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1
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Charles S, Edgar MP. Geometric Deep learning Prioritization and Validation of Cannabis Phytochemicals as Anti-HCV Non-nucleoside Direct-acting Inhibitors. Biomed Eng Comput Biol 2024; 15:11795972241306881. [PMID: 39678171 PMCID: PMC11638990 DOI: 10.1177/11795972241306881] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2024] [Accepted: 11/27/2024] [Indexed: 12/17/2024] Open
Abstract
Introduction The rate of acute hepatitis C increased by 7% between 2020 and 2021, after the number of cases doubled between 2014 and 2020. With the current adoption of pan-genotypic HCV therapy, there is a need for improved availability and accessibility of this therapy. However, double and triple DAA-resistant variants have been identified in genotypes 1 and 5 with resistance-associated amino acid substitutions (RAASs) in NS3/4A, NS5A, and NS5B. The role of this research was to screen for novel potential NS5B inhibitors from the cannabis compound database (CBD) using Deep Learning. Methods Virtual screening of the CBD compounds was performed using a trained Graph Neural Network (GNN) deep learning model. Re-docking and conventional docking were used to validate the results for these ligands since some had rotatable bonds >10. About 31 of the top 67 hits from virtual screening and docking were selected after ADMET screening. To verify their candidacy, 6 random hits were taken for FEP/MD and Molecular Simulation Dynamics to confirm their candidacy. Results The top 200 compounds from the deep learning virtual screening were selected, and the virtual screening results were validated by re-docking and conventional docking. The ADMET profiles were optimal for 31 hits. Simulated complexes indicate that these hits are likely inhibitors with suitable binding affinities and FEP energies. Phytil Diphosphate and glucaric acid were suggested as possible ligands against NS5B.
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Affiliation(s)
- Ssemuyiga Charles
- PharmaQsar Bioinformatics Firm, Kampala, Uganda
- Department of Microbiology, Kampala International University, School of Natural and Applied Sciences (SONAS), Kansanga, Kampala, Uganda
| | - Mulumba Pius Edgar
- PharmaQsar Bioinformatics Firm, Kampala, Uganda
- Department of Microbiology, Kampala International University, School of Natural and Applied Sciences (SONAS), Kansanga, Kampala, Uganda
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2
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Thakur S, Kumar V, Das R, Sharma V, Mehta DK. Biomarkers of Hepatic Toxicity: An Overview. CURRENT THERAPEUTIC RESEARCH 2024; 100:100737. [PMID: 38860148 PMCID: PMC11163176 DOI: 10.1016/j.curtheres.2024.100737] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/28/2023] [Accepted: 01/31/2024] [Indexed: 06/12/2024]
Abstract
Background Hepatotoxicity is the foremost issue for clinicians and the primary reason for pharmaceutical product recalls. A biomarker is a measurable and quantifiable attribute used to evaluate the efficacy of a treatment or to diagnose a disease. There are various biomarkers which are used for the detection of liver disease and the intent of liver damage. Objective This review aims to investigate the current state of hepatotoxicity biomarkers and their utility in clinical settings. Using hepatic biomarkers, the presence of liver injury, its severity, prognosis, causative agent, and type of hepatotoxicity can all be determined. Methods Relevant published articles up to 2022 were systematically retrieved from MEDLINE/PubMed, SCOPUS, EMBASE, and WOS databases using keywords such as drug toxicity, hepatotoxicity biomarkers, biochemical parameters, and nonalcoholic fatty liver disease. Results In clinical trials and everyday practice, biomarkers of drug-induced liver injury are essential for spotting the most severe cases of hepatotoxicity. Hence, developing novel biomarker approaches to enhance hepatotoxicity diagnosis will increase specificity and/or identify the person at risk. Importantly, early clinical studies on patients with liver illness have proved that some biomarkers such as aminotransferase, bilirubin, albumin, and bile acids are even therapeutically beneficial. Conclusions By assessing the unique signs of liver injury, health care professionals can rapidly and accurately detect liver damage and evaluate its severity. These measures contribute to ensuring prompt and effective medical intervention, hence reducing the risk of long-term liver damage and other major health concerns.
