Inhibit HCV Replication Research Articles

HCV Interplay With Mir34a: Implications in Hepatocellular Carcinoma.

Since its identification, HCV has been considered one of the main causes of hepatitis and liver cancer. Currently, the molecular mechanisms of HCC development induced by HCV infection have not been sufficiently clarified. The recent discovery of novel treatments that inhibit HCV replication gave rise to new questions concerning HCC mechanisms. In particular, the HCV eradication mediated by new direct-acting antiviral (DAAs) drugs does not exclude the possibility of de novo HCC development; this finding opened more questions on the interplay between liver cells and the virus. Different groups have investigated the pathways leading to cancer recurrence in patients treated with DAAs. For this reason, we tried to gain molecular insights into the changes induced by HCV infection in the target liver cells. In particular, we observed an increase in microRNA34a (miR34a) expression following HCV infection of HCC cell line Huh7.5. In addition, Huh7.5 treated with extracellular vesicles (EVs) from the previously HCV-infected Huh7.5 underwent apoptosis. Since miR34 expression was increased in Huh7.5 EVs, we hypothesized a paracrine mechanism of viral infection mediated by miR34a cargo of EVs. The balance between viral infection and cell transformation may raise some questions on the possible use of antiviral drugs in association with antineoplastic treatment.

Interferon α2-Thymosin α1 Fusion Protein (IFNα2-Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect.

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2–thymosin α1 fusion protein (IFNα2–Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2–Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2–Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2–Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2–Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.

The combination of the NS5A and cyclophilin inhibitors results in an additive anti-HCV inhibition in humanized mice without development of resistance.

We and others previously reported that the direct-acting agents (DAA) NS5A inhibitors (NS5Ai) and the host-targeting agents cyclophilin inhibitors (CypIs) inhibit HCV replication in vitro. In this study, we investigated whether the combination of NS5Ai and CypI offers a potent anti-HCV effect in vivo. A single administration of NS5Ai or CypI alone to HCV-infected humanized-mice inhibits HCV replication. The combination of NS5Ai with CypI suppresses HCV (GT1a, GT2a, GT3a and GT4a) replication in an additive manner. NS5Ai/CypI combinations provide a statistically more profound anti-HCV inhibition for GT2a and GT3a than GT1a and GT4a, leading to a fastest and deepest inhibition of GT2a and GT3a replications. Combining CypI with NS5Ai prevents the viral rebound normally observed in mice treated with NS5Ai alone. Results were confirmed in mice implanted with human hepatocytes from different donors. Therefore, the combination of NS5Ai with CypI may serve as a regimen for the treatment of HCV patients with specific genotypes and disorder conditions, which diminish sustain viral response levels to DAA, such as GT3a infection, cirrhosis, and DAA resistance associated with the selection of resistance-associated substitutions present at baseline or are acquired during treatment.

Inhibition of Long Noncoding RNA Linc‐Pint by Hepatitis C Virus in Infected Hepatocytes Enhances Lipogenesis

Background and Aims HCV often causes chronic infection in liver, cirrhosis, and, in some instances, HCC. HCV encodes several factors' those impair host genes for establishment of chronic infection. The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. However, their role in virus replication and underlying diseases is poorly understood. In this study, we have shown that HCV exploits lncRNA long intergenic nonprotein‐coding RNA, p53 induced transcript (Linc‐Pint) in hepatocytes for enhancement of lipogenesis. Approach and Results We identified a lncRNA, Linc‐Pint, which is significantly down‐regulated in HCV‐replicating hepatocytes and liver specimens from HCV infected patients. Using RNA pull‐down proteomics, we identified serine/arginine protein specific kinase 2 (SRPK2) as an interacting partner of Linc‐Pint. A subsequent study demonstrated that overexpression of Linc‐Pint inhibits the expression of lipogenesis‐related genes, such as fatty acid synthase and ATP‐citrate lyase. We also observed that Linc‐Pint significantly inhibits HCV replication. Furthermore, HCV‐mediated enhanced lipogenesis can be controlled by exogenous Linc‐Pint expression. Together, our results suggested that HCV‐mediated down‐regulation of Linc‐Pint enhances lipogenesis favoring virus replication and liver disease progression. Conclusions We have shown that SRPK2 is a direct target of Linc‐Pint and that depletion of SRPK2 inhibits lipogenesis. Our study contributes to the mechanistic understanding of the role of Linc‐Pint in HCV‐associated liver pathogenesis.

