Promotes Hepatitis C Virus Replication Research Articles

Location specific annealing of miR-122 and other small RNAs defines an Hepatitis C Virus 5' UTR regulatory element with distinct impacts on virus translation and genome stability.

Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5′ untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1–44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19–37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.

Effect of P-body component Mov10 on HCV virus production and infectivity.

Mov10 is a processing body (P-body) protein and an interferon-stimulated gene that can affect replication of retroviruses, hepatitis B virus, and hepatitis C virus (HCV). The mechanism of HCV inhibition by Mov10 is unknown. Here, we investigate the effect of Mov10 on HCV infection and determine the virus life cycle steps affected by changes in Mov10 overexpression. Mov10 overexpression suppresses HCV RNA in both infectious virus and subgenomic replicon systems. Additionally, Mov10 overexpression decreases the infectivity of released virus, unlike control P-body protein DCP1a that has no effect on HCV RNA production or infectivity of progeny virus. Confocal imaging of uninfected cells shows endogenous Mov10 localized at P-bodies. However, in HCV-infected cells, Mov10 localizes in circular structures surrounding cytoplasmic lipid droplets with NS5A and core protein. Mutagenesis experiments show that the RNA binding activity of Mov10 is required for HCV inhibition, while its P-body localization, helicase, and ATP-binding functions are not required. Unexpectedly, endogenous Mov10 promotes HCV replication, as CRISPR-Cas9-based Mov10 depletion decreases HCV replication and infection levels. Our data reveal an important and complex role for Mov10 in HCV replication, which can be perturbed by excess or insufficient Mov10.

Sam68 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 2 of Viral 5' Untranslated Region.

The Src-associated in mitosis 68-kDa (Sam68) protein is a highly conserved nuclear protein and is involved in a series of cellular processes, including transcription and signal transduction. Sam68 is comprised of 443 amino acids and contains an RGG box domain, a KH domain, and a tyrosine-rich domain. Its role in hepatitis C virus (HCV) replication is unknown. Here, we find that Sam68 promotes HCV replication without affecting viral translation. The RNA immunoprecipitation experiments show that the positive strand of HCV RNA interacts with Sam68. HCV infection triggers the translocation of the Sam68 protein from the nucleus to the cytoplasm, where it interacts with the HCV genome. Further study shows that the region of Sam68 spanning amino acids 1 to 157 is the pivotal domain to interact with the stem-loop 2 of the HCV 5' untranslated region (5' UTR) and is responsible for the enhancement of HCV replication. These data suggested that Sam68 may serve as a proviral factor of HCV to facilitate viral replication through interaction with the viral genome.IMPORTANCE Hepatitis C virus (HCV) is a member of the Flaviviridae family, and its infection causes chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma. No vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. This study discloses a new host factor that binds to the HCV 5' UTR and promotes HCV replication. Sam68 may play an important role in HCV-related diseases, and further investigation is highly encouraged to explore its specific actions in HCV pathogenesis.

Activation of the oncogenic miR-21-5p promotes HCV replication and steatosis induced by the viral core 3a protein.

miR-21-5p is a potent oncogenic microRNA targeting many key tumour suppressors including phosphatase and tensin homolog (PTEN). We recently identified PTEN as a key factor modulated by hepatitis C virus (HCV) to promote virion egress. In hepatocytes, expression of HCV-3a core protein was sufficient to downregulate PTEN and to trigger lipid droplet accumulation. Here, we investigated whether HCV controls PTEN expression through miR-21-5p-dependent mechanisms to trigger steatosis in hepatocytes and to promote HCV life cycle. MiR-21-5p expression in HCV-infected patients was evaluated by transcriptome meta-analysis. HCV replication and viral particle production were investigated in Jc1-infected Huh-7 cells after miR-21-5p inhibition. PTEN expression and steatosis were assessed in HCV-3a core protein-expressing Huh-7 cells and in mouse primary hepatocytes having miR-21-5p inhibited or genetically deleted respectively. HCV-3a core-induced steatosis was assessed in vivo in Mir21a knockout mice. MiR-21-5p expression was significantly increased in hepatic tissues from HCV-infected patients. Infection by HCV-Jc1, or transduction with HCV-3a core, upregulated miR-21-5p expression and/or activity in Huh-7 cells. miR-21-5p inhibition decreased HCV replication and release of infectious virions by Huh-7 cells. HCV-3a core-induced PTEN downregulation and steatosis were further prevented in Huh-7 cells following miR-21-5p inhibition or in Mir21a knockout mouse primary hepatocytes. Finally, steatosis induction by AAV8-mediated HCV-3a core expression was reduced in vivo in Mir21a knockout mice. MiR-21-5p activation by HCV is a key molecular step, promoting both HCV life cycle and HCV-3a core-induced steatosis and may be among the molecular changes induced by HCV-3a to promote carcinogenesis.

