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Mohamadi M, Azarbayjani K, Mozhgani SH, Bamdad T, Alamdary A, Nikoo HR, Hashempour T, Hedayat Yaghoobi M, Ajorloo M. Hepatitis C virus alternative reading frame protein (ARFP): Production, features, and pathogenesis. J Med Virol 2020; 92:2930-2937. [PMID: 32470157 DOI: 10.1002/jmv.26091] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2020] [Accepted: 05/28/2020] [Indexed: 01/01/2023]
Abstract
Earlier observation suggests that hepatitis C virus (HCV) is a single-stranded RNA virus which encodes at least 10 viral proteins. F protein is a novel protein which has been discovered recently. These studies suggest three mechanisms for the production of this protein concerning ribosomal frameshift at codon 10, initial translation at codons 26 and 85 or 87. In this study, the association between protein F and chronicity of hepatocellular carcinoma (HCC) has been reviewed. Evidence suggests that humoral immune system can recognize this protein and produce antibodies against it. By detecting antibodies in infected people, investigators found that F protein might have a role in HCV infection causing chronic cirrhosis and HCC as higher prevalence was found in patients with mentioned complications. The increment of CD4+, CD25+, and FoxP3+ T cells, along with CD8+ T cells with low expression of granzyme B, also leads to weaker responses of the immune system which helps the infection to become chronic. Moreover, it contributes to the survival of the virus in the body through affecting the production of interferon. F protein also might play roles in the disease development, resulting in HCC. The existence of F protein affects cellular pathways through upregulating p53, c-myc, cyclin D1, and phosphorylating Rb. This review will summarize these effects on immune system and related mechanisms in cellular pathways.
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Affiliation(s)
- Mahdi Mohamadi
- Student Research Committee, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
| | - Kimia Azarbayjani
- Student Research Committee, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
| | - Sayed-Hamidreza Mozhgani
- Non-Communicable Diseases Research Center, Alborz University of Medical Sciences, Karaj, Iran
- Department of Microbiology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran
| | - Taravat Bamdad
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Ashkan Alamdary
- Department of Biology, Science, and Research Branch, Islamic Azad University, Tehran, Iran
| | - Hadi Razavi Nikoo
- Infectious Disease Research Center, Golestan University of Medical Sciences, Gorgan, Iran
- Department of Microbiology, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
| | - Tayebeh Hashempour
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mojtaba Hedayat Yaghoobi
- Department of Infectious Disease, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran
| | - Mehdi Ajorloo
- Hepatitis Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran
- Department of Clinical Laboratory Sciences, School of Allied Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
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Genotypic Regulation of Type I Interferon Induction Pathways by Frameshift (F) Proteins of Hepatitis C Virus. J Virol 2020; 94:JVI.00312-20. [PMID: 32434887 DOI: 10.1128/jvi.00312-20] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Accepted: 05/14/2020] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) has evolved mechanisms to evade innate immunity that are leading to chronic infections. The immunological function of the HCV frameshift (F) protein, which is a frameshift product of core coding sequences, has not been well characterized. The HCV F protein is produced during natural HCV infections and is found most commonly in genotype 1 HCV. In this study, we investigated whether the F protein plays a role in type I interferon (IFN) induction pathways. We engineered F expression constructs from core coding sequences of 4 genotypes (1a, 2a, 3a, and 4a) of HCV as well as the sequences which would only be able to produce core proteins. The peptide lengths and amino acids sequences of F proteins are highly variable. We hypothesized that F proteins from different genotypes might control the type I IFN production and response differently. We found that both IFN-beta (IFN-β) promoter activities are significantly higher in genotype 1a F protein (F1a)-expressing cells. Conversely, the IFN-β promoter activities are lower in genotype 2a F (F2a) protein-expressing cells. We also used real-time PCR to confirm IFN-β mRNA expression levels. By generating chimera F proteins, we discovered that the effects of F proteins were determined by the amino acid sequence 40 to 57 of genotype 1a. The regulation of type I IFN induction pathway is related but not limited to the activity of F1a to interact with proteasome subunits and to disturb the proteasome activity. Further molecular mechanisms of how F proteins from different genotypes of HCV control these pathways differently remain to be investigated.IMPORTANCE Although naturally present in HCV infection patient serum, the virological or immunological functions of the HCV F protein, which is a frameshift product of core coding sequences, remain unclear. Here, we report the effects of the HCV F protein between genotypes and discuss a potential explanation for the differential responses to type I IFN-based therapy among patients infected with different genotypes of HCV. Our study provides one step forward to understanding the host response during HCV infection and new insights for the prediction of the outcome of IFN-based therapy in HCV patients.