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Affiliation(s)
- Simran Thakur
- Department of Pharmacy Practice, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
| | - Vishal Kumar
- Department of Pharmacy Practice, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
| | - Rina Das
- Department of Pharmaceutical Chemistry, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
| | - Vishal Sharma
- Department of Pharmaceutical Chemistry, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
| | - Dinesh Kumar Mehta
- Department of Pharmaceutical Chemistry, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India
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3
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Vrazas V, Moustafa S, Makridakis M, Karakasiliotis I, Vlahou A, Mavromara P, Katsani KR. A Proteomic Approach to Study the Biological Role of Hepatitis C Virus Protein Core+1/ARFP. Viruses 2022; 14:v14081694. [PMID: 36016316 PMCID: PMC9518822 DOI: 10.3390/v14081694] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2022] [Revised: 07/26/2022] [Accepted: 07/28/2022] [Indexed: 11/16/2022] Open
Abstract
Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein-known as ARFP (alternative reading frame protein) or Core+1 protein-is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.
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Affiliation(s)
- Vasileios Vrazas
- Laboratory of Biochemistry and Molecular Virology, Department of Molecular Biology and Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece; (V.V.); (P.M.)
| | - Savvina Moustafa
- Clinical Immunology-Rheumatology Unit, 2nd Department of Medicine and Laboratory, Hippokration General Hospital of Athens, 11527 Athens, Greece;
| | - Manousos Makridakis
- Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece; (A.V.); (M.M.)
| | - Ioannis Karakasiliotis
- Laboratory of Biology, Department of Medicine, Democritus University of Thrace, 68100 Alexandroupolis, Greece;
| | - Antonia Vlahou
- Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece; (A.V.); (M.M.)
| | - Penelope Mavromara
- Laboratory of Biochemistry and Molecular Virology, Department of Molecular Biology and Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece; (V.V.); (P.M.)
| | - Katerina R. Katsani
- Laboratory of Biochemistry and Molecular Virology, Department of Molecular Biology and Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece; (V.V.); (P.M.)
- Correspondence:
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4
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Elsheikh MEA, McClure CP, Tarr AW, Irving WL. Sero-reactivity to three distinct regions within the hepatitis C virus alternative reading frame protein (ARFP/core+1) in patients with chronic HCV genotype-3 infection. J Gen Virol 2022; 103:001727. [PMID: 35230930 PMCID: PMC9176264 DOI: 10.1099/jgv.0.001727] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022] Open
Abstract
Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5 % of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three -20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47 % for ARFP-P2, 5.9 % for ARFP-P3 and 100 % for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.
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Affiliation(s)
- Mosaab E A Elsheikh
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK
| | - C Patrick McClure
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK.,Wolfson Centre for Global Virus Infections, The University of Nottingham, Nottingham, UK
| | - Alexander W Tarr
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK.,Wolfson Centre for Global Virus Infections, The University of Nottingham, Nottingham, UK.,NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK
| | - William L Irving
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK.,Wolfson Centre for Global Virus Infections, The University of Nottingham, Nottingham, UK.,NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK
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Computational based design and tracking of synthetic variants of Porcine circovirus reveal relations between silent genomic information and viral fitness. Sci Rep 2021; 11:10620. [PMID: 34012100 PMCID: PMC8134455 DOI: 10.1038/s41598-021-89918-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Accepted: 04/29/2021] [Indexed: 12/17/2022] Open
Abstract
Viral genomes not only code the protein content, but also include silent, overlapping codes which are important to the regulation of the viral life cycle and affect its evolution. Due to the high density of these codes, their non-modular nature and the complex intracellular processes they encode, the ability of current approaches to decipher them is very limited. We describe the first computational-experimental pipeline for studying the effects of viral silent and non-silent information on its fitness. The pipeline was implemented to study the Porcine Circovirus type 2 (PCV2), the shortest known eukaryotic virus, and includes the following steps: (1) Based on the analyses of 2100 variants of PCV, suspected silent codes were inferred. (2) Five hundred variants of the PCV2 were designed to include various ‘smart’ silent mutations. (3) Using state of the art synthetic biology approaches, the genomes of these five hundred variants were generated. (4) Competition experiments between the variants were performed in Porcine kidney-15 (PK15) cell-lines. (5) The variant titers were analyzed based on novel next-generation sequencing (NGS) experiments. (6) The features related to the titer of the variants were inferred and their analyses enabled detection of various novel silent functional sequence and structural motifs. Furthermore, we demonstrate that 50 of the silent variants exhibit higher fitness than the wildtype in the analyzed conditions.