TRIM27 Promotes Hepatitis C Virus Replication by Suppressing Type I Interferon Response.

Type I interferon (IFN) response is central for host defense against viral infection. Tripartite motif 27 (TRIM27) is implicated in antiviral innate immune response; however, whether it affects the replication of hepatitis C virus (HCV) and the underlying mechanisms remain uncharacterized. Here, we show that TRIM27 expression is induced in Huh7.5 human hepatoma cells infected with HCV or stimulated with type I IFNs in vitro. In addition, TRIM27 overexpression increases and its knockdown decreases viral RNA and protein levels, suggesting that TRIM27 positively regulates HCV replication. Mechanistically, TRIM27 inhibits type I IFN response against HCV infection through inhibiting IRF3 and NF-κB pathways, since TRIM27 mutant unable to inhibit these two inflammatory pathways fails to promote HCV replication. Taken together, this study identifies TRIM27 as a novel positive regulator of HCV replication, and also implicates that targeting TRIM27 may serve as a therapeutic strategy for controlling HCV replication.

Alcohol-induced autophagy via upregulation of PIASy promotes HCV replication in human hepatoma cells

Both alcohol and hepatitis C virus (HCV) infection could induce cellular autophagy in liver cells, which is considered to be essential for productive HCV replication. However, whether alcohol-induced autophagy is involved in the pathogenesis of HCV infection is still poorly understood. Alcohol treatment could induce autophagy in Huh7 cells (a hepatoma cell line that supports HCV JFH-1 replication), evidenced by the increase of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the decrease of p62 level in alcohol-treated cells compared with control cells. Alcohol treatment also significantly increased PIASy (a member of the PIAS family) expression, which can act as a SUMO (small ubiquitin-like modifier protein) E3 ligase to regulate a broader range of cellular processes including autophagy. Overexpression or the silencing expression of PIASy in alcohol-treated Huh7 cells could increase or decrease autophagic activation caused by alcohol treatment, respectively, and thus affect HCV replication correspondingly. In the absence of alcohol, overexpression or silencing expression of PIASy increase or decrease the level of cellular autophagy, judged by the changes of LC3B-II and p62 levels in the presence or absence of chloroquine (CQ), a lysosome inhibitor. More importantly, in the presence of 3-methyladenine (3-MA), an inhibitor in the early stage of autophagy, the effects of overexpression or silencing expression of PIASy on HCV replication were largely blocked. Furthermore, PIASy could selectively drive the accumulation of SUMO1-conjugated proteins, along with upregulation of the expression of several important autophagy factors, including ATG7 and ATG5–ATG12. In conclusion, alcohol promotes HCV replication through activation of autophagy in Huh7 cells, which partly attributes to its induction of PIASy expression. PIASy-enhanced accumulation of SUMO1-conjugated proteins may contribute to its inducing effect of autophagy. Our findings provide a novel mechanism for the action of alcohol-promoting HCV replication in the context of cellular autophagy.

Establishment and Application of a High Throughput Screening System Targeting the Interaction between HCV Internal Ribosome Entry Site and Human Eukaryotic Translation Initiation Factor 3.