Structurally distinct cyclosporin and sanglifehrin analogs CRV431 and NV556 suppress established HCV infection in humanized-liver mice.

We previously reported that the non-immunosuppressive cyclophilin inhibitors (CypIs)—cyclosporin A analog CRV431 and sanglifehrin analog NV556—efficiently inhibit HCV replication in vitro. In this study, we asked whether they can also reduce HCV replication in vivo. We found that a single oral administration of CRV431 and NV556 to HCV-infected humanized-liver mice drastically reduced HCV blood levels. The antiviral effect was observed when CRV431 or NV556 were each individually administered with HCV, 3, 6 weeks or even 3 months post-infection when viral replication is robust. These results were confirmed in chimeric mice implanted with human hepatocytes isolated from three distinct donors. Remarkably, no viral rebound was observed 5 months after a single dose administration of 50 mg/kg of CRV431 or NV556 four weeks post-HCV infection, indicating the possibility of suppression of an established viral infection. Since we recently demonstrated that both CRV431 and NV556 also inhibit the development of liver fibrosis and hepatocellular carcinoma in nonviral-induced non-alcoholic steatohepatitis mouse models, our present data suggest that the two entirely structurally different CypIs—CRV431 and NV556—derived from unrelated natural products, represent attractive partners to current direct-acting agent (DAA) regimens for the treatment of hepatitis C and liver diseases.

SAR study of bisamides as cyclophilin a inhibitors for the development of host-targeting therapy for hepatitis C virus infection

The therapy of chronic hepatitis C virus infections has significantly improved with the development of direct-acting antivirals (DAAs), which contain NS3/4A protease, NS5A, and NS5B polymerase inhibitors. However, mutations in specific residues in these viral target genes are associated with resistance to the DAAs. Especially inhibitors of NS3/4A protease and NS5A, such as grazoprevir and velpatasvir, have a low barrier to resistant mutations. As a result, the mutations influence the virological outcomes after DAA treatment. CypA inhibitors, as host-targeted agents, act on host factors to inhibit HCV replication, exhibiting a high resistance barrier and pan-genotype activities against HCV. Therefore, they can be developed into alternative, more effective anti-HCV agents. However, CypA inhibitors are natural products and analogs. Based on previous studies, bisamide derivatives were designed and synthesized to develop a novel class of CypA inhibitors. Bisamide derivative 7c is a promising compound with potent anti-HCV activity at subtoxic concentrations. Surface plasmon resonance experiments revealed that 7c directly binds to CypA. All these studies indicated that the derivative 7c is a potent CypA inhibitor, which can be used as a host-targeted agent in combination with other antiviral agents for anti-HCV treatment.

MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication.

The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 μM and a maximum HCV RNA level reduction of approximatively 1 log10 MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

IL-1β Enhances the Antiviral Effect of IFN-α on HCV Replication by Negatively Modulating ERK2 Activation.

Chronic hepatitis C infection is a leading cause of liver cirrhosis, which is linked to chronic hepatic inflammation. While there are multiple studies detailing the proinflammatory role of interleukin-1β (IL-1β) in HCV-induced inflammasome signaling, the antiviral capacity of this cytokine has not been adequately investigated in the context of HCV infection or other members of Flaviridae. Our data indicated that IL-1β alone does not inhibit HCV replication, yet when in combination with IFN-α, it can boost the anti-HCV activity of IFN-α, which is mediated by augmented STAT1 tyrosine 701 phosphorylation. Through signaling inhibitor screening, we found that ERK2 kinase is directly linked to the enhanced activation of the STAT1 complex. Our study found that IL-1β negatively affects ERK2 phosphorylation, which suggests that IL-1β-mediated STAT1 tyrosine 701 phosphorylation employed kinase machinery of ERK2 other than JNK or P38 kinase. Our results identify IL-1β as a proinflammatory cytokine possessing wide spectrum synergistic antiviral capability via enhancing IFN-α-induced interferon-stimulated genes (ISGs) expression. A more nuanced understanding of the antiviral mechanisms of this important cytokine could facilitate the development of new therapeutic options.

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

HCV is a positive-strand RNA virus thatprimarily infects human hepatocytes. Recent studies havereported 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. 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. 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. C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection invivo. 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. 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.