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Hepatitis C Virus Translation Regulation. Int J Mol Sci 2020; 21:ijms21072328. [PMID: 32230899 PMCID: PMC7178104 DOI: 10.3390/ijms21072328] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 03/18/2020] [Accepted: 03/25/2020] [Indexed: 12/12/2022] Open
Abstract
Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.
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Majzoub K, Hafirassou ML, Meignin C, Goto A, Marzi S, Fedorova A, Verdier Y, Vinh J, Hoffmann JA, Martin F, Baumert TF, Schuster C, Imler JL. RACK1 controls IRES-mediated translation of viruses. Cell 2015; 159:1086-1095. [PMID: 25416947 DOI: 10.1016/j.cell.2014.10.041] [Citation(s) in RCA: 131] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2014] [Revised: 09/16/2014] [Accepted: 10/20/2014] [Indexed: 01/31/2023]
Abstract
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
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Affiliation(s)
- Karim Majzoub
- CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France
| | - Mohamed Lamine Hafirassou
- Université de Strasbourg, 67000 Strasbourg, France; Inserm UMR1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France
| | - Carine Meignin
- CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France
| | - Akira Goto
- CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France
| | - Stefano Marzi
- CNRS UPR9002, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France
| | - Antonina Fedorova
- Université de Strasbourg, 67000 Strasbourg, France; Inserm UMR1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France
| | | | - Joëlle Vinh
- USR3149, ESPCI ParisTech, 75005 Paris, France
| | - Jules A Hoffmann
- CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France; Institut d'Etudes Avancées de l'Université de Strasbourg, 67000 Strasbourg, France
| | - Franck Martin
- CNRS UPR9002, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France
| | - Thomas F Baumert
- Université de Strasbourg, 67000 Strasbourg, France; Inserm UMR1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France; Institut Hospitalo-Universitaire (IHU), Pôle hépato-digestif, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France
| | - Catherine Schuster
- Université de Strasbourg, 67000 Strasbourg, France; Inserm UMR1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France.
| | - Jean-Luc Imler
- CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France.
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Li HC, Ma HC, Yang CH, Lo SY. Production and pathogenicity of hepatitis C virus core gene products. World J Gastroenterol 2014; 20:7104-7122. [PMID: 24966583 PMCID: PMC4064058 DOI: 10.3748/wjg.v20.i23.7104] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Revised: 12/05/2013] [Accepted: 04/03/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and its infection is also associated with insulin resistance and type 2 diabetes mellitus. HCV, belonging to the Flaviviridae family, is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein. The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein (a.a. 1-177) and the cleaved peptide (a.a. 178-191). Core protein could induce insulin resistance, steatosis and even hepatocellular carcinoma through various mechanisms. The peptide (a.a. 178-191) may play a role in the immune response. The polymorphism of this peptide is associated with the cellular lipid drop accumulation, contributing to steatosis development. In addition to the conventional open reading frame (ORF), in the +1 frame, an ORF overlaps with the core protein-coding sequence and encodes the alternative reading frame proteins (ARFP or core+1). ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism. In this review, we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis.
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Xu X, Yue M, Jiang L, Deng X, Zhang Y, Zhang Y, Zhu D, Xiao W, Zhou Z, Yao W, Kong J, Yu X, Wei J. Genetic variants in human leukocyte antigen-DP influence both hepatitis C virus persistence and hepatitis C virus F protein generation in the Chinese Han population. Int J Mol Sci 2014; 15:9826-43. [PMID: 24897020 PMCID: PMC4100124 DOI: 10.3390/ijms15069826] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2014] [Revised: 05/19/2014] [Accepted: 05/21/2014] [Indexed: 12/16/2022] Open
Abstract
Chronic hepatitis C is a serious liver disease that often results in cirrhosis or hepatocellular carcinoma. The aim of this study was to assess the association of human leukocyte antigen-DP (HLA-DP) variants with risk of chronic hepatitis C virus (HCV) or anti-F antibody generation. We selected two single nucleotide polymorphisms (SNPs) in a region including HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277534) and genotyped SNPs in 702 cases and 342 healthy controls from the Chinese population using TaqMan SNP genotyping assay. Moreover, the exon 2 of the HLA-DPA1 and HLA-DPB1 genes were amplified and determined by sequencing-based typing (SBT). The results showed that rs3077 significantly increased the risk of chronic HCV infection in additive models and dominant models (odds ratio (OR) = 1.32 and 1.53). The rs3077 also contributed to decrease the risk of anti-F antibody generation in additive models and dominant models (OR = 0.46 and 0.56). Subsequent analyses revealed the risk haplotypes (DPA1*0103-DPB1*0501 and DPA1*0103-DPB1*0201) and protective haplotypes (DPA1*0202-DPB1*0501 and DPA1*0202-DPB1*0202) to chronic HCV infection. Moreover, we also found that the haplotype of DPA1*0103-DPB1*0201 and DPA1*0202-DPB1*0202 were associated with the anti-F antibody generation. Our findings show that genetic variants in HLA-DP gene are associated with chronic HCV infection and anti-F antibody generation.
