Hepatitis C Viral Replication Complex.
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ABSTRACT:
SUBMITTER: Li HC
PROVIDER: S-EPMC8004249 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV+ hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection.IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) that can become a long-term, chronic infection and lead to cirrhosis or liver cancer. HBV is a small DNA virus that belongs to the hepadnavirus family, with virions and subviral forms of particles that lack a core. MicroRNA (miRNA), a small (∼22-nt) noncoding RNA, was recently found to be an important regulator of gene expression. We found that HBV encodes miRNA (HBV-miR-3). More importantly, we revealed that HBV-miR-3 targets its transcripts to attenuate HBV replication. This may contribute to explaining how HBV infection leads to mild damage in liver cells and the subsequent establishment/maintenance of persistent infection. Our findings highlight a mechanism by which HBV-encoded miRNA controls the process of self-replication by regulating the virus itself during infection and might provide new biomarkers for diagnosis and treatment of hepatitis B.
| S-EPMC5411615 | biostudies-literature
Project description:Hepatitis E Virus (HEV) is a positively oriented RNA virus having a 7.2 kb genome. HEV consists of three open reading frames (ORF1-3). Of these, ORF1 codes for the enzymes Methyltransferase (Mtase), Papain-like cysteine protease (PCP), RNA helicase, and RNA-dependent RNA polymerase (RdRp). Unavailability of a vaccine or effective drug against HEV and considering the side effects associated with the off-label use of ribavirin (RBV) and pegylated interferons, an alternative approach is required by the modulation of specific enzymes to prevent the infection. HEV helicase is involved in unwinding the double-stranded RNA, RNA processing, transcriptional regulation, and pre-mRNA processing. Therefore, we screened FDA-approved compounds from the ZINC15 database against the modelled 3D structure of HEV helicase and found that methotrexate and compound A (Pubchem ID BTB07890) inhibit the NTPase and dsRNA unwinding activity leading to inhibition of HEV RNA replication. This may be further authenticated by in vivo study.
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Project description:The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.
| S-EPMC1262582 | biostudies-literature
Project description:Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.
| S-EPMC4183590 | biostudies-literature
Project description:BackgroundHepatitis B core-related antigen (HBcrAg) is a promising disease-monitoring marker for chronic hepatitis B (CHB). We investigated correlations between HBcrAg with antiviral efficacy and virological and histological variables.MethodsOne hundred and forty-five CHB patients from the mainland of China between August 2013 and September 2016 who underwent liver biopsy received entecavir therapy and had paired liver biopsy at 78 weeks. We analyzed correlations between HBcrAg and virological and histological variables in hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients. We also explored the predictors of HBeAg loss after 78 weeks of antiviral therapy. Pearson correlation analysis and logistic forward stepwise regression were the main statistic methods.ResultsHBeAg-positive patients (n = 93) had higher baseline HBcrAg (median 7.4 vs. 5.3 log10 U/mL P < 0.001) and greater HBcrAg declines (median 1.6 vs. 0.9 log10 U/mL P = 0.007) than HBeAg-negative patients after 78 weeks of therapy. At baseline, HBcrAg correlated with hepatitis B virus (HBV) DNA in both HBeAg-positive (r = 0.641, P < 0.001) and -negative patients (r = 0.616, P < 0.001), with hepatitis B surface antigen (HBsAg) in HBeAg-positive patients (r = 0.495, P < 0.001), but not with anti-hepatitis B virus core antibody (anti-HBc). Weak correlations existed between HBcrAg, histology activity index (HAI; r = 0.232, P = 0.025), and Ishak fibrosis score (r = -0.292, P = 0.005) in HBeAg-positive patients. At 78 weeks, significant correlations existed only between HBcrAg and anti-HBc in HBeAg-positive (r = -0.263, P = 0.014) and HBeAg-negative patients (r = -0.291, P = 0.045). Decreased HBcrAg significantly correlated with reduced HBV DNA (r = 0.366, P = 0.001; r = 0.626, P < 0.001) and HBsAg (r = 0.526, P = 0.001; r = 0.289, P = 0.044) in HBeAg-positive and -negative patients, respectively, and with reduced HAI in HBeAg-positive patients (r = 0.329, P = 0.001). Patients with HBeAg loss (n = 29) showed a larger reduction in HBcrAg than those without (median 2.3 vs. 1.3 log10 U/mL, P = 0.001). In multivariate analysis, decreased HBcrAg was an independent predictor of HBeAg loss (P = 0.005).ConclusionsHBcrAg reflects viral replication and protein production. Decreased HBcrAg could predict HBeAg loss after antiviral therapy.Trial registrationClinical Trials.gov: NCT01962155; https://www.clinicaltrials.gov/ct2/show/NCT01962155?term=NCT01962155&draw=2&rank=1.