Application of an integrated cheminformatics-molecular docking approach for discovery for physicochemically similar analogs of fluoroquinolones as putative HCV inhibitors

BackgroundHepatitis C Virus (HCV) infection is a major public health concern across the globe. At present, direct-acting antivirals are the treatment of choice. However, the long-term effect of this therapy has yet to be ascertained. Previously, fluoroquinolones have been reported to inhibit HCV replication by targeting NS3 protein. Therefore, it is logical to hypothesize that the natural analogs of fluoroquinolones will exhibit NS3 inhibitory activity with substantially lesser side effects. MethodIn this study, we tested the application of a recently devised integrated in-silico Cheminformatics-Molecular Docking approach to identify physicochemically similar natural analogs of fluoroquinolones from the available databases (Ambinter, Analyticon, Indofines, Specs, and TimTec). Molecular docking and ROC curve analyses were performed, using PatchDock and Graphpad software, respectively, to compare and analyze drug-protein interactions between active natural analogs, Fluoroquinolones, and HCV NS3 protein. ResultIn our analysis, we were able to shortlist 18 active natural analogs, out of 10,399, that shared physicochemical properties with the template drugs (fluoroquinolones). These analogs showed comparable binding efficacy with fluoroquinolones in targeting 32 amino acids in the HCV NS3 active site that are crucial for NS3 activity. Our approach had around 80 % sensitivity and 70 % specificity in identifying physicochemically similar analogs of fluoroquinolones. ConclusionOur current data suggest that our approach can be efficiently applied to identify putative HCV drug inhibitors that can be taken for in vitro testing. This approach can be applied to discover physicochemically similar analogs of virtually any drug, thus providing a speedy and inexpensive approach to complement drug discovery and design, which can tremendously economize on time and money spent on the screening of putative drugs.

Direct antiviral agents upregulate natural killer cell potential activity in chronic hepatitis C patients.

Direct antiviral agents (DAAs) can eliminate hepatitis C virus rapidly and make chronic hepatitis C (CHC) curable. The changes in the innate immune system during treatment with DAAs are still in dispute. To investigate how the functions of natural killer (NK) cells change during and after treatment with DAAs in each NK cell subset. Thirteen CHC patients were treated with sofosbuvir/ledipasvir, and the expression levels of NKp46 and NKG2A were tested via flow cytometry at baseline, at 2, 4, 8 and 12weeks during the therapy and 12 and 24weeks after the end of treatment; expression levels were compared between CHC patients and 13 healthy controls. A redirected killing assay was used to detect the cytotoxicity of NK cells. After coculturing NK cells with JFH-Huh7 cells for 72h, HCV RNA was tested to analyze the inhibition ability of NK cells. All patients achieved sustained virologic response. The expression of the activating receptor NKp46 was decreased first at week 8 during therapy with DAAs and then increased and normalized to levels in healthy controls after treatment with DAAs. The expression of the inhibitory receptor NKG2A was decreased during and after treatment with DAAs. Each NK cell subset has a similar changing trend during and after treatment with DAAs, although some differences can be found earlier and later. The ratio of NKp46 and NKG2A was upregulated after treatment with DAAs. CD56bright NK cells have less amplitude in the frequency ratio changes after treatment with DAAs. The coculture results showed that both the specific lysis and the inhibition of HCV replication were significantly upregulated after treatment with DAAs. DAA treatments can affect patients' NK cell function. After DAA treatments, the expression of functional markers is downregulated, but the potential activity of NK cells is upregulated. The function of NK cells is normalized to levels in healthy controls. CD56bright NK cells play an important role in this process.

Atorvastatin in Combination with Pegylated Interferon and Ribavirin Provided High Rate of Sustained Virological Response in Patients with Genotype 3 Hepatitis C Virus