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Affiliation(s)
- Xiaodong Xu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing 210029, China.
| | - Ming Yue
- School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
| | - Longfeng Jiang
- Department of Infectious Diseases, the First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China.
| | - Xiaozhao Deng
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing 210029, China.
| | - Yongxiang Zhang
- Department of Infectious Diseases, the First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China.
| | - Yun Zhang
- Institute of Disease Control and Prevention, Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China.
| | - Danyan Zhu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing 210029, China.
| | - Wen Xiao
- School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
| | - Zhenxian Zhou
- Department of Clinical Laboratory, Nanjing Second Hospital, Nanjing 210003, China.
| | - Wenjuan Yao
- Department of Pharmacology, Nantong University Medical College, Nantong 226019, China.
| | - Jing Kong
- School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian 223003, China.
| | - Xiaojie Yu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing 210029, China.
| | - Juan Wei
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing 210029, China.
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Li W, Zhang Y, Kao CC. The classic swine fever virus (CSFV) core protein can enhance de novo-initiated RNA synthesis by the CSFV polymerase NS5B. Virus Genes 2014; 49:106-15. [DOI: 10.1007/s11262-014-1080-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 04/23/2014] [Indexed: 12/28/2022]
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Lefèvre M, Felmlee DJ, Parnot M, Baumert TF, Schuster C. Syndecan 4 is involved in mediating HCV entry through interaction with lipoviral particle-associated apolipoprotein E. PLoS One 2014; 9:e95550. [PMID: 24751902 PMCID: PMC3994096 DOI: 10.1371/journal.pone.0095550] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2013] [Accepted: 03/28/2014] [Indexed: 12/16/2022] Open
Abstract
Hepatitis C virus (HCV) is a major cause of liver disease worldwide and HCV infection represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. We sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. Using adenoviral vectors and siRNA to modulate apoE expression we show a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein expression. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Furthermore, a novel synthetic peptide that mimics apoE’s HSPG-BD directly and competitively inhibits HCV infection. Genetic knockdown of the HSPG proteins syndecan (SDC) 1 and 4 revealed that SDC4 principally mediates HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatoma cells and establish infection. Targeting apoE-SDC interactions could be an alternative strategy for blocking HCV entry, a critical step in maintaining chronic HCV infection.
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Affiliation(s)
- Mathieu Lefèvre
- Inserm, U1110, Research Institute on Viral and Hepatic Disease, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
| | - Daniel J. Felmlee
- Inserm, U1110, Research Institute on Viral and Hepatic Disease, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
| | - Marie Parnot
- Inserm, U1110, Research Institute on Viral and Hepatic Disease, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
| | - Thomas F. Baumert
- Inserm, U1110, Research Institute on Viral and Hepatic Disease, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
- Pôle hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
| | - Catherine Schuster
- Inserm, U1110, Research Institute on Viral and Hepatic Disease, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
- * E-mail:
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Positive ratio of specific antibodies to F protein in serum samples from chronic HCV-infected patients using an enzyme-linked immunosorbent assay: systematic review and meta-analysis. Eur J Gastroenterol Hepatol 2013; 25:1152-8. [PMID: 23603785 DOI: 10.1097/meg.0b013e328360fa2e] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
AIMS AND BACKGROUND Although some studies have reported a positive ratio of specific antibodies to the alternative reading frame protein in an enzyme-linked immunosorbent assay test, our data from meta-analysis provide evidence supporting the presence of circulating anti-F protein antibodies. METHODS We collected studies focused on hepatitis C virus (HCV) and F protein. From an initial identification of 460 articles, we selected 16 studies that were randomized-controlled trials (RCTs). RESULTS The results of the Mantel-Haenszel test showed that a statistically significant number of studies reported an effective value in chronic HCV-infected individuals (P<0.00001). We concluded that compared with healthy individuals, the positive ratio of F protein detection was higher in chronic HCV-infected individuals; the odds ratio was 63.61 [95% confidence interval (CI)=28.69, 141.06]. The values for chronic HCV-infected individuals were significantly different from those for non-HCV-infected individuals; the odds ratio was 53.43 (95% CI=23.33, 122.35). The positive ratio of the core protein was higher than that of F protein (rate difference=-38%, 95% CI=-42, -35%). CONCLUSION We concluded that F protein elicits specific antibodies in most chronic HCV-infected individuals. Further, we confirmed the results of previous reports. The relationship between anti-F protein antibody and HCV coinfection still needs to be confirmed with further studies. Considering the high polymorphism rate of HCV, further studies are still needed for the selection of synthetic peptides from F protein that can coat the wells on microplates and serve as a commercial reagent.