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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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7
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Vassilaki N, Frakolaki E, Kalliampakou KI, Sakellariou P, Kotta-Loizou I, Bartenschlager R, Mavromara P. A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF. Int J Mol Sci 2020; 21:ijms21186974. [PMID: 32972019 PMCID: PMC7554737 DOI: 10.3390/ijms21186974] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 09/04/2020] [Accepted: 09/18/2020] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.
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Affiliation(s)
- Niki Vassilaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
- Correspondence: (N.V.); (P.M.)
| | - Efseveia Frakolaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Katerina I. Kalliampakou
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Panagiotis Sakellariou
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Ioly Kotta-Loizou
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany;
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Penelope Mavromara
- Molecular Virology Laboratory, Hellenic Pasteur Institute (HPI), 11521 Athens, Greece; (E.F.); (K.I.K.); (P.S.); (I.K.-L.)
- Laboratory of Biochemistry and Molecular Virology, Department of Molecular Biology and Genetics, Democritus University of Thrace, 68100 Thrace, Greece
- Correspondence: (N.V.); (P.M.)
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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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Romero-López C, Berzal-Herranz A. The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle. Int J Mol Sci 2020; 21:ijms21041479. [PMID: 32098260 PMCID: PMC7073135 DOI: 10.3390/ijms21041479] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 02/05/2023] Open
Abstract
RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.
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Musavi Z, Hashempour T, Moayedi J, Dehghani B, Ghassabi F, Hallaji M, Hosseini SY, Yaghoubi R, Gholami S, Dehyadegari MA, Merat S. Antibody Development to HCV Alternate Reading Frame Protein in Liver Transplant Candidate and its Computational Analysis. CURR PROTEOMICS 2020. [DOI: 10.2174/1570164617666190822103329] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Background::
HCV Alternate Reading Frame Protein (ARFP) is a frameshift product of
HCV-core encoding. Here, we characterized specific anti-ARFP antibodies in Liver Transplant Candidate
(LTC) and chronic HCV-infected patients.
Methods::
The ARFP gene was cloned and the recombinant protein was purified using Nickel chromatography
and confirmed by western blotting. ELISA was developed using recombinant core-1a, core-
1b, ARFP-1a protein, and 99-residue synthetic ARFP 1b peptide. By several Bioinformatics tools,
general properties, immunogenic epitopes, and structures of these proteins were obtained.
Results::
The seroprevalence of anti-core and anti-ARFP antibodies was 100% in LTC patients, but only
75.2% and 94.3% of chronic patients had evidence of anti-ARFP and anti-core antibodies, respectively.
In-silico results demonstrated physicochemical features, antigen properties and potential interactors
that could describe progression toward advanced liver disease.
Conclusion::
As the first report, the prevalence of anti-ARFP antibodies in LTC patients is of the order
of 100% and titer of anti-ARFP antibody was significantly higher in LTC patients compared to chronic
individuals, suggesting the possible role of ARFP in the progression toward advanced liver disease. In
addition, docking analysis determined several interactor proteins such as prefoldin 2, cathepsin B, vitronectin,
and angiotensinogen that have an important role in progression to chronic infection and liver
disease development.
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Affiliation(s)
- Zahra Musavi
- Clinical Microbiology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Tayebeh Hashempour
- Clinical Microbiology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Javad Moayedi
- Clinical Microbiology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Behzad Dehghani
- Shiraz HIV/AIDS Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Farzaneh Ghassabi
- Clinical Microbiology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mehrdad Hallaji
- Clinical Microbiology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Seyed Younes Hosseini
- Department of Bacteriology and Virology, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ramin Yaghoubi
- Shiraz Transplant Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Siavash Gholami
- Shiraz Organ Transplant Unit, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mohamad Ali Dehyadegari
- Clinical Microbiology Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Shahin Merat
- Liver and Pancreatobiliary Diseases Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran
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Chu L, Jin M, Feng C, Wang X, Zhang D. A highly divergent hepacivirus-like flavivirus in domestic ducks. J Gen Virol 2019; 100:1234-1240. [PMID: 31282853 DOI: 10.1099/jgv.0.001298] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Using random amplification and reverse transcription-PCR, a novel RNA virus was detected in sera of domestic ducks. The full genome of the virus was determined for three strains, identifying the first hepacivirus-like flavivirus in birds. The virus, that we tentatively named duck hepacivirus-like virus (DuHV), possesses several unique molecular features, such as possession of the largest hepacivirus-like polyprotein gene and a Pegivirus A-like internal ribosome entry site. Sequence comparisons and phylogenetic and sliding-window analyses indicated that DuHV is most closely related to, but highly divergent from, the known hepaciviruses. DuHV was detected in 69.7 % of 185 serum samples from four duck species and in 31 of 33 flocks from five provinces of China, reflecting a high prevalence in duck populations and a wide geographical distribution. The detection of DuHV in the same flock in November 2018 and April 2019 suggested that persistent infection can be established in the infected ducks.
