JFH1 Virus Research Articles

Antiviral Activities of Curcuma Genus against Hepatitis C Virus

Hepatitis C virus (HCV) infection is one of the major public health problems in the world. Even though the new agents are shown to increase the sustained virology response, however, there are still many people who cannot access the therapy due to the high cost. Moreover, the emergence of resistance and side effects presented the necessity to develop alternative treatment agents for HCV infection. Plants of the genus of curcuma are popular among traditional medicines in the world, including Indonesia. They have been used for many herb remedies and reported to possess many biological activities. Several plants from the curcuma genus were known as treatment agents in liver disease and jaundice. Our current study determines antiviral activities of Curcuma domestica, Curcuma xanthorrhiza, and Curcuma heyneana against HCV and further examines the mechanism of actions. Antiviral activity was performed by in vitro culture cells using Huh 7.5it cells and treated with the mixture of extract and virus JFH1. The effects of extracts in HCV life cycle were determined by mode of action analysis to examine the action of substances in the entry or post entry steps. The results revealed that ethanol extract of C. domestica, C. xanthorrhiza, and C. heyneana showed strong anti-HCV activities with IC50 values of 1.68 ± 0.05, 4.93 ± 0.42 and 5.49 ± 0.59 μg/mL, respectively without any cytotoxicity effect. Mode of action analysis demonstrated that of C. domestica and C. heyneana exhibit HCV in the entry step, while C. xanthorrhiza inhibit in the entry and post entry steps of HCV life cycle. Docking analysis to predict the interaction of curcumin, the main compound of curcuma genus, revealed a strong interaction between curcumin and 4GAG receptor, a protein involved in the entry step of HCV infection. Moreover, it was also reported to possess good interaction with 4EAW, an HCV NS5B, which plays an important role in HCV replication. These results suggested that C. domestica, C. xanthorrhiza, and C. Heyneana possessed strong inhibition against hepatitis C virus, therefore they may be good candidates for anti-HCV agents.

Alcohol attenuates anti‐HCV function of IFN‐λ1 through up‐regulation of PLASy expression in human hepatic cells

Alcohol could compromise the anti-hepatitis C virus (HCV) function of interferon-alpha (IFN-α). However, little information is available about the effect of alcohol on interferon-lambda (IFN-λ, type III IFN), a novel candidate for development of therapy for HCV infection. Huh7 cells were infected with HCV JFH-1 virus, then treated with alcohol, and/or IFN-λ1. RT-PCR and Western blot were used to detect the levels of HCV and key cellular factors. Overexpression or silencing expression was performed to verify the role of key factors in alcohol-attenuated anti-HCV function of IFN-λ1. Alcohol treatment compromised anti-HCV effect of IFN-λ1 in HCV JFH-1-infected Huh7 cells, evidenced by the significantly increased levels of HCV RNA, and HCV core protein in alcohol-/IFN-λ1-treated cells compared to cells with IFN-λ1 treatment alone. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol enhanced the expression of protein inhibitor of activated STAT (PIASy). Overexpression of PIASy compromised anti-HCV ability of IFN-λ1, whereas silencing expression of PIASy partly restored the alcohol-attenuated anti-HCV effect of IFN-λ1. More importantly, overexpression of PIASy significantly down-regulated the level of IFN-λ1-indcued phosphorylation of STAT1 (p-STAT1), an important adaptor in IFN-λ pathway, as well as reduced the expression of IFN-λ1-induced IFN-stimulated genes 56 (ISG56), and myxovirus resistance 1 (Mx1), two antiviral effectors in in IFN-λ pathway. These findings indicate that alcohol, through inducing the expression of negative regulator in IFN-λ pathway, inhibits IFN-λ-mediated anti-HCV action in human hepatic cells, which may lead to the poor efficacy of IFN-λ-based therapy against HCV infection.

17,β-estradiol inhibits hepatitis C virus mainly by interference with the release phase of its life cycle.