BACKGROUND:Chronic hepatitis C virus infection represents a more frequent cause of liver cirrhosis and hepatocellular carcinoma. Statins, inhibit HCV replication in vitro, enhance the antiviral effect of the already known antiviral drugs and reduce their resistance.AIM:To determine the impact of additional therapy (treatment with Atorvastatin 20 mg) to the standard antiviral therapy (pegylated interferon alpha-peg-IFN α and ribavirin) on achieving sustained virological response (SVR).MATERIAL AND METHODS:In the study which is comparative, open-label, prospective-retrospective, 70 patients diagnosed with chronic hepatitis C virus infection who met criteria for treatment with standard antiviral therapy combined with anti-lipemic therapy (Atorvastatin 20 mg) were included. Patients in the study were divided into two groups: one group of 35 patients receiving combination therapy (Atorvastatin + peg-IFN α + Ribavirin) and another group of 35 patients received only standard antiviral therapy. Those parameters were followed in all patients: genotyping, quantification of the virus, histological assessment of liver inflammation and fibrosis degree (before starting treatment), the presence of steatosis, laboratory analysis: hematology, liver, lipid and carbohydrate status, insulin blood level (the calculation of HOMA-IR) and body mass index (BMI) calculation. The overall treatment of the patients depends from the virus genotype, thus, patients with genotype 1 and 4 received 48 weeks standard antiviral therapy, but patients with genotypes 2 and 3 received 24 weeks of antiviral therapy. SVR was considered an undetectable level of HCV RNA levels 24 weeks after completion of antiviral therapy. The results were statistically analysed, and all results for p < 0.05 were considered statistically significant.RESULTS:Combination therapy leads to a slightly higher percentage of SVR (85.71%) in patients with chronic hepatitis C versus standard therapy (74.29%), but in a group of patients with genotype 3 this rate of SVR amounting to 95.83%. Combination therapy leads to significant improvement of lipid and glucose status after treatment, and in terms of side effects, there was no appearance of serious adverse events that would be a reason for discontinuation of the therapy.CONCLUSION:Combination therapy Atorvastatin + pegylated interferon alpha + Ribavirin leads to high rate of SVR of 95.83% in patients with chronic hepatitis C, genotype 3. Statins can be used safely in patients with chronic hepatitis C.

Integrated Cheminformatics-Molecular Docking Approach to Drug Discovery Against Viruses.

In the current study, we present an integrated in silico cheminformaticsmolecular docking approach to screen and test potential therapeutic compounds against viruses. Fluoroquinolones have been shown to inhibit HCV replication by targeting HCV NS3-helicase. Based on this observation, we hypothesized that natural analogs of fluoroquinolones will have similar or superior inhibitory potential while having potentially fewer adverse effects. To screen for natural analogs of fluoroquinolones, we devised an integrated in silico Cheminformatics-Molecular Docking approach. We used 17 fluoroquinolones as bait reference, to screen large databases of natural analogs. 10399 natural compounds and their derivatives were retrieved from the databases. From these compounds, molecules bearing physicochemical similarities with fluoroquinolones were analyzed using a cheminformatics-docking approach. From the 10399 compounds screened using our cheminformatics approach, only 20 compounds were found to share physicochemical similarities with fluoroquinolones, while the remaining 10379 compounds were physiochemically different from fluoroquinolones. Molecular docking analysis showed 32 amino acids in the HCV NS3 active site that were most frequently targeted by fluoroquinolones and their natural analogues, indicating a functional similarity between the two groups of compounds. This study describes a speedy and inexpensive approach to complement drug discovery and design against viral agents. The in silico analyses we used here can be employed to shortlist promising compounds/putative drugs that can be further tested in wet-lab.

Design, Synthesis, Molecular Modeling Studies and Biological Evaluation of N'-Arylidene-6-(benzyloxy)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide Derivatives as Novel Anti-HCV Agents.

HCV-induced hepatitis is one of the most debilitating diseases. The limited number of anti-HCV drugs and drug-resistance necessitate developing of new scaffolds with different mode of actions. HCV non-structural protein 5B (NS5B) is an attractive target for development of novel inhibitors of HCV replication. In this paper, new N′-arylidene-6-(benzyloxy)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide derivatives were designed based on the pharmacophores of HCV NS5B active site binding inhibitors. Designed compounds were synthesized and evaluated for their inhibitory activities in a cell-based HCV replicon system assay. Among tested compounds, compounds 18 and 20 were found to be the most active (EC50 = 35 and 70 µM, respectively) with good selectivity index (SI > 2) in the corresponding series. Molecular modeling studies showed that the designed compounds are capable of forming key coordination with the two magnesium ions as well as interactions with other key residues at the active site of HCV NS5B.

