNA-130a in Hepatitis C Virus Replication Independent of Type I Interferon Signaling Pathway.

Calcitriol, the bioactive metabolite of vitamin D, was reported to inhibit HCV production in a synergistic fashion with interferon, a treatment in vitro. Our previous study established that miR-130a inhibits HCV replication by restoring the host innate immune response. We aimed to determine whether there is additive inhibitory effect of calcitriol and miR-130a on HCV replication. Here we showed that calcitriol potentiates the anti-HCV effect of miR-130a in both Con1b replicon and J6/JFH1 culture systems. Intriguingly, this potentiating effect of calcitriol on miR-130a was not through upregulating the expression of cellular miR-130a or through increasing the miR-130a-mediated IFNα/β production. All these findings may contribute to the development of novel anti-HCV therapeutic strategies although the antiviral mechanism needs to be further investigated.

Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro

Hepatitis B virus (HBV) is an important account of infectious hepatitis and interferon (IFN) remains one of the best treatment options. Activation of type I IFN signaling pathway leads to expressions of IFN-stimulated genes (ISGs) which play important roles in antiviral and immunomodulatory responses to HBV or hepatitis C virus (HCV) infection. Our previous studies indicated that ISG15 and its conjugation (ISGylation) were exploited by HCV to benefit its replication and persistent infection. This study was designed to assess the role of ISG15 and ISGylation in HBV infection in vitro. The levels of ISG15 and ISGylation were upregulated by ISG15 plasmid transfection into HepG2.2.15 cells. Decreased ISGylation was achieved by siRNA targeting UBE1L, the only E1 activating enzyme for ISGylation. Overexpression of ISG15 and subsequent ISGylation significantly increased the levels of HBV DNA in the culture supernatants although the intracellular viral replication remained unaffected. Silencing UBE1L, with decreased ISGylation achieved, abrogated this ISGylation-mediated promoting effect. Our data indicated that overexpression of ISG15 stimulated HBV production in an ISGylation-dependent manner. Identification of ISG15-conjugated proteins (either HBV viral or host proteins) may reveal promising candidates for further antiviral drug development.

Ubiquitin-like protein modifiers and their potential for antiviral and anti-HCV therapy

All viral infections subvert the host immune response. Targeting the host mechanisms that are modulated by viral infection offers new avenues for antiviral drug development. Host ubiquitin and multiple ubiquitin-like modifiers (Ubls) are commonly altered by, or important for, viral infection. Protein modification by ubiquitin or Ubls contributes to numerous cellular processes, such as protein degradation, signal transduction, protein relocalization and pathogen–host interactions. This post-translational modification plays an essential role for viral life cycles and host antiviral mechanisms. Some Ubls, such as ISG15 and SUMO, have been shown to modulate virus infections and are potential targets for therapeutic manipulation. Hepatitis C virus (HCV) is a positive-stranded RNA virus that predominantly infects hepatocytes. Recent data suggest that ISG15 might be a potential drug target for anti-HCV therapy. Inhibition of ISG15 expression and/or ISG15 conjugation (ISGylation) provides a rationale for the design of new anti-HCV drugs.

MicroRNA 130a Regulates both Hepatitis C Virus and Hepatitis B Virus Replication through a Central Metabolic Pathway

ABSTRACT Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.

A novel lncRNA regulates HCV infection through IFI6

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon‐stimulated genes (ISGs) were detected by the secrete‐pair dual luminescence assay. We constructed the full‐length and four deletion mutants of an interferon‐induced lncRNA RP11‐288L9.4 (lncRNA‐IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real‐time PCR (qRT‐PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host‐encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA‐IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA‐IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)–infected Huh7.5.1 cells and PHHs. We observed that lncRNA‐IFI6 regulation of HCV was independent of Janus kinase‐signal transducer and activator of transcription (JAK‐STAT) signaling. lncRNA‐IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full‐length lncRNA‐IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA‐IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA‐IFI6 regulates HCV infection independently of the JAK‐STAT pathway. lncRNA‐IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.