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.     

Hepatitis C virus induces interferon-λ and interferon-stimulated genes in primary liver cultures

Hepatitis C virus (HCV) replication in primary liver cells is less robust than that in hepatoma cell lines, suggesting that innate antiviral mechanisms in primary cells may limit HCV replication or spread. Here we analyzed the expression of 47 genes associated with interferon (IFN) induction and signaling following HCV infection of primary human fetal liver cell (HFLC) cultures from 18 different donors. We report that cell culture-produced HCV (HCVcc) induced expression of Type III (λ) IFNs and of IFN-stimulated genes (ISGs). Little expression of Type I IFNs was detected. Levels of IFNλ and ISG induction varied among donors and, often, between adapted and nonadapted HCV chimeric constructs. Higher levels of viral replication were associated with greater induction of ISGs and of λ IFNs. Gene induction was dependent on HCV replication, as ultraviolet light-inactivated virus was not stimulatory and an antiviral drug, 2'-C-methyladenosine, reduced induction of λ IFNs and ISGs. The level of IFNλ protein induced was sufficient to inhibit HCVcc infection of na?ve cultures. Conclusion: Together, these results indicate that despite its reported abilities to blunt the induction of an IFN response, HCV infection is capable of inducing antiviral cytokines and pathways in primary liver cell cultures. Induction of ISGs and λ IFNs may limit the growth and spread of HCV in primary cell cultures and in the infected liver. HCV infection of HFLC may provide a useful model for the study of gene induction by HCV in vivo.     

Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs

RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of approximately 22-nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes. We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions. We report the efficient silencing of both cellular lamin AC and HCV RNAs in Huh-7 hepatoma cell lines supporting HCV replication. Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each. siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80-fold decrease in HCV RNA after 4 days. The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication-competent HCV RNAs. These data support the principle of siRNA-based HCV antiviral therapy.     

Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells

Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well-characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV-infected cells. V protein-transduced HFLC supported enhanced (10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV-inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein-transduced cultures. Conclusion: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population.     

Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins

BACKGROUND & AIMS: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. METHODS: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. RESULTS: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA-expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. CONCLUSIONS: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.     

Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response

BACKGROUND & AIMS: Persistent infection with hepatitis C virus (HCV) leads to chronic hepatitis and hepatocellular carcinoma (HCC). RNA interference (RNAi) may act as a host antiviral response against viral RNA. METHODS: The effects of RNAi on both the replicative intermediates and the internal ribosome entry site (IRES) of HCV were studied by using HCV-related short interfering RNA (siRNA) detection assay. The mechanism that permits HCV to escape RNAi was studied by using RNAi assay materials. RESULTS: These studies demonstrate that the Dicer, an RNase enzyme that generates short siRNA, can target and digest both the IRES and the replicative intermediate of HCV into siRNA of approximately 22 nucleotides. Further studies also show that Dicer can inhibit the replication of the HCV subgenomic replicon. However, the HCV core protein inhibits this RNAi and rescues the replication of the HCV subgenomic replicon through a direct interaction with Dicer. CONCLUSIONS: RNAi is a limiting factor for HCV infection, and the core protein suppresses the RNA silencing-based antiviral response. This ability of the core protein to counteract the host defense may lead to a persistent viral infection and may contribute to the pathogenesis of HCV.     

Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling

Hepatitis C virus (HCV) is a major human pathogen that infects 170 million people. A hallmark of HCV is its ability to establish persistent infections reflecting the evasion of host immunity and interference with alpha/beta-IFN innate immune defenses. We demonstrate that disruption of retinoic acid-inducible gene I (RIG-I) signaling by the viral NS3/4A protease contributes to the ability of HCV to control innate antiviral defenses. RIG-I was essential for virus or HCV RNA-induced signaling to the IFN-beta promoter in human hepatoma cells. This signaling was disrupted by the protease activity of NS3/4A, which ablates RIG-I signaling of downstream IFN regulatory factor 3 and NF-kappaB activation, attenuating expression of host antiviral defense genes and interrupting an IFN amplification loop that otherwise suppresses HCV replication. Treatment of cells with an active site inhibitor of the NS3/4A protease relieved this suppression and restored intracellular antiviral defenses. Thus, NS3/4A control of RIG-I supports HCV persistence by preventing IFN regulatory factor 3 and NF-kappaB activation. Our results demonstrate that these processes are amenable to restoration through pharmacologic inhibition of viral protease function.     