Viruses are intracellular obligate parasites and the host cellular machinery is usually recruited for their replication. Human eukaryotic translation initiation factor 3 (eIF3) could be directly recruited by the hepatitis C virus (HCV) internal ribosome entry site (IRES) to promote the translation of viral proteins. In this study, we establish a fluorescence polarization (FP) based high throughput screening (HTS) system targeting the interaction between HCV IRES and eIF3. By screening a total of 894 compounds with this HTS system, two compounds (Mucl39526 and NP39) are found to disturb the interaction between HCV IRES and eIF3. And these two compounds are further demonstrated to inhibit the HCV IRES-dependent translation in vitro. Thus, this HTS system is functional to screen the potential HCV replication inhibitors targeting human eIF3, which is helpful to overcome the problem of viral resistance. Surprisingly, one compound HP-3, a kind of oxytocin antagonist, is discovered to significantly enhance the interaction between HCV IRES and eIF3 by this HTS system. HP-3 is demonstrated to directly interact with HCV IRES and promote the HCV IRES-dependent translation both in vitro and in vivo, which strongly suggests that HP-3 has potentials to promote HCV replication. Therefore, this HTS system is also useful to screen the potential HCV replication enhancers, which is meaningful for understanding the viral replication and screening novel antiviral drugs. To our knowledge, this is the first HTS system targeting the interaction between eIF3 and HCV IRES, which could be applied to screen both potential HCV replication inhibitors and enhancers.

Studies on interaction between host thioredoxin-interacting protein and hepatitis C virus infection

Objective To explore the relationship between host cell thioredoxin-interacting protein (TXNIP) and hepatitis C virus (HCV) replication, as well as the mechanism of how TXNIP promotes HCV replication. Methods The transcriptome shift of Huh7 cells after cell cultrued HCV (HCVcc) infection was analyzed by using Glue Grant Human Transcriptome Microarray. The differential expression of TXNIP was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput sequencing. The effect of TXNIP on HCV infection was tested by TXNIP overexpression and TXNIP knockdown. Results Transcriptome array demonstrated a 3.7 times fold upregulation of TXNIP mRNA in HCVcc infected Huh7 cell higher than those without HCVcc infection, which was further verified by high-throughput deep sequencing. The TXNIP mRNA level was significantly up-regulated at 36 h and 60 h post HCV infection (t=24.90 and 8.27, respectively, both P<0.01). In cell level analysis, knockdown of TXNIP by small interfering RNA (siRNA) could reduce HCV infection, while TXNIP overexpression could enhance HCV replication. Infection with HCVcc could increase the expression of TXNIP in Huh7 but not in Huh7.5.1 cells, and interferons α, β, and γ treatment could up-regulate TXNIP expressions in both cell lines. Conclusions HCV infection induces host cell TXNIP expression, and upregulated TXNIP enhances HCV replication. Host cell TXNIP plays an important role in HCV replication, infection and immune escape. Key words: Hepacivirus; Thioredoxin-interacting protein; Interferons

HIV-1 Vpr increases HCV replication through VprBP in cell culture

Coinfection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs at a high frequency, in which HIV shows a promotion of HCV-derived liver diseases. However, the mechanism of how this occurs is not well understood. Our previous work has demonstrated that the HIV-1 accessory protein Vpr enhances HCV RNA replication in cell culture. Because Vpr performs most of its functions through host protein VprBP (DCAF1), the role of VprBP in the regulation of HCV by Vpr was investigated in this study. We found that the Vpr mutant Q65R, which is deficient in VprBP binding, could not enhance HCV replication. Furthermore, Vpr-mediated enhancement of HCV replication was severely diminished in VprBP knockdown cells. In addition, an inhibitor of Cullin RING E3 ligases, MLN4924, impaired the function of Vpr during HCV replication. Together, these results suggest that Vpr promotes HCV replication in a VprBP-dependent manner, and that the activity of Cullin RING E3 ligases is essential to this process. In conclusion, our findings demonstrate that HIV-1 Vpr makes the cellular environment more suitable for HCV replication, which might relate with the host ubiquitination system.

Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells.

Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3′ NTR and domain IIId of the HCV-IRES in the 5′ NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.

Hepatitis C virus addiction to liver miR-122 takes its Toll on the host.

Hepatitis C virus addiction to liver miR-122 takes its Toll on the host.

FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis. It is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with the CHC background suggests that FBP1 has an important role in promoting HCV infection and HCC tumors by suppressing p53.