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Affiliation(s)
- Lili Chu
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, PR China
| | - Meiling Jin
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, PR China
| | - Chonglun Feng
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, PR China
| | - Xiaoyan Wang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, PR China
| | - Dabing Zhang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, PR China
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13
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Pol S, Lagaye S. The remarkable history of the hepatitis C virus. Microbes Infect 2019; 21:263-270. [PMID: 31295571 DOI: 10.1016/j.micinf.2019.06.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 03/25/2019] [Accepted: 04/01/2019] [Indexed: 12/23/2022]
Abstract
The infection with the hepatitis C virus (HCV) is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological and virological tools for diagnosis but also for prognosis (the non-invasive biochemical or morphological fibrosis tests, the predictors of the specific immune response including genetic IL28B polymorphisms). Finally, well-tolerated and effective treatments with oral antivirals inhibiting HCV non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. HCV chronic infection, which is a public health issue, is a hepatic disease which may lead to a cirrhosis and an hepatocellular carcinoma (HCC) but also a systemic disease with extra-hepatic manifestations either associated with a cryoglobulinemic vasculitis or chronic inflammation. The HCV infection is the only chronic viral infection which may be cured: the so-called sustained virologic response, defined by undetectable HCV RNA 12 weeks after the end of the treatment, significantly reduces the risk of morbidity and mortality associated with hepatic and extra-hepatic manifestations which are mainly reversible. The history of HCV ends with the pangenotypic efficacy of the multiple combinations, easy to use for 8-12 weeks with one to three pills per day and little problems of tolerance. This explains the short 30 years from the virus discovery to the viral hepatitis elimination policy proposed by the World Health Organization (WHO) in 2016.
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Affiliation(s)
- Stanislas Pol
- Université Paris Descartes, Paris, France; Département d'Hépatologie, Hôpital Cochin, APHP, Paris, France; INSERM UMS-20, Institut Pasteur, Paris, France; Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France; INSERM U1223, Institut Pasteur, Paris, France.
| | - Sylvie Lagaye
- Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France; INSERM U1223, Institut Pasteur, Paris, France.
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14
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Detection and characterization of a novel hepacivirus in long-tailed ground squirrels (Spermophilus undulatus) in China. Arch Virol 2019; 164:2401-2410. [PMID: 31243554 DOI: 10.1007/s00705-019-04303-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2018] [Accepted: 05/02/2019] [Indexed: 12/13/2022]
Abstract
Rodent populations are known to be reservoirs of viruses with the potential to infect humans. However, a large number of such viruses remain undiscovered. In this study, we investigated the shedding of unknown viruses in long-tailed ground squirrel (Spermophilus undulatus) feces by high-throughput sequencing. A novel and highly divergent virus related to members of the genus Hepacivirus was identified in ground squirrel liver. This virus, tentatively named RHV-GS2015, was found to have a genome organization that is typical of hepaciviruses, including a long open reading frame encoding a polyprotein of 2763 aa. Sequence alignment of RHV-GS2015 with the most closely related hepaciviruses yielded p-distances of the NS3 and NS5B regions of 0.546 and 0.476, respectively, supporting the conclusion that RHV-GS2015 is a member of a new hepacivirus species, which we propose to be named "Hepacivirus P". Phylogenetic analysis of the NS3 and NS5B regions indicated that RHV-GS2015 shares common ancestry with other rodent hepaciviruses (species Hepacivirus E, and species Hepacivirus F), Norway rat hepacivirus 1 (species Hepacivirus G), and Norway rat hepacivirus 2 (species Hepacivirus H). A phylogenetic tree including the seven previously identified rodent hepaciviruses revealed extreme genetic heterogeneity among these viruses. RHV-GS2015 was detected in 7 out of 12 ground squirrel pools and was present in liver, lung, and spleen tissues. Furthermore, livers showed extremely high viral loads of RHV-GS2015, ranging from 2.5 × 106 to 2.0 × 108 copies/g. It is reasonable to assume that this novel virus is hepatotropic, like hepatitis C virus. The discovery of RHV-GS2015 extends our knowledge of the genetic diversity and host range of hepaciviruses, helping to elucidate their origins and evolution.