Mycophenolic acid inhibits hepatitis C virus replication and acts in synergy with cyclosporin A and interferon-alpha

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Clinical evidence suggests that particular immunosuppressive agents can have an influence on HCV recurrence. Cyclosporine A (CsA) specifically inhibits HCV replication through blocking the viral RNA polymerase enzyme NS5B. In this study, we investigated the effect of mycophenolic acid (MPA) and other immunosuppressants on HCV replication. METHODS: MPA and other compounds were tested in vitro using an HCV-replication model containing a luciferase reporter gene. RESULTS: At clinically relevant concentrations (1.0-6.0 microg/mL), MPA inhibited HCV replication to approximately 75%. CsA and interferon (IFN)-alpha also showed inhibition in a dose-dependent manner. In these short-term (18 hours) experiments, MPA did not inhibit cell proliferation or induce cell death, which could have accounted for the antiviral effect. In contrast to the antiviral activity of MPA against West Nile virus, the effect of MPA on HCV replication was guanosine independent. When combined, MPA and CsA showed significant synergistic inhibition of replication, reaching maximum inhibition of approximately 90% at the highest doses. Synergistic effects were observed with suboptimal concentrations of IFN-alpha with MPA or CsA. The kinetics of HCV inhibition by MPA, CsA, and IFN-alpha were clearly distinct, with earliest effects seen with IFN-alpha. No specific inhibitory effects were observed with tacrolimus or rapamycin. CONCLUSIONS: The immunosuppressive drug MPA is as potent as CsA as an inhibitor of HCV replication. MPA was shown to have a distinct anti-HCV mechanism of action, independent of cell proliferation and guanosine depletion.     

Morphine inhibits intrahepatic interferon- alpha expression and enhances complete hepatitis C virus replication

Heroin addicts are a high-risk group for hepatitis C virus (HCV) infection and the development of chronic HCV disease. We thus examined whether morphine, the active metabolite of heroin, has the ability to inhibit intrahepatic interferon (IFN)- alpha expression, facilitating HCV replication in human hepatocytes. Morphine inhibited intrahepatic IFN- alpha expression, which was associated with an increase in HCV replication in hepatocytes. Moreover, morphine compromised the anti-HCV effect of recombinant IFN- alpha . Investigation of the mechanism responsible for the morphine action revealed that morphine inhibited expression of IFN regulatory factor 5 in the hepatocytes. In addition, morphine suppressed the expression of p38, an important signal-transducing molecule involved in IFN- alpha -mediated anti-HCV activity. These findings indicate that morphine plays a cofactor role in facilitating HCV persistence in human hepatocytes.     

Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation.

Interferon (IFN)-alpha is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN-alpha, -beta and -gamma inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5' untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full-length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES-dependent mechanisms (pCITE-GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN-alpha, -beta and -gamma inhibit replication of sub-genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV- and EMCV-IRES sequences present in the dicistronic HCV sub-genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES-mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.     

Impact of viral amino acid substitutions and host interleukin-28b polymorphism on replication and susceptibility to interferon of hepatitis C virus

Amino acid (aa) substitutions of core 70 and 91 and in the NS5A (nonstructural protein 5A) interferon sensitivity determining region (ISDR) as well as genetic polymorphisms in the host interleukin-28B (IL28B) locus affect the outcome of interferon (IFN)-based therapies for patients with chronic hepatitis C. The combination of these factors and the quasispecies nature of the virus complicate understanding of the underlying mechanism. Using infectious hepatitis C virus (HCV) genotype 1b clone HCV-KT9, we introduced substitutions at both core aa70 (Arg to Gln) and aa91 (Leu to Met). We also introduced four and nine ISDR aa substitutions into core mutant HCV-KT9. Using human hepatocyte chimeric mice with different IL28B genotypes, we examined the infectivity, replication ability, and susceptibility to IFN of these clones. Although aa substitutions in the ISDR significantly impaired infectivity and replication ability of the virus, core aa70 and 91 substitutions did not. The effect of IFN treatment was similar in core wild-type and mutant viruses. Interestingly, virus titer was significantly higher in mice with the favorable IL28B allele (rs8099917 TT and rs12979860 CC) in the transplanted hepatocytes than in mice with hepatocytes from rs8099917 TG and rs12979860 TT donors (P < 0.001). However, the effect of IFN was significantly greater, and intrahepatic expression levels of IFN-stimulated genes were significantly higher in mice with the favorable IL28B allele. Conclusion: Our data suggest that HCV replication levels and response to IFN are affected by human hepatocyte IL28B single-nucleotide polymorphism genotype and mutations in the ISDR. The mechanism underlying the clinically observed association of wild-type core protein in eradication-favorable host cells should be investigated further.     