FUSE Binding Protein1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53 and p53‐Regulatory Factors

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP expression in HCC tumors with CHC background. Oncomine data analysis of normal versus HCC tumors with CHC background indicated a 4‐fold increase in FBP expression with a concomitant 2.5‐fold decrease in the expression of p53.We found that FBP promotes HCV replication by inhibiting p53, and regulating BCCIP and TCTP, which, respectively, are positive and negative regulators of p53. The severe inhibition of HCV replication in FBP‐knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP, but significantly activated upon knockdown of FBP. Transient expression of FBP in FBP‐knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using EMSA and ITC, we found no significant difference in in‐vitro target DNA binding affinity of recombinant wild‐type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP, DNA binding ability of p53 is strongly inhibited. We confirmed that FBP downregulates BCCIP, p21, and p53, and upregulates TCTP under radiation‐induced stress. Since FBP is overexpressed in most HCC tumors with HCV background, it may have a role in promoting persistent virus infection and tumorigenesis.

Investigating the antiviral role of cell death‐inducing DFF45‐like effector B in HCV replication

Cell-death-inducing DFF45-like effector B (CIDEB) is an apoptotic host factor, which was recently found to also regulate hepatic lipid homeostasis. Herein we delineate the relevance of these dual roles of CIDEB in apoptosis and lipid metabolism in the context of hepatitis C virus (HCV) replication. We demonstrate that HCV upregulates CIDEB expression in human serum differentiated hepatoma cells. CIDEB overexpression inhibits HCV replication in HCV replicon expressing Huh7.5 cells, while small interfering RNA knockdown of CIDEB expression in human serum differentiated hepatoma cells promotes HCV replication and secretion of viral proteins. Furthermore, we characterize a CIDEB mutant, KRRA, which is deficient in lipid droplet clustering and fusion and demonstrate that CIDEB-mediated inhibition of HCV is independent of the protein's lipid droplet fusogenic role. Our results suggest that higher levels of CIDEB expression, which favour an apoptotic role for the host factor, inhibit HCV. Collectively, our data demonstrate that CIDEB can act as an anti-HCV host factor and contribute to altered triglyceride homeostasis.

Effects of alcohol on HCV replication and its mechanism

Objective To investigate the effects of alcohol on hepatitis C virus（ HCV） replication and type I interferon signaling pathway in human hepatocytes .Methods Primary hepatocytes were treated with different concentrations of alcohol , and then infected with HCV .The infected cells were collected to measure the level of HCV RNA .The alcohol-treated hepatocytes were also collected to detect the expression of HCV Core, IFN-α, IFN-β, IRF-7, suppressor of cytokine signaling SOCS-2 and SOCS-3 at mRNA and protein levels by real-time quantitative PCR and ELISA or Western blot , respectively .Results Alcohol treatment enhanced HCV infection and replication in primary hepatocytes at concentrations higher than 10 mmol/L in a dose-dependent manner （P<0.05）.Treatment with 40 mmol/L of alcohol significantly reduced the expression of IFN-α, IFN-βand IRF-7 at mRNA and protein levels , and increased the expression of SOCS-2 and SOCS-3 at mRNA and protein levels .Conclusion Alcohol treatment could damage the host in-nate immunity in human hepatocytes and promote HCV replication by reducing the expression of type Ⅰinter-feron （ IFN-αand IFN-β） and IRF-7 and increasing the expression of negative regulators including SOCS-2 and SOCS-3.These results demonstrated that the impairment of innate immunity in liver of alcohol abusers might contribute to the enhancement of HCV infection and result in poor therapeutic effect of IFN -α. Key words: Hepatitis C virus; Alcohol; Type I interferon; Innate immunity

Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication

OPN (osteopontin)) is a Hh (Hedgehog)-regulated cytokine that is up-regulated during chronic liver injury and directly promotes fibrosis. We have reported that Hh signalling enhances viral permissiveness and replication in HCV (hepatitis C virus)-infected cells. Hence we hypothesized that OPN directly promotes HCV replication, and that targeting OPN could be beneficial in HCV. In the present study, we compared the expression of OPN mRNA and protein in HCV (JFH1)-infected Huh7 and Huh7.5 cells, and evaluated whether modulating OPN levels using exogenous OPN ligands (up-regulate OPN) or OPN-specific RNA-aptamers (neutralize OPN) leads to changes in HCV expression. Sera and livers from patients with chronic HCV were analysed to determine whether OPN levels were associated with disease severity or response to therapy. Compared with Huh7 cells, Huh7.5 cells support higher levels of HCV replication (15-fold) and expressed significantly more OPN mRNA (30-fold) and protein. Treating Huh7 cells with OPN ligands led to a dose-related increase in HCV (15-fold) and OPN (8-fold) mRNA. Conversely, treating Huh7.5 cells with OPN-specific RNA aptamers inhibited HCV RNA and protein by >50% and repressed OPN mRNA to basal levels. Liver OPN expression was significantly higher (3-fold) in patients with advanced fibrosis. Serum OPN positively correlated with fibrosis-stage (P=0.009), but negatively correlated with ETBCR (end-of-treatment biochemical response), ETVR (end-of-treatment virological response), SBCR (sustained biochemical response) and SVR (sustained virological response) (P=0.007). The OPN fibrosis score (serum OPN and presence of fibrosis ≥F2) may be a predictor of SVR. In conclusion, OPN is up-regulated in the liver and serum of patients with chronic hepatitis C, and supports increased viral replication. OPN neutralization may be a novel therapeutic strategy in chronic hepatitis C.