Toll-Like Receptor 7 (TLR-7) and TLR-9 Agonists Improve Hepatitis C Virus Replication and Infectivity Inhibition by Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) are innate immune cells with high antiviral activity triggered by Toll-like receptor 7 (TLR-7) and TLR-9 stimulation. Moreover, they are important mediators between innate and adaptive immunity. Although nowadays there is available an effective therapeutic arsenal against hepatitis C virus (HCV), a protective vaccine is not available. We have analyzed the pDCs' response to HCV infection in a hepatitis C virus (HCV)-Huh7.5 virus-cell system, which allows completion of the virus infectious cycle. pDCs were cocultured following human immunodeficiency virus (HIV) aldrithiol-2 (AT-2 [TLR-7 agonist]) inactivation and CpG (TLR-9 agonist) stimulation. We employed three virus derivatives-wild-type Jc1, interferon (IFN)-resistant virus IR, and high-replicative-fitness virus P100-in order to explore additional IFN-α-related virus inhibition mechanisms. pDCs inhibited HCV infectivity and replication and produced IFN-α. After TLR-7 and TLR-9 stimulation, inhibition of infectivity and IFN-α production by pDCs were enhanced. TLR-7 stimulation drove higher TNF-related apoptosis-inducing ligand (TRAIL) expression in pDCs. Additionally, TLR-7- and TLR-9-stimulated pDCs exhibited a mature phenotype, improving the antigen presentation and lymph node homing-related markers. In conclusion, pDCs could serve as a drug target against HCV in order to improve antiviral activity and as an enhancer of viral immunization.IMPORTANCE We implemented a coculture system of pDCs with HCV-infected hepatoma cell line, Huh7.5. We used three HCV derivatives in order to gain insight into pDCs' behavior against HCV and associated antiviral mechanisms. The results with this cell coculture system support the capacity of pDCs to inhibit HCV replication and infectivity mainly via IFN-α, but also through additional mechanisms associated with pDC maturation. We provided evidence that TLR agonists can enhance antiviral pDCs' function and can induce phenotypic changes that may facilitate the interplay with other immune cells. These findings suggest the possibility of including TLR agonists in the strategies of HCV vaccine development.

Long Noncoding RNA ITPRIP-1 Positively Regulates the Innate Immune Response through Promotion of Oligomerization and Activation of MDA5.

Emerging evidence indicates that long noncoding RNAs (lncRNAs) regulate various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and the interferon (IFN) pathway remains largely unknown. Here, we report that lncRNA ITPRIP-1 (lncITPRIP-1) is involved in viral infection and plays a crucial role in the virus-triggered IFN signaling pathway through the targeting of melanoma differentiation-associated gene 5 (MDA5). LncITPRIP-1 can be induced by viral infection, which is not entirely dependent on the IFN signal. Besides, there is no coding potential found in the lncITPRIP-1 transcript. LncITPRIP-1 binds to the C terminus of MDA5, and it possesses the ability to boost the oligomerization of both the full length and the 2 caspase activation and recruitment domains of MDA5 in a K63-linked polyubiquitination-independent manner. Amazingly, we also found that MDA5 can suppress hepatitis C virus (HCV) replication independently of IFN signaling through its C-terminal-deficient domain bound to viral RNA, in which lncITPRIP-1 plays a role as an assistant. In addition, the expression of lncITPRIP-1 is highly consistent with MDA5 expression, indicating that lncITPRIP-1 may function as a cofactor of MDA5. All the data suggest that lncITPRIP-1 enhances the innate immune response to viral infection through the promotion of oligomerization and activation of MDA5. Our study discovers the first lncRNA ITPRIP-1 involved in MDA5 activation.IMPORTANCE Hepatitis C virus infection is a global health issue, and there is still no available vaccine, which makes it urgent to reveal the underlying mechanisms of HCV and host factors. Although RIG-I has been recognized as the leading cytoplasmic sensor against HCV for a long time, recent findings that MDA5 regulates the IFN response to HCV have emerged. Our work validates the significant role of MDA5 in IFN signaling and HCV infection and proposes the first lncRNA inhibiting HCV replication by promoting the activation of MDA5 and mediating the association between MDA5 and HCV RNA, the study of which may shed light on the MDA5 function and treatment for hepatitis C patients. Our suggested model of how lncITPRIP-1 orchestrates signal transduction for IFN production illustrates the essential role of lncRNAs in virus elimination.

Opposite Effects of Two Human ATG10 Isoforms on Replication of a HCV Sub-genomic Replicon Are Mediated via Regulating Autophagy Flux in Zebrafish.