Cellular cofactors affecting hepatitis C virus infection and replication

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.     

Hepatitis C virus expression suppresses interferon signaling by degrading STAT1

BACKGROUND & AIMS: The molecular mechanisms by which hepatitis C virus (HCV) antagonizes the antiviral actions of interferon (IFN) have not been fully characterized. Specifically, how HCV proteins impact on IFN signaling components has yet to be elucidated. We used an HCV cell-based expression model to examine the interaction between HCV protein expression and host type I IFN signaling components in the Jak-STAT kinase pathway. METHODS: Full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into Huh-T7 cells. HCV expression was monitored by an HCV core antigen enzyme-linked immunosorbent assay. STAT1, P-STAT1, and HCV protein expression was investigated with immunoprecipitation and Western blots. RESULTS: Overexpression and small interfering RNA studies showed that STAT1 was indispensable for control of HCV expression. STAT1 and P-STAT1 expression were markedly reduced in HCV-transfected cells. Full-length HCV, HCV core/E1/E2, and NS3-4A were each associated with decreased STAT1 expression, which was attributable to proteasome-dependent degradation of STAT1. HCV core, but not HCV E1, E2, NS3, NS4, or NS5, bound to STAT1. STAT2 expression was not affected by HCV. CONCLUSIONS: HCV expression selectively degrades STAT1 and reduces P-STAT1 accumulation in the nucleus in a proteasome-dependent manner. HCV core protein binds STAT1, suggesting that this viral protein is associated with STAT1 degradation. STAT1 plays an indispensable role in innate antiviral immunity against HCV expression. In turn, HCV subverts the Jak-STAT kinase by selectively inducing STAT1 degradation.     

Major Vault Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection in CRL2843CD163 Cell Lines and Primary Porcine Alveolar Macrophages

Porcine reproductive and respiratory syndrome (PRRS), a significant viral infectious disease that commonly occurs among farmed pigs, leads to considerable economic losses to the swine industry worldwide. Major vault protein (MVP) is a host factor that induces type Ⅰ interferon (IFN) production. In this study, we evaluated the effect of MVP on PRRSV infection in CRL2843^CD163 cell lines and porcine alveolar macrophages (PAMs). Our results showed that MVP expression was downregulated by PRRSV infection. Adenoviral overexpression of MVP inhibited PRRSV replication, whereas the siRNA knockdown of MVP promoted PRRSV replication. In addition, MVP knockdown has an adverse effect on the inhibitive role of MVP overexpression on PRRSV replication. Moreover, MVP could induce the expression of type Ⅰ IFNs and IFN-stimulated gene 15 (ISG15) in PRRSV-infected PAMs. Based on these results, MVP may be a potential molecular target of drugs for the effective prevention and treatment of PRRSV infection.     

RNAi: a powerful tool to unravel hepatitis C virus-host interactions within the infectious life cycle

Cellular cofactors affecting hepatitis C virus infection and replication. Randall G, Panis M, Cooper JD, Tellinghuisen TL, Sukhodolets KE, Pfeffer S, Landthaler M, Landgraf P, Kan S, Lindenbach BD, Chien M, Weir DB, Russo JJ, Ju J, Brownstein MJ, Sheridan R, Sander C, Zavolan M, Tuschl T, Rice CM. Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target DICER, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target DICER inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh7.5 cells, and Huh7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV. [Abstract reproduced by permission of Proc Natl Acad Sci USA 2007;104:12884-12889]     

Griseofulvin, an oral antifungal agent, suppresses hepatitis C virus replication in vitro

Aim: Hepatitis C virus (HCV), which infects an estimated 170 million people worldwide, is a major cause of chronic liver disease. The current standard therapy for chronic hepatitis C is based on pegylated interferon (IFN)alpha in combination with ribavirin. However, the success rate remains at approximately 50%. Therefore, alternative agents are needed for the treatment of HCV infection. Methods: Using an HCV-1b subgenomic replicon cell culture system (Huh7/Rep-Feo), we found that griseofulvin, an oral antifungal agent, suppressed HCV-RNA replication and protein expression in a dose-dependent manner. We also found that griseofulvin suppressed the replication of infectious HCV JFH-1. A combination of IFNalpha and griseofulvin exhibited a synergistic inhibitory effect in Huh7/Rep-Feo cells. Results: We found that griseofulvin blocked the cell cycle at the G(2)/M phase in the HCV subgenomic replicon cells, but did not inhibit HCV internal ribosome entry site-dependent translation. Conclusion: Our results suggest that griseofulvin may represent a new approach to the development of a novel therapy for HCV infection.