scholarly journals A host cell RNA-binding protein, Staufen1, has a role in hepatitis C virus replication before virus assembly

2013 ◽  
Vol 94 (11) ◽  
pp. 2429-2436 ◽  
Author(s):  
Samantha L. Blackham ◽  
Michael J. McGarvey
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Core Protein ◽  
Virus Assembly ◽  
Binding Protein ◽  
Replication Cycle ◽  
Hcv Rna ◽  
Huh7 Cells ◽  
Hiv 1

Staufen1 is a dsRNA-binding protein involved in the regulation of translation and the trafficking and degradation of cellular RNAs. Staufen1 has also been shown to stimulate translation of human immunodeficiency virus type 1 (HIV-1) RNA, regulate HIV-1 and influenza A virus assembly, and there is also indication that it can interact with hepatitis C virus (HCV) RNA. To investigate the role of Staufen1 in the HCV replication cycle, the effects of small interfering RNA knockout of Staufen1 on HCV strain JFH-1 replication and the intracellular distribution of the Staufen1 protein during HCV infection were examined. Silencing Staufen1 in HCV-infected Huh7 cells reduced virus secretion by around 70 %, intracellular HCV RNA levels by around 40 %, and core and NS3 proteins by around 95 and 45 %, respectively. Staufen1 appeared to be predominantly localized in the endoplasmic reticulum at the nuclear periphery in both uninfected and HCV-infected Huh7 cells. However, Staufen1 showed significant co-localization with NS3 and dsRNA, indicating that it may bind to replicating HCV RNA that is associated with the non-structural proteins. Staufen1 and HCV core protein localized very closely to one another during infection, but did not appear to overlap, indicating that Staufen1 may not bind to core protein or localize to the core-coated lipid droplets, suggesting that it may not be directly involved in HCV virus assembly. These findings indicate that Staufen1 is an important factor in HCV replication and that it might play a role early in the HCV replication cycle, e.g. in translation, replication or trafficking of the HCV genome, rather than in virion morphogenesis.

scholarly journals Adaptive mutations promote hepatitis C virus assembly by accelerating Core translocation to the endoplasmic reticulum

2020 ◽  
pp. jbc.RA120.016010
Author(s):  
Fuxiang Zheng ◽  
Ni Li ◽  
Yi Xu ◽  
Yuanping Zhou ◽  
Yi-Ping Li
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Assembly ◽  
Buoyant Density ◽  
Virus Production ◽  
Adaptive Mutations ◽  
Huh7 Cells ◽  
Density Analysis

The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved in are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ~1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain, and that mutations I853V, C2865F, I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.

scholarly journals Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

2015 ◽  
Vol 89 (8) ◽  
pp. 4562-4574 ◽  
Author(s):  
Saravanabalaji Shanmugam ◽  
Dhanaranjani Saravanabalaji ◽  
MinKyung Yi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Core Protein ◽  
Virus Assembly ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Short Term Treatment ◽  
Particle Assembly ◽  
Detergent Resistant Membranes

ABSTRACTPreviously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production.IMPORTANCEThe biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process.

The prevalence of hepatitis B and C co-infections among people with HIV-1 in Bulgaria: 2010–2015

2019 ◽  
Vol 14 (12) ◽  
pp. 791-798
Author(s):  
Ivailo Alexiev ◽  
Elitsa Golkocheva-Markova ◽  
Asya Kostadinova ◽  
Reneta Dimitrova ◽  
Lora Nikolova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatitis B ◽  
Hepatitis B Surface Antigen ◽  
Hcv Rna ◽  
B Virus ◽  
Sex With Men ◽  
Multiple Risk Behavior ◽  
Hiv 1 ◽  
Hiv Aids

Aim: To evaluate hepatitis B virus (HBV) and hepatitis C virus (HCV) among individuals with HIV/AIDS in Bulgaria diagnosed between 2010 and 2015. Materials & methods: A total of 1158 individuals were diagnosed with HIV/AIDS during the study period. Different transmission groups were tested with ELISA and real-time PCR for HBV and HCV markers. Results: Hepatitis B surface antigen and hepatitis C virus antiboby were found in 9.3 and 23.2% of the tested. HBV DNA and HCV RNA has been found in 47.4 and 69.6%. Hepatitis B and C co-infections were predominant in multiple risk behavior groups, including people who inject drugs, men who have sex with men, prisoners and Roma individuals. Conclusion: HIV prevalence in Bulgaria is low but the rates of hepatitis B and C co-infections among these patients fall within the upper range reported in Europe.

scholarly journals RNA binding protein 24 regulates the translation and replication of hepatitis C virus

2018 ◽  
Vol 9 (11) ◽  
pp. 930-944 ◽  
Author(s):  
Huang Cao ◽  
Kaitao Zhao ◽  
Yongxuan Yao ◽  
Jing Guo ◽  
Xiaoxiao Gao ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein

Hepatitis C virus core protein interacts with p53-binding protein, 53BP2/Bbp/ASPP2, and inhibits p53-mediated apoptosis

2004 ◽  
Vol 315 (4) ◽  
pp. 788-795 ◽  
Author(s):  
Yongheng Cao ◽  
Tomio Hamada ◽  
Tadashi Matsui ◽  
Takayasu Date ◽  
Kuniyoshi Iwabuchi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Binding Protein ◽  
Virus Core

scholarly journals Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus

2018 ◽  
Vol 19 (9) ◽  
pp. 2771 ◽  
Author(s):  
Yoo Cho ◽  
Hwan Lee ◽  
Hyojeung Kang ◽  
Hyosun Cho
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nk Cells ◽  
Natural Killer ◽  
Core Protein ◽  
Lymphoid Cells ◽  
Hcv Rna ◽  
Cell Infection ◽  
Hcv Core Protein

HCV genotype 2a strain JFH-1 replicates and produces viral particles efficiently in human hepatocellular carcinoma (huh) 7.5 cells, which provide a stable in vitro cell infection system for the hepatitis C virus (HCVcc system). Natural killer (NK) cells are large lymphoid cells that recognize and kill virus-infected cells. In this study, we investigated the interaction between NK cells and the HCVcc system. IL-10 is a typical immune regulatory cytokine that is produced mostly by NK cells and macrophages. IL-21 is one of the main cytokines that stimulate the activation of NK cells. First, we used anti-IL-10 to neutralize IL-10 in a coculture of NK cells and HCVcc. Anti-IL-10 treatment increased the maturation of NK cells by enhancing the frequency of the CD56+dim population in NK-92 cells. However, with anti-IL-10 treatment of NK cells in coculture with J6/JFH-1-huh 7.5 cells, there was a significant decrease in the expression of STAT1 and STAT5 proteins in NK-92 cells and an increase in the HCV Core and NS3 proteins. In addition, rIL-21 treatment increased the frequency of the CD56+dim population in NK-92 cells, Also, there was a dramatic increase in the expression of STAT1 and STAT5 proteins in rIL-21 pre-stimulated NK cells and a decrease in the expression of HCV Core protein in coculture with J6/JFH-1-huh 7.5 cells. In summary, we found that the functional activation of NK cells can be modulated by anti-IL-10 or rIL-21, which controls the expression of HCV proteins as well as HCV RNA replication.

scholarly journals Efficient Virus Assembly, but Not Infectivity, Determines the Magnitude of Hepatitis C Virus-Induced Interferon Alpha Responses of Plasmacytoid Dendritic Cells

2014 ◽  
Vol 89 (6) ◽  
pp. 3200-3208 ◽  
Author(s):  
Elena Grabski ◽  
Ilka Wappler ◽  
Stephanie Pfaender ◽  
Eike Steinmann ◽  
Sibylle Haid ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Assembly ◽  
Plasmacytoid Dendritic Cells ◽  
Type I ◽  
Hepatitis C Virus Infection ◽  
Infected Cells ◽  
Interferon Responses

ABSTRACTWorldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV.IMPORTANCEAlthough pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the nonstructural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that, not the virus infectivity, but rather, the efficiency of virus assembly and core protein envelopment critically determined the magnitude of interferon responses. To our knowledge, this is the first example of hepatitis C virus-associated genetic variations that determine the magnitude of innate host responses.

scholarly journals A Disulfide-Bonded Dimer of the Core Protein of Hepatitis C Virus Is Important for Virus-Like Particle Production

2010 ◽  
Vol 84 (18) ◽  
pp. 9118-9127 ◽  
Author(s):  
Yukihiro Kushima ◽  
Takaji Wakita ◽  
Makoto Hijikata
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Rna Binding ◽  
Core Protein ◽  
Dominant Negative ◽  
Proteinase K ◽  
Dominant Negative Effect ◽  
The Core ◽  
Virus Like Particle

ABSTRACT Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

scholarly journals Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

2008 ◽  
Vol 52 (6) ◽  
pp. 2097-2110 ◽  
Author(s):  
Pantxika Bellecave ◽  
Christian Cazenave ◽  
Julie Rumi ◽  
Cathy Staedel ◽  
Ophélie Cosnefroy ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Selection Procedure ◽  
Inhibitory Effect ◽  
Hcv Rna ◽  
Dna Aptamers ◽  
Huh7 Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

scholarly journals HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

2015 ◽  
Vol 90 (5) ◽  
pp. 2332-2344 ◽  
Author(s):  
Rong Yu ◽  
Darong Yang ◽  
Shaohua Lei ◽  
Xiaohong Wang ◽  
Xianghe Meng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Untranslated Region ◽  
Rna Binding ◽  
High Mobility ◽  
Hcv Rna ◽  
Functional Domain ◽  
Hmgb1 Protein ◽  
Stem Loop

ABSTRACTHigh-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome.IMPORTANCEHepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is warranted to elucidate the specific actions of HMGB1 in HCV pathogenesis.

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