Silent information regulator 1 inhibition induces lipid metabolism disorders of hepatocytes and enhances hepatitis C virus replication

Hepatic steatosis is an important histopathological feature of chronic hepatitis C virus (HCV) infection. Silent information regulator 1 (SIRT1) plays key role in regulation of hepatic lipid metabolism. We investigated the possible effect of HCV replication on lipid metabolism of hepatocytes and expression of SIRT1 using Huh-7.5 cells harboring HCV replicon. The level of reactive oxygen species (ROS) and malondialdehyde (MDA), the activity of superoxide dismutase (SOD), and the value of nicotinamide adenine dinucleotide (NAD(+) )/NADH was detected. The level of triacylglycerol (TG), total cholesterol (TC) and fatty acid β-oxidation rate was detected. The activity and expression levels of SIRT1 and expression of its downstream lipid-metabolism genes were measured. In replicon cells, the level of ROS and MDA increased, SOD activity and the value of NAD(+) /NADH decreased, then the activity and expression level of mRNA and protein of SIRT1 reduced. Inhibition of SIRT1 decreased phosphorylation of forkhead box O1 (FoxO1), which not only upregulated SREBP-1c, FAS, ACC, SREBP-2, HMGR and HMGS genes and increased fatty acid synthesis; but also downregulated PPAR-α and CPT1A genes and decreased fatty acid β-oxidation. Interferon treatment restored aforementioned changes. SIRT1 activator improved lipid metabolism disorders by an increase in fatty acid β-oxidation and a decrease in TG and TC synthesis and inhibited HCV replication. HCV replication decreasing NAD(+) /NADH ratio may downregulate the activity and the expression of SIRT1, then change the expression profile of lipid metabolism-related genes, thereby cause lipid metabolism disorders of hepatocytes and promote HCV replication. Treatment with SIRT1 activator ameliorates lipid metabolic disorders and inhibits HCV replication.

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.

New targets for antiviral therapy of chronic hepatitis C

Until recently, chronic hepatitis C caused by persistent infection with the hepatitis C virus (HCV) has been treated with a combination of pegylated interferon-alpha (PEG-IFNα) and ribavirin (RBV). This situation has changed with the development of two drugs targeting the NS3/4A protease, approved for combination therapy with PEG-IFNα/RBV for patients infected with genotype 1 viruses. Moreover, two additional viral proteins, the RNA-dependent RNA polymerase (residing in NS5B) and the NS5A protein have emerged as promising drug targets and a large number of antivirals targeting these proteins are at different stages of clinical development. Although this progress is very promising, it is not clear whether these new compounds will suffice to eradicate the virus in an infected individual, ideally by using a PEG-IFNα/RBV-free regimen, or whether additional compounds targeting other factors that promote HCV replication are required. In this respect, host cell factors have emerged as a promising alternative. They reduce the risk of development of antiviral resistance and they increase the chance for broad-spectrum activity, ideally covering all HCV genotypes. Work in the last few years has identified several host cell factors used by HCV for productive replication. These include, amongst others, cyclophilins, especially cyclophilinA (cypA), microRNA-122 (miR-122) or phosphatidylinositol-4-kinase III alpha. For instance, cypA inhibitors have shown to be effective in combination therapy with PEG-IFN/RBV in increasing the sustained viral response (SVR) rate significantly compared to PEG-IFN/RBV. This review briefly summarizes recent advances in the development of novel antivirals against HCV.