Autophagy is a host mechanism for cellular homeostatic control. Intracellular stresses are symptoms of, and responses to, dysregulation of the physiological environment of the cell. Alternative gene transcription splicing is a mechanism potentially used by a host to respond to physiological or pathological challenges. Here, we aimed to confirm opposite effects of two isoforms of the human autophagy-related protein ATG10 on an HCV subgenomic replicon in zebrafish. A liver-specific HCV subreplicon model was established and exhibited several changes in gene expression typically induced by HCV infection, including overexpression of several HCV-dependent genes (argsyn, leugpcr, rasgbd, and scaf-2), as well as overexpression of several ER stress related genes (atf4, chop, atf6, and bip). Autophagy flux was blocked in the HCV model. Our results indicated that the replication of the HCV subreplicon was suppressed via a decrease in autophagosome formation caused by the autophagy inhibitor 3MA, but enhanced via dysfunction in the lysosomal degradation caused by another autophagy inhibitor CQ. Human ATG10, a canonical isoform in autophagy, facilitated the amplification of the HCV-subgenomic replicon via promoting autophagosome formation. ATG10S, a non-canonical short isoform of the ATG10 protein, promoted autophagy flux, leading to lysosomal degradation of the HCV-subgenomic replicon. Human ATG10S may therefore inhibit HCV replication, and may be an appropriate target for future antiviral drug screening.

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

Background: The RNAi-based transient therapeutic approach has been well explored for its potential against the hepatitis V virus (HCV). However, to achieve a sustained virological response, a consistent presence of siRNA is needed and it can be achieved by constitutively expressing shRNAs. In this context, the lentiviral vector has emerged as an attractive tool for shRNA delivery against HCV. Methods: We monitored HCV inhibition after single and multiple rounds of siRNA treatments against La autoantigen and HCV-NS5B in Huh-7.5 cells infected with the FL-J6/JFH chimeric HCV strain. A bicistronic self-inactivating third-generation lentiviral vector expressing shRNA under U6 and H1 promoters was constructed. To ascertain the long-term HCV inhibition, cells were transduced with lentiviral vectors and HCV inhibition was monitored by RT-PCR and Western blotting at regular intervals. Results: We observed transient antiviral activity after a single round of siRNA treatment, and consecutive rounds of treatments with siRNA demonstrated a sustained HCV inhibition. Delivery of duplex shRNA expressing lentiviral vectors provided constant expression of shRNA leading to synergistic and sustained HCV inhibition. Conclusion: A lentiviral vector-based delivery system is a “single-shot” therapeutic strategy. It can express duplex shRNA for long-term synergistic inhibition of HCV and qualify as a promising therapeutic approach for sustained inhibition of HCV replication.

Human single chain-transbodies that bound to domain-I of non-structural protein 5A (NS5A) of hepatitis C virus

A safe and broadly effective direct acting anti-hepatitis C virus (HCV) agent that can withstand the viral mutation is needed. In this study, human single chain antibody variable fragments (HuscFvs) to conserved non-structural protein-5A (NS5A) of HCV were produced by phage display technology. Recombinant NS5A was used as bait for fishing-out the protein bound-phages from the HuscFv-phage display library. NS5A-bound HuscFvs produced by five phage transfected-E. coli clones were linked molecularly to nonaarginine (R9) for making them cell penetrable (become transbodies). The human monoclonal transbodies inhibited HCV replication in the HCVcc infected human hepatic cells and also rescued the cellular antiviral immune response from the viral suppression. Computerized simulation verified by immunoassays indicated that the transbodies used several residues in their multiple complementarity determining regions (CDRs) to form contact interface with many residues of the NS5A domain-I which is important for HCV replication complex formation and RNA binding as well as for interacting with several host proteins for viral immune evasion and regulation of cellular physiology. The human monoclonal transbodies have high potential for testing further as a new ramification of direct acting anti-HCV agent, either alone or in combination with their cognates that target other HCV proteins.

Hypericin inhibits hepatitis C virus replication via deacetylation and down-regulation of heme oxygenase-1

BackgroundHepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity. Traditional therapy with pegylated interferon-<alpha> and ribavirin has had only limited success, with some adverse effects. Direct-acting antivirals (DAAs) are effective in suppressing HCV replication, but are expensive. PurposeHypericin has been reported to be a good antiviral agent for inhibiting HCV replication, however, little is known about its mechanisms of action. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA. MethodsTo determine the non-structure protein 5A (NS5A) mRNA and NS3 protein expression levels, real-time PCR and Western blot analysis were performed, respectively. To investigate how hypericin inhibits HCV replication, 5-aza-2′-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression. ResultsHypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication. ConclusionIn conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation.