HCV Life Cycle Research Articles

Involvement of ribosomal protein L17 and Y-box binding protein 1 in the assembly of hepatitis C virus potentially via their interaction with the 3' untranslated region of the viral genome.

Although basic research on the HCV life cycle has progressed significantly over the past two decades, our understanding of the molecular mechanisms that regulate the process of particle formation, in particular encapsidation of the genome or nucleocapsid assembly, has been limited. We present here, for the first time, that two RNA-binding proteins, RPL17 and YBX1, bind to the 3'X in the 3'UTR of the HCV genome, which potentially acts as a packaging signal, and facilitates the viral particle assembly. Our study revealed that RPL17 and YBX1 exert a positive effect on the interaction between HCV RNA and Core protein, suggesting that the presence of both host factors modulate an RNA structure or conformation suitable for packaging the viral genome. These findings help us to elucidate not only the regulatory mechanism of the particle assembly of HCV but also the function of host RNA-binding proteins during viral infection.

Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

Akt1 is involved in HCV release by promoting endoplasmic reticulum-to-endosome transition of infectious virions

AimsHepatitis C virus (HCV) relies on the viral and host factors to complete its life cycle. It has evolved to profit from Akt activation at some stage in its life cycle through various mechanisms, notably by activating lipogenesis, which is crucial for infectious virions production. Materials and methodsBy employing an Akt-specific inhibitor, the impact of Akt on intracellular and extracellular infectivity was investigated. To ascertain the role of Akt in the HCV life cycle, the two-part cell culture-derived HCV infection protocol utilizing Akt1 small interfering RNAs (siRNAs) was implemented. The impact of Akt1 on intracellular HCV transition was determined using membrane flotation assay and proximity ligation assay coupled with Anti-Rab7 immunoprecipitation and immunofluorescence. Key findingsAkt1 silencing reduced infectious virions release to a degree comparable to that of ApoE, a host component involved in the HCV assembly and release, suggesting Akt1 was critical in the late stage of the HCV life cycle. Extracellular infectivity of HCV was inhibited by brefeldin A, and the inhibitory effect was augmented by Akt1 silencing and partially restored by ectopic Akt1 expression. Immunofluorescence revealed that Akt1 inhibition suppressed the interaction between HCV core protein and lipid droplet. Akt1 silencing impeded the transition of HCV from the endoplasmic reticulum to the endosome and hence inhibited the secretion of HCV infectious virions from the late endosome. SignificanceOur study demonstrates that Akt1 has an impact on the lipogenesis pathway and plays a critical role in the assembly and secretion of infectious HCV.

Co-infection of Hepatitis C among HIV-infected patients: A cross-sectional study from A University Teaching hospital in Anambra State, Nigeria

Hepatitis C virus co-infection (HCV) with HIV is regarded as a significant public health risk despite being less frequent than hepatitis B co-infection with HIV. This observation results from HIV's impact on the HCV life cycle and, ultimately, the hepatic system. In this study, potential HCV co-infection was examined in connection to the socio-demographic characteristics of HIV-positive patients attending an HIV clinic at a University Teaching Hospital in Awka, Anambra State, Nigeria. Between September 2021 and June 2022, 255 HIV patients who consented to participate in the study had their HCV levels checked using the anti-HCV antibody ELISA kit (DIA.PRO, Italy). Using the Partec CyFlow, CD4 counts were calculated. During routine investigations, blood samples (approximately 5ml) were aseptically collected into sterile EDTA vials, and plasma samples were obtained by centrifugation. With the aid of Abbott Real-Time HIV-1 Assay US methodology, plasma viral loads (PVL) were also calculated. A total of 11 (4.3%) HIV-positive people were also confirmed to have HCV infection, with women making up the majority (4.8%). Most of those affected were between the ages of 31 and 40 (6.8%). With marital status, HCV co-infection was found more amongst married patients, with a rate of 4.8%. The prevalence of HIV/HCV co-infection was higher (4.8%) in those with CD4 cell counts under >350 cells per ml than others. Most patients (3.1%) did not have their viral RNA detected (TND). Self-employed infected persons and those with secondary school educational backgrounds had the highest HIV/HCV prevalence rate of 6.6% and 7.6%, respectively. The study's findings show no statistically significant relationships between the patients' socio-demographic traits and HCV. Also, HCV co-infection prevalence was significantly low in the study participants.

PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication

BackgroundHepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection.MethodsWild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc.ResultsMass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B’delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B.ConclusionsPPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Discovery and Optimization of a 4-Aminopiperidine Scaffold for Inhibition of Hepatitis C Virus Assembly

The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced in vitro toxicity, as well as improved in vitro and in vivo ADME properties.

Current therapeutics against HCV.

Hepatitis C is a positive stranded enveloped RNA virus belonging to the Flaviviridae family. HCV infection leads to severe liver diseases, cirrhosis and hepatocellular carcinoma worldwide. Although treatments have been available for a while, due to its complexity and genetic diversity, only few are reported to be effective against all HCV genotypes. Here, we review the HCV life cycle and its immunogenic potential and various mechanisms via which the virus interferes in the signalling process. A comprehensive overview of current anti-HCV therapeutics, such as, Direct Acting Antiviral (DAA) as well as Host Targeting Agents (HTA), along with their scope, known mechanism of action and limitations are presented.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13337-021-00697-0.

Evaluating serum levels of miR-181a and miR-let7a in HCV infected Iranian patients compared with healthy individuals

IntroductionHepatitis C virus (HCV) is a single-stranded RNA virus which causes acute liver disorder that could develop cirrhosis and hepatocellular carcinoma of the HCV life cycle, and their different expression. Currently, the prevalence of hepatitis C disorder among the general population of Iran is estimated to be 0.3%. MiR-let7a and miR-181a have effects on multiple steps of the HCV life cycle and their differential expression levels have been observed in different stages of hepatitis C infection. At the current study, we investigated the serum levels of miR-let7a and miR-181a in HCV-infected individuals compared to the healthy ones. Materials and methods97 serum samples of hepatitis C-infected patients (case group) and 97 serum samples of healthy individuals (control group) were used for total RNA extraction, cDNA synthesis, real-time polymerase chain reaction (PCR) performance, and final evaluation of serum levels of miR-let7a and miR-181a in the case group, compared with the healthy ones. ResultsLevels of miR-let7a in serum samples of the case group compared with the healthy group showed a considerable decrease with a P value <0.001, while there was no significant difference in the serum levels of miR-181a between these two groups (P value = 0.064). ConclusionsThe serum levels of miR-let7a in the case group had a decrease compared with the control group, and the serum levels of miR-181a in these two groups did not differ significantly. Accordingly, further studies on the possible role of miR-let7a levels in different stages of HCV infection are necessary.

The Hepatitis C virus NS5A and core proteins exert antagonistic effects on HAMP gene expression: the hidden interplay with the MTF-1/MRE pathway.

Hepcidin, a 25-amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron-regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down-regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A-expressing stable cell line, which also exhibited down-regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well-characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A-driven reduction of hepcidin levels, we ruled out any NS5A-induced alterations in the expression of the well-known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF-1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.

ROLE OF MODERN TECHNOLOGY FOR TREATMENT OF HCV

HCV is the main reason of the liver disease and worldwide it is one of the major issues of health due to its development into cirrhosis, failure and cancer of liver. The transference of HCV is mainly through the parental but people who use drug like intravenous are also at greatest threat. The life cycle of HCV is now understood in a more precise way due to extensive studies. Due to more understanding of this virus there is establishment of more effectual antiviral medications and also diagnostic devices. Test of nucleic acids are suggested for the validation of active HCV. Serology tests are suggested for the groups that are at the greatest risk. Earlier for the standard medications of HCV interferon (IFN-a) and ribavirin are used. Later FDA approved a number of drugs such as harvoni, simeprevir and boceprevir etc. for the proper treatment of HCV. Antiviral medications will be utilized to treat the infections of HCV. In the management of certain severe viral infection, therapeutic option has improved in a better way. There is need of follow-up and careful consideration as well as there are many new technologies that have developed for the quantitative measurement of viral genome concentration in the body fluid of patients. Initially this measurement led to important insight in the viral infection pathogenesis as well as these test also revolutionized natural history of HCV. In addition viral load test are pure tool for research, these are used in routine viral diagnosis. Viral load test are used in clinical virology for diagnosis and prognosis of patient’s.

Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus

Objective and designHuman stem cell-derived hepatocyte-like cells (HLCs) have shown high potential as authentic model for dissection of the HCV life cycle and virus-induced pathogenesis. However, modest HCV replication, possibly...

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.

Incidence of Acute Kidney Injury in HCV Infected Patients Receiving Direct-Acting Antivirals

Background: Egypt had high burden of HCV infection worldwide. The new direct-acting antivirals (DAAs) can target almost all steps of HCV life cycle. Objective: Study of the incidence of acute kidney injury (AKI) in HCV infected patients receiving DAAs. Patients and methods: A prospective study that was conducted in the Virology Clinic in Kobry El-Koba Military Hospital on 63 male patients who were eligible to treatment by DAAs, who received treatment with sofosbuvir, daclatasvir and ribavirin combination. They were divided into Group A: 33 patients with eGFR > 90 ml/min and Group B: 30 patients with chronic kidney disease (CKD) stage II-III (eGFR 30 mL/min). The fluctuations in serum creatinine and eGFR were measured while on-therapy and for 3 months follow up after end of treatment. Results: total of 63 male patients, treatment-naive were included. Group A (33 patients) with eGFR > 90 mL/min. Group B (30 patients) with eGFR 90 ml/min in comparison with those with eGFR 45 ml/min, but with no statistical significance. Conclusion: AKI events during and after end of therapy with DAAs were observed especially in patients with normal baseline serum creatinine. Patients with CKD stage II-III experienced improvement in their kidney functions during and after end of therapy.

Study of the incidence of acute kidney injury in HCV infected patients receiving DAAs

Abstract Background Egypt had the highest burden of hepatitis C virus infection worldwide with genotype 4. The new direct-acting antivirals DAAs can target almost all steps of the HCV life cycle. The objective of our study was to detect AKI events during and on follow up after the end of treatment with combination DAAs. Aim of the work to study the incidence of acute kidney injury in HCV infected patients receiving IFN-free combination new DAAs. Patients and Methods A longitudinal prospective study that was conducted in the virology clinic in Kobry El-Koba Military Armed Forces Hospital on 63 male patients (&amp;gt;18years) who are HCV antibody and PCR positive, treatment-naïve, who received treatment with sofosbuvir, daclatasvir and ribavirin. They were divided into Group A: 33 patients with normal kidney functions stage I and Group B: 30 patients with chronic kidney disease (CKD) with estimated GFR (eGFR) &amp;gt;30 mL/min and &amp;lt; 90 mL/min stage II, III. The fluctuations in serum creatinine and eGFR were measured while on-therapy and for 3 months follow up after end of treatment. Results Sixty three male patients, treatment-naive were included. Thirty of them had eGFR of less than 90 mL/min/1.73 m2 (25 with eGFR = 60-89 mL/min) &amp; (5 with eGFR = 45-59 mL/min) of mean age 62.4 ± 7.4. These were compared to thirty-three patients with eGFR of more than 90 mL/min/1.73 m2 of mean age 53.8 ± 11 (P = 0.001). Our included treatment regimen was daclatasvir/sofosbuvir/RBV. A total of 54% had liver cirrhosis, 62/63 (98.5%) of the patients achieved SVR at 12th week; One CKD stage II patient continued treatment for 6 months because he had a detectable HCV RNA after 1st month of starting treatment. All other patients completed the treatment for 3 months. There is a statistical difference between different readings of serum creatinine in both groups on therapy and on follow up during the next 3 months after the end of therapy with a P value of 0.001 in group A and a P value of 0.005 in group B. There is a rise in serum creatinine in group A during the three months of therapy with slight improvement during follow up after the end of therapy without reaching the baseline. In group B it was noticed that there is slight increase in serum creatinine after 1st month of start of therapy then there is significant improvement after 3rd and 4th months of start of therapy to come to a plateau by the end of 6th month. The incidence of AKI was more observed in patients with eGFR &amp;gt; 90 ml/min per 1.73 m2 by being 13.65% by the end of treatment but decreases to 6.1% by the end of the three months follow up after the end of treatment. This was compared to 3.3% in those with eGFR &amp;lt; 90 ml/min per 1.73 m2 and &amp;gt;45 ml/min per 1.73 m2 that was the same all through the follow up, but of no statistical significant difference. Conclusion daclatasvir/sofosbuvir/ribavirin antiviral therapy is effective in patients with stage 1-3 CKD with SVR of 98.5%. Although adverse effects are common, serious adverse effects and treatment discontinuations are rare. There were AKI events during and after the end of therapy especially in patients with normal baseline serum creatinine by 12.76% in comparison to 3.3% in already CKD patients stage 2 and 3. Patients with CKD stage 2 and 3 experienced improvement in their kidney functions during and after end of therapy . Future studies are needed to determine predictors of kidney recovery with HCV eradication and confirm the long-term effects of HCV eradication on kidney function.

Treatment of chronic viral hepatitis C with direct acting antiviral agent: review

HCV infection treatment regimens are viewed from positions of HCV life cycle and replication, effects of NS3/4A protease inhibitors and NS5A/NS5B inhibitors on HCV strain replication. Evolution of HCV treatment regimens from its discovery to modern DAA agents had led to substantial progress although drug resistance poses a new issue to be addressed.

Serine 229 Balances the Hepatitis C Virus Nonstructural Protein NS5A between Hypo- and Hyperphosphorylated States.

The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle.IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.

Human apolipoprotein E promotes hepatitis B virus infection and production.

Hepatitis B virus (HBV) is a common cause of liver diseases, including chronic hepatitis, steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV chronically infects about 240 million people worldwide, posing a major global health problem. The current standard antiviral therapy effectively inhibits HBV replication but does not eliminate the virus unlike direct-acting antivirals (DAA) for curing hepatitis C. Our previous studies have demonstrated that human apolipoprotein E (apoE) plays important roles in hepatitis C virus infection and morphogenesis. In the present study, we have found that apoE is also associated with HBV and is required for efficient HBV infection. An apoE-specific monoclonal antibody was able to capture HBV similar to anti-HBs. More importantly, apoE monoclonal antibody could effectively block HBV infection, resulting in a greater than 90% reduction of HBV infectivity. Likewise, silencing of apoE expression or knockout of apoE gene by CRISPR/Cas9 resulted in a greater than 90% reduction of HBV infection and more than 80% decrease of HBV production, which could be fully restored by ectopic apoE expression. However, apoE silencing or knockout did not significantly affect HBV DNA replication or the production of nonenveloped (naked) nucleocapsids. These findings demonstrate that human apoE promotes HBV infection and production. We speculate that apoE may also play a role in persistent HBV infection by evading host immune response similar to its role in the HCV life cycle and pathogenesis. Inhibitors interfering with apoE biogenesis, secretion, and/or binding to receptors may serve as antivirals for elimination of chronic HBV infection.

Overview of HCV Life Cycle with a Special Focus on Current and Possible Future Antiviral Targets.

Hepatitis C infection is the leading cause of liver diseases worldwide and a major health concern that affects an estimated 3% of the global population. Novel therapies available since 2014 and 2017 are very efficient and the WHO considers HCV eradication possible by the year 2030. These treatments are based on the so-called direct acting antivirals (DAAs) that have been developed through research efforts by academia and industry since the 1990s. After a brief overview of the HCV life cycle, we describe here the functions of the different targets of current DAAs, the mode of action of these DAAs and potential future inhibitors.

Functional Characterization of Apolipoproteins in the HCV Life Cycle.

Apolipoprotein E (apoE) plays dual functions in the HCV life cycle by promoting HCV infection and virion assembly and production. ApoE is a structural component on the HCV envelope. It mediates HCV cell attachment through specific interactions with the cell surface receptors such as syndecan-1 (SDC-1) and SDC-2 heparan sulfate proteoglycans (HSPGs). It also interacts with NS5A and E2, resulting in an enhancement of HCV morphogenesis. It can bind HCV extracellularly and promotes HCV infection. It is critical for HCV cell-to-cell transmission and may alsoplay a role in HCV persistence by interfering with the action of HCV-neutralizing antibodies. Other apolipoproteins particularly apoB and apoC1 were also found on the HCV envelope, but their roles in the HCV life cycle remain unclear. In the last decade, a number of genomic, immunological, structural, and cell biology methodologies have been developed and used for determining the importance of apoE in the HCV life cycle. These methods and protocols will continue to be valuable to further understand the importance and the underlying molecular mechanism of various apolipoproteins in HCV infection and pathogenesis.

Harzianoic acids A and B, new natural scaffolds with inhibitory effects against hepatitis C virus.

Two new sesquiterpene-based analogues, namely harzianoic acids A (1) and B (2), were isolated from a sponge-associated fungus Trichoderma harzianum. Their structures were determined on the basis of the extensive spectroscopic analyses in association with the ECD data for the configurational assignment. Harzianoic acids A and B were structurally characterized as a sesquiterpene and a norsesquiterpene with a cyclobutane nucleus, which is uncommonly found from nature. Both compounds exhibited the inhibitory activity to reduce the HCV RNA levels with low cytotoxicity. The preliminary investigation of the mode of action revealed that the compounds blocked the entry step in the HCV life cycle, while the viral E1/E2 and the host cell CD81 were the potential target proteins.