Background & AimsOestrogen and oestrogen‐mediated signalling protect from hepatitis C virus through incompletely understood mechanisms. We aimed to ascertain which phase(s) of hepatitis C virus life cycle is/are affected by oestrogens.MethodsHuh7 cells infected with the JFH1 virus (genotype 2a) were exposed to dehydroepiandrosterone, testosterone, progesterone and 17β‐estradiol (tested with/without its receptor antagonist fulvestrant). Dose–response curves were established to calculate half maximal inhibitory concentration values. To dissect how 17β‐estradiol interferes with phases of hepatitis C virus life cycle, its effects were measured on the hepatitis C virus pseudo‐particle system (viral entry), the subgenomic replicon N17/JFH1 and the replicon cell line Huh7‐J17 (viral replication). Finally, in a dual‐step infection model, infectious supernatants, collected from infected cells exposed to hormones, were used to infect naïve cells.ResultsProgesterone and testosterone showed no inhibitory effect on hepatitis C virus; dehydroepiandrosterone was only mildly inhibitory. In contrast, 17β‐estradiol inhibited infection by 64%‐67% (IC 50 values 140‐160 nmol/L). Fulvestrant reverted the inhibition by 17β‐estradiol in a dose‐dependent manner. 17β‐estradiol exerted only a slight inhibition (<20%) on hepatitis C virus pseudo‐particles, and had no effect on cells either transiently or stably (Huh7‐J17 cells) expressing the N17/JFH1 replicon. In the dual‐step infection model, a significant half maximal inhibitory concentration decline occurred between primary (134 nmol/L) and secondary (100 nmol/L) infections (P=.02), with extracellular hepatitis C virus RNA and infectivity being reduced to a higher degree in comparison to its intracellular counterpart.Conclusions17β‐estradiol inhibits hepatitis C virus acting through its intracellular receptors, mainly interfering with late phases (assembly/release) of the hepatitis C virus life cycle.

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region.

BackgroundThe E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions.ObjectivesThe aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it.MethodsSpecific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequencedResultsQuantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture.ConclusionsReal-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines.

Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15–20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.

Hepatitis C Virus NS4B Can Suppress STING Accumulation To Evade Innate Immune Responses.

The cyclic dinucleotide 2',3'-cGAMP can bind the adaptor protein STING (stimulator of interferon [IFN] genes) to activate the production of type I IFNs and proinflammatory cytokines. We found that cGAMP added to the culture medium could suppress the replication of the hepatitis C virus (HCV) genotype 1b strain Con1 subgenomic replicon in human hepatoma cells. Knockdown of STING expression diminished the inhibitory effect on replicon replication, while overexpression of STING enhanced the inhibitory effects of cGAMP. The addition of cGAMP into 1b/Con1 replicon cells significantly increased the expression of type I IFNs and antiviral interferon-stimulated genes. Unexpectedly, replication of the genotype 2a JFH1 replicon and infectious JFH1 virus was less sensitive to the inhibitory effect of cGAMP than was that of 1b/Con1 replicon. Using chimeric replicons, 2a NS4B was identified to confer resistance to cGAMP. Transient expression of 2a NS4B resulted in a pronounced inhibitory effect on STING-mediated beta IFN (IFN-β) reporter activation compared to that of 1b NS4B. 2a NS4B was found to suppress STING accumulation in a dose-dependent manner. The predicted transmembrane domain of 2a NS4B was required to inhibit STING accumulation. These results demonstrate a novel genotype-specific inhibition of the STING-mediated host antiviral immune response. The cyclic dinucleotide cGAMP was found to potently inhibit the replication of HCV genotype 1b Con1 replicon but was less effective for the 2a/JFH1 replicon and infectious JFH1 virus. The predicted transmembrane domain in 2a NS4B was shown to be responsible for the decreased sensitivity to cGAMP. The N terminus of NS4B has been reported to suppress STING-mediated signaling by disrupting the interaction of STING and TBK1 and/or MAVS. We show that 2a/JFH1 NS4B has an additional mechanism to evade STING signaling through suppressing STING accumulation.

Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro

Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50=2.3μM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3∗43 (EC50=4.0μM), 3∗20 (EC50=8.2μM) and 5∗362 (EC50=38.9μM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.

Casein kinase II inhibitor enhances production of infectious genotype 1a hepatitis C virus (H77S).