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15
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Pol S, Lagaye S. The remarkable history of the hepatitis C virus. Genes Immun 2019; 20:436-446. [PMID: 31019253 DOI: 10.1038/s41435-019-0066-z] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 03/25/2019] [Accepted: 04/01/2019] [Indexed: 02/06/2023]
Abstract
The infection with the hepatitis C virus (HCV) is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological and virological tools for diagnosis but also for prognosis (the non-invasive biochemical or morphological fibrosis tests, the predictors of the specific immune response including genetic IL28B polymorphisms). Finally, well-tolerated and effective treatments with oral antivirals inhibiting HCV non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. HCV chronic infection, which is a public health issue, is a hepatic disease, which may lead to a cirrhosis and an hepatocellular carcinoma (HCC) but also a systemic disease with extra-hepatic manifestations either associated with a cryoglobulinemic vasculitis or chronic inflammation. The HCV infection is the only chronic viral infection, which may be cured: the so-called sustained virologic response, defined by undetectable HCV RNA 12 weeks after the end of the treatment, significantly reduces the risk of morbidity and mortality associated with hepatic and extra-hepatic manifestations, which are mainly reversible. The history of HCV ends with the pangenotypic efficacy of the multiple combinations, easy to use for 8-12 weeks with one to three pills per day and little problems of tolerance. This explains the short 30 years from the virus discovery to the viral hepatitis elimination policy proposed by the World Health Organization (WHO) in 2016.
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Affiliation(s)
- Stanislas Pol
- Université Paris Descartes, Paris, France. .,Département d'Hépatologie, Hôpital Cochin, APHP, Paris, France. .,INSERM UMS-20, Institut Pasteur, Paris, France. .,Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France. .,INSERM U1223, Institut Pasteur, Paris, France.
| | - Sylvie Lagaye
- Immunobiologie des Cellules Dendritiques, Institut Pasteur, Paris, France. .,INSERM U1223, Institut Pasteur, Paris, France.
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16
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Ashraf MU, Iman K, Khalid MF, Salman HM, Shafi T, Rafi M, Javaid N, Hussain R, Ahmad F, Shahzad-Ul-Hussan S, Mirza S, Shafiq M, Afzal S, Hamera S, Anwar S, Qazi R, Idrees M, Qureshi SA, Chaudhary SU. Evolution of efficacious pangenotypic hepatitis C virus therapies. Med Res Rev 2018; 39:1091-1136. [PMID: 30506705 DOI: 10.1002/med.21554] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2018] [Revised: 10/11/2018] [Accepted: 10/11/2018] [Indexed: 12/12/2022]
Abstract
Hepatitis C compromises the quality of life of more than 350 million individuals worldwide. Over the last decade, therapeutic regimens for treating hepatitis C virus (HCV) infections have undergone rapid advancements. Initially, structure-based drug design was used to develop molecules that inhibit viral enzymes. Subsequently, establishment of cell-based replicon systems enabled investigations into various stages of HCV life cycle including its entry, replication, translation, and assembly, as well as role of host proteins. Collectively, these approaches have facilitated identification of important molecules that are deemed essential for HCV life cycle. The expanded set of putative virus and host-encoded targets has brought us one step closer to developing robust strategies for efficacious, pangenotypic, and well-tolerated medicines against HCV. Herein, we provide an overview of the development of various classes of virus and host-directed therapies that are currently in use along with others that are undergoing clinical evaluation.