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Yue M, Deng X, Zhai X, Xu K, Kong J, Zhang J, Zhou Z, Yu X, Xu X, Liu Y, Zhu D, Zhang Y. Th1 and Th2 cytokine profiles induced by hepatitis C virus F protein in peripheral blood mononuclear cells from chronic hepatitis C patients. Immunol Lett 2013; 152:89-95. [PMID: 23680070 DOI: 10.1016/j.imlet.2013.05.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2013] [Revised: 05/04/2013] [Accepted: 05/05/2013] [Indexed: 12/28/2022]
Abstract
Th1 and Th2 cytokine response has been confirmed to be correlated with the pathogenesis of HCV infection. The aim of the study is to investigate the Th1 and Th2 cytokine profiles induced by HCV alternate reading frame protein (F protein) in chronic hepatitis C patients. We assessed the immune responses specific to HCV F protein in 55 chronic HCV patients. IFN-γ, IL-2, IL-4 and IL-5 secretion by peripheral blood mononuclear cells (PBMC) post F protein stimulation were compared among HCV patients and healthy donors. Finally, the associations between HCV F protein and HLA class II alleles were explored. We found that the seroprevalence of anti-F antibodies in HCV-related hepatocellular carcinoma (HCC) patients was significantly higher than that of patients without HCC, but such a significant difference in humoral immune responses to F protein was not observed in HCV 1b-infected- and non-HCV 1b-infected-patients. Additionally, the PBMC proliferation of HCC patients was significantly lower than that of patients without HCC. Furthermore, F protein stimulation of PBMCs from F-seropositive patients resulted in Th2 biased cytokine responses (significantly decreased IFN-γ and/or IL-2 and significantly increased IL-4 and/or IL-5 levels) that reportedly may contribute to HCC progression and pathogenesis. However, no significant difference in the association between HCV F protein and HLA-DRB1*0201, 0301, 0405, 1001 and HLA-DQB1*0201, 0401, 0502, 0602 was observed in this study. These findings suggest that F protein may contribute to the HCV-associated bias in Th1/Th2 responses of chronic hepatitis C patients including the progress of HCC pathogenesis.
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Affiliation(s)
- Ming Yue
- School of Life Science and Technology, China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu Province, China
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Hu WT, Li HC, Lee SK, Ma HC, Yang CH, Chen HL, Lo SY. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice. Biochem Biophys Res Commun 2013; 435:147-52. [PMID: 23628415 DOI: 10.1016/j.bbrc.2013.04.059] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2013] [Accepted: 04/17/2013] [Indexed: 12/27/2022]
Abstract
The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.
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Affiliation(s)
- Wen-Ta Hu
- Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan
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12
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Norovirus RNA synthesis is modulated by an interaction between the viral RNA-dependent RNA polymerase and the major capsid protein, VP1. J Virol 2012; 86:10138-49. [PMID: 22787222 DOI: 10.1128/jvi.01208-12] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027-13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRps in vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR). Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.
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Effect of Hepatitis C F Protein and Core Secondary Structure on Viral Replication and Infection*. PROG BIOCHEM BIOPHYS 2012. [DOI: 10.3724/sp.j.1206.2011.00272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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Qureshi H, Qazi R, Hamid S, Qureshi SA. Identification of immunogenic regions within the alternative reading frame protein of hepatitis C virus (genotype 3). Eur J Clin Microbiol Infect Dis 2011; 30:1075-83. [DOI: 10.1007/s10096-011-1194-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2010] [Accepted: 01/25/2011] [Indexed: 01/29/2023]
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Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b. Virus Res 2010; 155:213-20. [PMID: 20959129 DOI: 10.1016/j.virusres.2010.10.007] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2010] [Revised: 10/01/2010] [Accepted: 10/06/2010] [Indexed: 01/02/2023]
Abstract
The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9-11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1(342-770) region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG(85), similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG(58) or under certain conditions GUG(26) internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.