Genotype 2a JFH1 virus has substantially contributed to the progress of HCV biology by allowing entire viral life cycle of HCV in cell culture. Using this genotype 2a virus, casein kinase II (CKII) was previously identified as a crucial host factor in virus assembly by phosphorylating NS5A. Since most of the prior studies employed genotype 2a JFH1 or JFH1-based intragenotypic chimera, we used genotype 1a H77S to study virus assembly. CKII inhibition by chemical inhibitors enhanced H77S virus production in contrast to that of JFH1 virus, but genetic inhibition of CKII by siRNA did not change H77S virus titer significantly. The different outcomes from these two approaches of CKII inhibition suggested that nonspecific target kinase of CKII inhibitors plays a role in increasing H77S virus production and both viral and host factors were investigated in this study. Our results emphasize substantial differences among the HCV genotypes that should be considered in both basic research and clinical practices.

2-Alkyloxazoles as potent and selective PI4KIIIβ inhibitors demonstrating inhibition of HCV replication

Synthesis and SAR of 2-alkyloxazoles as class III phosphatidylinositol-4-kinase beta (PI4KIIIβ) inhibitors is described. These compounds demonstrate that inhibition of PI4KIIIβ leads to potent inhibition of HCV replication as observed in genotype (GT) 1a and 1b replicon and GT2a JFH1 virus assays in vitro.

Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection.

The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.

Phenylboronic-Acid-Modified Nanoparticles: Potential Antiviral Therapeutics

Phenylboronic-acid-modified nanoparticles (NPs) are attracting considerable attention for biological and biomedical applications. We describe here a convenient and general protocol for attaching multiple copies of para-substituted phenylboronic acid moieties onto either iron-oxide-, silica- or diamond-derived NPs. The boronic acid functionalized NPs are all fabricated by first modifying the surface of each particle type with 4-azidobenzoic ester functions. These azide-terminated nanostructures were then reacted with 4-[1-oxo-4-pentyn-1-yl) amino]phenylboronic acid units via a Cu(I) catalyzed Huisgen cycloaddition to furnish, conveniently, the corresponding boronic-acid modified NPs (or "borono-lectins") targeted in this work. The potential of these novel "borono-lectins" as antiviral inhibitors was investigated against the Hepatitis C virus (HCV) exploiting a bioassay that measures the potential of drugs to interfere with the ability of cell-culture-derived JFH1 virus particles to infect healthy hepatocytes. As far as we are aware, this is the first report that describes NP-derived viral entry inhibitors and thus serves as a "proof-of-concept" study. The novel viral entry activity demonstrated, and the fact that the described boronic-acid-functionalized NPs all display much reduced cellular toxicities compared with alternate NPs, sets the stage for their further investigation. The data supports that NP-derived borono-lectins should be pursued as a potential therapeutic strategy for blocking viral entry of HCV.

Safety and Antiviral Activity of the HCV Entry Inhibitor ITX5061 in Treatment-Naive HCV-Infected Adults: A Randomized, Double-Blind, Phase 1b Study

Hepatitis C virus (HCV) entry involves scavenger receptor B1 (SRB1). In vitro, SRB1 inhibition by ITX5061 impedes HCV replication. Multicenter study to assess safety/activity of ITX5061 in previously untreated, noncirrhotic, HCV genotype 1 infected adults. Design included sequential cohorts of 10 subjects with ITX5061 (n = 8) or placebo (n = 2) to escalate duration (3 to 14 to 28 days) or deescalate dose (150 to 75 to 25 mg) based on predefined criteria for safety and activity (≥ 4 of 8 subjects with HCV RNA decline ≥ 1 log10 IU/mL). Thirty subjects enrolled in 3 cohorts: ITX5061 150 mg/day by mouth for 3 (A150), 14 (B150), and 28 (C150) days. Six subjects had grade ≥ 3 adverse events (one in placebo); none were treatment related. One of the 7 C150 subjects (14.3%, 95% confidence interval [CI], .7%-55.4%) had ≥ 1 log10 IU/mL decline in HCV RNA (1.49 log10 IU/mL), whereas none of the 6 placebo, 8 A150 or 8 B150 subjects showed such decline. Oral ITX5061 150 mg/day for up to 28 days was safe and well tolerated. In the 28-day cohort, 1 of 7 subjects showed antiviral activity; however, predefined criteria for antiviral activity were not met at the doses and durations studied.