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Project description:Interferon alpha (IFN-alpha)-based therapy is the currently approved treatment for chronic hepatitis C viral infection. The sustained antiviral response rate is approximately 50% for genotype-1 infection. The major challenge to the HCV community is to improve antiviral efficacy and to reduce the side effects typically seen in IFNalpha-based therapy. One of the strategies is to identify new interferons, which may have better efficacy and less undesirable side effects. In this report, we examined the role of IL-28A (IFN lambda2), a novel type I IFN, in suppression of human hepatitis C viral RNA replication. We have cloned both the human genomic DNA and cDNA of IL-28A, and evaluated their biological activity using HCV RNA replicon cell culture system. The results show that IL-28A effectively inhibits HCV subgenomic RNA replication in a dose-dependent manner. Treatment of human hepatoma cells with IL-28A activates the JAK-STAT signaling pathway and induces the expression of some interferon-stimulated genes (ISGs), such as 6-16 and 1-8U. We also demonstrate that IL-28A induces expression of HLA class I antigens in human hepatoma cells. Moreover, IL-28A appears to specifically suppress HCV IRES-mediated translation. Although IL-28A receptor shares one subunit with the IL-10 receptor, IL-10 treatment has no detectable effect on IL-28A-induced antiviral activity. Interestingly, IL-28A can synergistically enhance IFNalpha antiviral efficacy. Our results suggest that IL-28A antiviral activity is associated with the activation of the JAK-STAT signaling pathway and expression of ISGs. The effectiveness of IL-28A antiviral activity and its synergistic effect on IFN-alpha indicate that IL-28A may be potentially used to treat HCV chronic infection.
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Project description:Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb.
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Project description:The mechanism underlying the chronicity of hepatitis B virus (HBV) infection has long been an interesting question. However, this mechanism remains unclear largely due to the lack of an animal model that can support persistent HBV replication and allow for the investigation of the relevant immune responses. In this study, we used hydrodynamic injection to introduce HBV replicon DNA into the livers of three different mouse strains: BALB/c, C57BL/6, and FVB/N. Interestingly, we found that an HBV clone persistently replicated in the livers of FVB/N mice for up to 50 weeks but was rapidly cleared from the livers of BALB/c and C57BL/6 mice. Flow cytometric analysis and quantitative reverse transcription PCR analysis of the mouse livers indicated that after DNA injection, FVB/N mice had few intrahepatic activated cytotoxic T lymphocytes (CTLs) and produced low levels of alanine aminotransferase, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and the CXCL9 and CXCL10 chemokines. These findings were in sharp contrast with those observed in BALB/c and C57BL/6 mice, reflecting a strong correlation between the degree of liver inflammation and viral clearance. Mutational analysis further demonstrated that a change of Asn-214 to Ser-214 in the HBV surface antigen rendered the persistent HBV clone clearable in FVB/N mice, which was accompanied by increased levels of activated CTL and upregulated expression of IFN-γ, CXCL9, and CXCL10 in the livers. These results indicate that the heterogeneity of the host factors and viral sequences may influence the immune responses against HBV. An inadequate activation of immune or inflammatory responses can lead to persistent HBV replication in vivo.
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Project description:Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus-cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus-cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication.
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Project description:Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.
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