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Boumlic A, Nominé Y, Charbonnier S, Dalagiorgou G, Vassilaki N, Kieffer B, Travé G, Mavromara P, Orfanoudakis G. Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein. FEBS J 2010; 277:774-89. [PMID: 20067524 DOI: 10.1111/j.1742-4658.2009.07527.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The hepatitis C virus (HCV) Core+1/S polypeptide, also known as alternative reading frame protein (ARFP)/S, is an ARFP expressed from the Core coding region of the viral genome. Core+1/S is expressed as a result of internal initiation at AUG codons (85-87) located downstream of the polyprotein initiator codon, and corresponds to the C-terminal part of most ARFPs. Core+1/S is a highly basic polypeptide, and its function still remains unclear. In this work, untagged recombinant Core+1/S was expressed and purified from Escherichia coli in native conditions, and was shown to react with sera of HCV-positive patients. We subsequently undertook the biochemical and biophysical characterization of Core+1/S. The conformation and oligomeric state of Core+1/S were investigated using size exclusion chromatography, dynamic light scattering, fluorescence, CD, and NMR. Consistent with sequence-based disorder predictions, Core+1/S lacks significant secondary structure in vitro, which might be relevant for the recognition of diverse molecular partners and/or for the assembly of Core+1/S. This study is the first reported structural characterization of an HCV ARFP/Core+1 protein, and provides evidence that ARFP/Core+1/S is highly disordered under native conditions, with a tendency for self-association.
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Affiliation(s)
- Anissa Boumlic
- Université de Strasbourg, CNRS FRE 3211, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France
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Benga WJA, Krieger SE, Dimitrova M, Zeisel MB, Parnot M, Lupberger J, Hildt E, Luo G, McLauchlan J, Baumert TF, Schuster C. Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles. Hepatology 2010; 51:43-53. [PMID: 20014138 DOI: 10.1002/hep.23278] [Citation(s) in RCA: 178] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
UNLABELLED Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. Restriction of HCV infection to human hepatocytes suggests that liver-specific host factors play a role in the viral life cycle. Using a yeast-two-hybrid system, we identified apolipoprotein E (apoE) as a liver-derived host factor specifically interacting with HCV nonstructural protein 5A (NS5A) but not with other viral proteins. The relevance of apoE-NS5A interaction for viral infection was confirmed by co-immunoprecipitation and co-localization studies of apoE and NS5A in an infectious HCV cell culture model system. Silencing apoE expression resulted in marked inhibition of infectious particle production without affecting viral entry and replication. Analysis of particle production in liver-derived cells with silenced apoE expression showed impairment of infectious particle assembly and release. The functional relevance of the apoE-NS5A interaction for production of viral particles was supported by loss or decrease of apoE-NS5A binding in assembly-defective viral mutants. CONCLUSION These results suggest that recruitment of apoE by NS5A is important for viral assembly and release of infectious viral particles. These findings have important implications for understanding the HCV life cycle and the development of novel antiviral strategies targeting HCV-lipoprotein interaction.