Sa1029 An Analysis of Response Rates by Fibrosis Stage in Patients Treated With Faldaprevir, Bi 207127 and Ribavirin in the Sound-C2 Study

Background: A high risk of chronicity is the major concern of HCV infection and chronic infection often leads to liver cirrhosis and hepatocellular carcinoma. Although proportion of patients achieving a sustained virological response has been increased by the introduction of combination therapy of pegylated-IFN-alpha and ribavirin, still half of the patients exhibits no response to the therapy. One of the mechanisms for the establishment of persistent infection of HCV is the escape from the host immune system. To eliminate HCV from the hepatocytes of the patients without possible cytotoxity due to an over induction of host immunity, we generated a therapeutic construct, cMR3, by which type I IFN is selectively induced in HCV infected cells. Methods: The cMR3 is composed of the N-terminal part of the interferon regulatory factor 7 (IRF7) possessing a dominant active function, the sequences specifically cleaved by HCV NS3/4A protease and an ER anchor, and exhibits a potent IFN inducing activity in HCV infected cells. After cleavage by the HCV protease, the processed cMR3 migrates into the nucleus and activates various IFN promoters including IFN-alpha6, IFN beta, and IFN stimulated response element. Furthermore, a recombinant baculovirus cMR3expressing vector was constructed. Results: The specific activation of the IFN promoters was observed in both HCV replicon cells and JFH1 virus infected cells upon introduction of the cMR3 but not in cells infected with JEV or DENV. Expression of viral protein and viral RNA replication were also impaired by the introduction of cMR3 into the HCV replicon cells. Furthermore, the recombinant baculovirus can delivery cMR3 into both HCV replicon cells and JFH1 virus infected cells efficiently. And the specific activation of the IFN promoters and suppression of viral replication was also observed. Conclusions: These results suggest that the selective expression of type I IFN in the hepatocytes infected with HCV by the introduction of the cMR3 might be feasible to eliminate HCV from the chronic hepatitis C patients without liver damage and baculoviral cMR3expressing vectors have the potential for therapeutic use against HCV infection.

Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy

Hepatitis C virus (HCV) is able to induce autophagy via endoplasmic reticulum (ER) stress, but the exact molecular signaling pathway is not well understood. We found that the activity of the mechanistic target of rapamycin complex 1 (MTORC1) was inhibited in Huh7 cells either harboring HCV-N (genotype 1b) full-genomic replicon or infected with JFH1 (genotype 2a) virus, which led to the activation of UNC-51-like kinase 1 (ULK1) and thus to autophagy. We then analyzed activity upstream of MTORC1, and found that both protein kinase, AMP-activated, α (PRKAA, including PRKAA1 and PRKAA2, also known as AMP-activated protein kinase, AMPKα) and AKT (refers to pan AKT, including three isoforms of AKT1-3, also known as protein kinase B, PKB) were inhibited by HCV infection. The inhibition of the AKT-TSC-MTORC1 pathway contributed to upregulating autophagy, but inhibition of PRKAA downregulated autophagy. The net effect on autophagy was from AKT, which overrode the inhibition effect from PRKAA. It was further found that HCV-induced ER stress was responsible for the inhibition of the AKT pathway. Metformin, a PRKAA agonist, inhibited HCV replication not only by activating PRKAA as previously reported, but also by activating AKT independently of the autophagy pathway. Taken together, our data suggested HCV inhibited the AKT-TSC-MTORC1 pathway via ER stress, resulting in autophagy, which may contribute to the establishment of the HCV-induced autophagy.

Oleanolic acid and ursolic acid: Novel hepatitis C virus antivirals that inhibit NS5B activity

Hepatitis C virus (HCV) infects up to 170 million people worldwide and causes significant morbidity and mortality. Unfortunately, current therapy is only curative in approximately 50% of HCV patients and has adverse side effects, which warrants the need to develop novel and effective antivirals against HCV. We have previously reported that the Chinese herb Fructus Ligustri Lucidi (FLL) directly inhibited HCV NS5B RNA-dependent RNA polymerase (RdRp) activity (Kong et al., 2007). In this study, we found that the FLL aqueous extract strongly suppressed HCV replication. Further high-performance liquid chromatography (HPLC) analysis combined with inhibitory assays indicates that oleanolic acid and ursolic acid are two antiviral components within FLL aqueous extract that significantly suppressed the replication of HCV genotype 1b replicon and HCV genotype 2a JFH1 virus. Moreover, oleanolic acid and ursolic acid exhibited anti-HCV activity at least partly through suppressing HCV NS5B RdRp activity as noncompetitive inhibitors. Therefore, our results for the first time demonstrated that natural products oleanolic acid and ursolic acid could be used as potential HCV antivirals that can be applied to clinic trials either as monotherapy or in combination with other HCV antivirals.

The C terminus of NS5A domain II is a key determinant of hepatitis C virus genome replication, but is not required for virion assembly and release.

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has been reported previously that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production; however, deletion of the entire domain II eliminated genome replication, pointing to a key role for the C terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and a JFH-1-derived subgenomic replicon. We showed that 12 of these residues were absolutely required for virus genome replication, whilst mutations of the remainder either had no phenotype or exhibited a partial reduction in genome replication. There was an absolute correlation between the datasets for virus and subgenomic replicon, indicating that this region is involved solely in the process of genome replication. Comparison of our data with a previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.

Antiviral activities of ISG20 against hepatitis C virus

To investigate the impact of interferon-stimulated exonuclease 20 kDa (ISG20) on replication of genotype 2a hepatitis C virus (HCV) subgenomic replicon RNA and infectivity of the cell culture-derived HCV strain JFH1 to determine the potential of exogenously expressed ISG20 as an anti-viral therapy of chronic hepatitis C. Plasma vectors containing wild-type (WT) ISG20 or a catalytically-inactive mutant ISG20m were transiently transfected into Huh7, Huh7.5 and HEK293 cells, and the replication of a monocistronic subgenomic JFH1 RNA replicon, SGRm-JFH1BlaRL, was measured. Huh7.5 cells stably expressing ISG20, ISG20m, or the control vector were established by transducing replication incompetent pCX4-Bsr-myc retroviruses encoding WT ISG20, D94G mutant ISG20, or the empty vector, respectively, and selecting with 5 mug/mL of blasticidin for approximately three weeks. The stable Huh7.5 cells were then transfected with HCV replicon RNA and infected with cell culture-derived HCV to investigate inhibition capacity of ISG20 against HCV. Huh7.5-ISG20, Huh7.5-ISG20m, and Huh7.5-Bsr controls cells stably expressing ISG20, ISG20m, or the control vector, respectively, were constructed successfully; the ectopically expressed ISG20 and ISG20m were distributed in both nucleus and cytoplasm, as detected by immuno uorescence. SGRm-JFH1BlaRL replicated efficiently and with similar kinetics in the Huh7.5-Bsr and Huh7.5-ISG20m cells, with expression levels plateauing at 48-96 h post-transfection. In contrast, at all time points examined, SGRm-JFH1BlaRL replication was 9.1% to 16.7% in the Huh7.5-ISG20 cells. The Huh7, Huh7.5 and HEK293 cells transiently expressing ISG20 also showed 16.7% to 25.0% of HCV replication that the respective controls. In addition, the amount of infectious progeny JFH1 virus released in culture supernatants was 9.1% to 12.5% from the Huh7.5-ISG20 cells than from the Huh7.5-Bsr and Huh7.5-ISG20m cells at 48-72 h post-infection, and the latter two cultures produced similar JFH1 virus yields. Finally, the expression of HCV core protein was also lower in the Huh7.5-ISG20 cells, as detected by immunoblot analysis. Exogenous expression of ISG20, either in a transient or stable manner, suppresses not only replication of genotype 2a HCV RNA replicons but also JFH1 virus propagation in cultured hepatocytes. The exonuclease activity of ISG20 is required for its antiviral activities against HCV.