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Affiliation(s)
- Wagane J A Benga
- Institut National de la Santé et de la Recherche Médicale (INSERM) Unite 748, Strasbourg, France
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Drouin C, Lamarche S, Bruneau J, Soudeyns H, Shoukry NH. Cell-mediated immune responses directed against hepatitis C virus (HCV) alternate reading frame protein (ARFP) are undetectable during acute infection. J Clin Virol 2009; 47:102-3. [PMID: 19955014 DOI: 10.1016/j.jcv.2009.10.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2009] [Revised: 10/28/2009] [Accepted: 10/28/2009] [Indexed: 01/29/2023]
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Vassilaki N, Mavromara P. The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product. IUBMB Life 2009; 61:739-52. [PMID: 19548320 DOI: 10.1002/iub.201] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus of the Flaviviridae family. It has a genome of about 9,600 nucleotides encoding a large polyprotein (about 3,000 amino acids) that is processed by cellular and viral proteases into at least 10 structural and nonstructural viral proteins. A novel HCV protein has also been identified by our laboratory and others. This protein--known as ARFP (alternative reading frame protein), F (for frameshift) or core+1 (to indicate the position) protein--is synthesized by an open reading frame overlapping the core gene at nucleotide +1 (core+1 ORF). However, almost 10 years after its discovery, we still know little of the biological role of the ARFP/F/core+1 protein. Abolishing core+1 protein production has no affect on HCV replication in cell culture or uPA-SCID mice, suggesting that core+1 protein is probably not important for the HCV reproductive cycle. However, the detection of specific anti-core+1 antibodies and T-cell responses in HCV-infected patients, as reported by many independent laboratories, provides strong evidence that this protein is produced in vivo. Furthermore, analyses of the HCV sequences isolated from patients with hepatocellular carcinoma and in vitro studies have provided strong preliminary evidence to suggest that core+1 protein plays a role in advanced liver disease and liver cancer. The available in vitro data also suggest that certain core function proteins may depend on production of the core+1 protein. We describe here the discovery of the various forms of the core+1 protein and what is currently known about the mechanisms of their production and their biochemical and functional properties. We also provide a detailed summary of the results of patient-based research.
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Affiliation(s)
- Niki Vassilaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, Greece.
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Yi G, Letteney E, Kim CH, Kao CC. Brome mosaic virus capsid protein regulates accumulation of viral replication proteins by binding to the replicase assembly RNA element. RNA (NEW YORK, N.Y.) 2009; 15:615-26. [PMID: 19237464 PMCID: PMC2661835 DOI: 10.1261/rna.1375509] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/17/2008] [Accepted: 01/16/2009] [Indexed: 05/20/2023]
Abstract
Viruses provide valuable insights into the regulation of molecular processes. Brome mosaic virus (BMV) is one of the simplest entities with four viral proteins and three genomic RNAs. Here we report that the BMV capsid protein (CP), which functions in RNA encapsidation and virus trafficking, also represses viral RNA replication in a concentration-dependent manner by inhibiting the accumulation of the RNA replication proteins. Expression of the replication protein 2a in trans can partially rescue BMV RNA accumulation. A mutation in the CP can decrease the repression of translation. Translation repression by the CP requires a hairpin RNA motif named the B Box that contains seven loop nucleotides (nt) within the 5' untranslated regions (UTR) of BMV RNA1 and RNA2. Purified CP can bind directly to the B Box RNA with a K (d) of 450 nM. The secondary structure of the B Box RNA was determined to contain a highly flexible 7-nt loop using NMR spectroscopy, native gel analysis, and thermal denaturation studies. The B Box is also recognized by the BMV 1a protein to assemble the BMV replicase, suggesting that the BMV CP can act to regulate several viral infection processes.
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Affiliation(s)
- Guanghui Yi
- Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77843, USA
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Internal initiation stimulates production of p8 minicore, a member of a newly discovered family of hepatitis C virus core protein isoforms. J Virol 2009; 83:3104-14. [PMID: 19129450 DOI: 10.1128/jvi.01679-08] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The hepatitis C virus (HCV) core gene is more conserved at the nucleic acid level than is necessary to preserve the sequence of the core protein, suggesting that it contains information for additional functions. We used a battery of anticore antibodies to test the hypothesis that the core gene directs the synthesis of core protein isoforms. Infectious viruses, replicons, and RNA transcripts expressed a p8 minicore containing the C-terminal portion of the p21 core protein and lacking the N-terminal portion. An interferon resistance mutation, U271A, which creates an AUG at codon 91, upregulated p8 expression in Con1 replicons, suggesting that p8 is produced by an internal initiation event and that 91-AUG is the preferred, but not the required, initiation codon. Synthesis of p8 was independent of p21, as shown by the abundant production of p8 from transcripts containing an UAG stop codon that blocked p21 production. Three infectious viruses, JFH-1 (2a core), J6/JFH (2a core), and H77/JFH (1a core), and a bicistronic construct, Bi-H77/JFH, all expressed both p8 and larger isoforms. The family of minicores ranges in size from 8 to 14 kDa. All lack the N-terminal portion of the p21 core. In conclusion, the core gene contains an internal signal that stimulates the initiation of protein synthesis at or near codon 91, leading to the production of p8. Infectious viruses of both genotype 1 and 2 HCV express a family of larger isoforms, in addition to p8. Minicores lack significant portions of the RNA binding domain of p21 core. Studies are under way to determine their functions.
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