The Arg719 residue at the C-terminal end of the stem region in hepatitis C virus JFH-1 E2 glycoprotein promotes viral infection

Hepatitis C virus (HCV) envelope glycoprotein E2 is involved in virus assembly and initial entry into host cells. The tertiary organization of the E2 ectodomain is mainly composed of domains I–III, followed by the stem (ST) region and transmembrane (TM) domain. The ST region is critical for reorganizing the envelope glycoproteins during the membrane fusion process. While this region is relatively flexible, the physicochemical properties of its amino acid residues are conserved. Whether and how this physicochemical conservation is required for HCV infection is still unclear. The last residue of the E2 ST region evolved to be either an arginine or lysine among different HCV strains, suggesting that the residues confer different functions during HCV infection. To address this possibility, we constructed an R719K point mutant in the JFH-1 strain (genotype 2a) in the context of the cell-culture derived HCV (HCVcc) system. Compared with wild-type (wt) HCV, the R719K mutant exhibited decreased growth, and its extracellular and intracellular infectivity were also significantly decreased at 48 and 72h post-electroporation. Correspondingly, less RNA and HCV core protein was observed in the supernatant for the R719K mutant, as well as less efficient RNA replication and protein expression. These findings indicate that the 719th residue of arginine on E2 is critical to promote HCV replication and infection. The data provide new clues for the biochemical function of E2, which is required for efficient HCV assembly and infection.

A laboratory-adapted HCV JFH-1 strain is sensitive to neutralization and can gradually escape under the selection pressure of neutralizing human plasma

Viral replication and neutralization of hepatitis C viruses (HCV) have been studied using the infectious molecular clone JFH-1. By passaging JFH-1 in hepatoma cells in the absence or presence of HCV neutralizing antibodies (nAbs), we investigated the molecular mechanisms of cell-culture adaptation and sensitivity to nAbs. The cell culture-adapted JFH-1 virus (JFH-1-CA) became more sensitive to nAbs than its parental virus. Sequence analysis revealed that the predominant viruses in the JFH-1-CA population carried two mutations in their envelopes (I414T and V293A). Plasma that could neutralize JFH-1-CA was found in 2 of 7 HCV-infected individuals who have cleared the virus in blood. Plasma 226233 with a higher 50% neutralization titer was used for in vitro selection of neutralization resistant viruses. Under the increasing selection pressure of plasma 226233, the neutralizing sensitivity of JFH-1-CA decreased gradually. Two mutations (T414I and P500S) in envelope were found in all but one sequenced clones in the viral population after eight rounds of selection. Interestingly, the cell-culture adapted mutation I414T reverted back to the wild-type residue (I414) under the selection pressure. By introducing mutations at positions 414 and 500 into the JFH-1 clone, we confirmed that the T414I mutation alone can confer neutralization resistance. The results of this current study suggest that nAbs are present in a subset of HCV-infected individuals who have cleared the virus in blood. Our data also provide the first evidence that, the E2 residue P500, located within a previously identified highly conserved polyclonal epitope, may be a target for neutralizing antibodies present in individual who have spontaneously resolved the HCV infection.

Hepatitis C Virus-Induced Autophagy Is Independent of the Unfolded Protein Response

Hepatitis C virus (HCV) has been shown to induce autophagy and the unfolded protein response (UPR), but the mechanistic link between the induction of these two cellular processes remains unclear. We demonstrate here that HCV infection induces autophagy, as judged by accumulation of lipidated LC3-II, and that this induction occurs rapidly after infection, preceding the stimulation of the UPR, which occurs only at later stages, after the viral envelope glycoproteins have been expressed to high levels. Furthermore, both genotype 1b and 2a subgenomic replicons expressing nonstructural (NS3-5B) proteins and JFH-1 virus lacking the envelope glycoproteins potently induced autophagy in the absence of detectable UPR. This ability was also shared by a subgenomic replicon derived from the related GB virus B (GBV-B). We also show that small interfering RNA (siRNA)-mediated silencing of the key UPR inducer, Ire1, has no effect on HCV genome replication or the induction of autophagy, further demonstrating that the UPR is not required for these processes. Lastly, we demonstrate that the HCV replicase does not colocalize with autophagosomes, suggesting that the induction of autophagy is not required to generate the membrane platform for HCV RNA replication.