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Affiliation(s)
- Muhammad Usman Ashraf
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan.,Virology Laboratory, Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Kanzal Iman
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan
| | - Muhammad Farhan Khalid
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan.,Department of Biomedical Engineering, University of Engineering and Technology, Lahore, Pakistan
| | - Hafiz Muhammad Salman
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan.,Plant Biotechnology Laboratory, Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan
| | - Talha Shafi
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan
| | - Momal Rafi
- Department of Statistics, University of Gujrat, Gujrat, Pakistan
| | - Nida Javaid
- Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan
| | - Rashid Hussain
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan
| | - Fayyaz Ahmad
- Department of Statistics, University of Gujrat, Gujrat, Pakistan
| | | | - Shaper Mirza
- Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan
| | - Muhammad Shafiq
- Plant Biotechnology Laboratory, Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan
| | - Samia Afzal
- Virology Laboratory, Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Sadia Hamera
- Department of Plant Genetics, Institute of Life Sciences, University of Rostock, Germany
| | - Saima Anwar
- Department of Biomedical Engineering, University of Engineering and Technology, Lahore, Pakistan
| | - Romena Qazi
- Department of Pathology, Shaukat Khanum Memorial Cancer Hospital & Research Centre, Lahore, Pakistan
| | - Muhammad Idrees
- Virology Laboratory, Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.,Hazara University, Mansehra, Pakistan
| | - Sohail A Qureshi
- Institute of Integrative Biosciences, CECOS-University of Information Technology and Emerging Sciences, Peshawar, Pakistan
| | - Safee Ullah Chaudhary
- Biomedical Informatics Research Laboratory, Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan
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17
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Mylopoulou T, Papadopoulos V, Kassela K, Karakasiliotis I, Souvalidou F, Mimidis P, Veletza S, Mavromara P, Mimidis K. Relationship between antibodies to hepatitis C virus core+1 protein and treatment outcome. Ann Gastroenterol 2018; 31:593-597. [PMID: 30174396 PMCID: PMC6102464 DOI: 10.20524/aog.2018.0290] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2018] [Accepted: 06/01/2018] [Indexed: 01/02/2023] Open
Abstract
Background It has been suggested that hepatitis C virus (HCV) core+1 protein plays a crucial role in the viral life cycle, potentially affecting liver cirrhosis and the development of hepatocellular carcinoma. Methods To investigate its relationship with the outcome of HCV standard combination therapy with peginterferon-α plus ribavirin, we screened 139 consecutive HCV patients (119 with chronic HCV infection and 20 who spontaneously cleared HCV) for the presence of anti-core+1 antibodies (Abs). In addition, liver fibrosis was determined by FibroScan in all but one patients. Results Twenty-nine patients were cirrhotic (stiffness >12.5 kPa, F4 METAVIR), all of them with mild liver cirrhosis (Child-Pugh score A). Eighty-six of 139 patients were treatment-experienced with standard combination therapy. Fifty of them had achieved a sustained virological response, while 36 were non-responders. The prevalence of anti-core+1 Abs in patients with chronic HCV infection was 22.69% (27/119 patients): 18% (9/50 patients) in responders and 36.11% (13/36 patients) in non-responders (P=0.050). Five (17.24%) of the 29 cirrhotic patients and 22 (24.72%) of the 89 non-cirrhotic patients were positive for anti-core+1 Abs (P=0.405). Furthermore, the presence of anti-core+1 Abs correlated with the poor response interleukin (IL) 28B genotype TT (P=0.040). No correlation between spontaneous clearance and anti-core+1 Abs was observed (P=0.088). Conclusion The presence of anti-core+1 Abs might be correlated with the poor response IL28B TT genotype and may negatively affect the outcome of standard combination treatments in HCV patients, suggesting that core+1 may play a biological role in the course of HCV infection.
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Affiliation(s)
- Theodora Mylopoulou
- First Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis (Theodora Mylopoulou, Konstantinos Mimidis), Greece
| | | | - Katerina Kassela
- Laboratory of Molecular Virology, Hellenic Pasteur Institute, Athens (Katerina Kassela, Penelope Mavromara), Greece
| | - Ioannis Karakasiliotis
- Laboratory of Medical Biology, Department of Medicine, Democritus University of Thrace, Alexandroupolis (Ioannis Karakasiliotis, Stavroula Veletza), Greece
| | - Fani Souvalidou
- Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis (Fani Souvalidou, Panagiotis Mimidis, Penelope Mavromara), Greece
| | - Panagiotis Mimidis
- Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis (Fani Souvalidou, Panagiotis Mimidis, Penelope Mavromara), Greece
| | - Stavroula Veletza
- Laboratory of Medical Biology, Department of Medicine, Democritus University of Thrace, Alexandroupolis (Ioannis Karakasiliotis, Stavroula Veletza), Greece
| | - Penelope Mavromara
- Laboratory of Molecular Virology, Hellenic Pasteur Institute, Athens (Katerina Kassela, Penelope Mavromara), Greece.,Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis (Fani Souvalidou, Panagiotis Mimidis, Penelope Mavromara), Greece
| | - Konstantinos Mimidis
- First Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis (Theodora Mylopoulou, Konstantinos Mimidis), Greece
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18
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Forni D, Cagliani R, Pontremoli C, Pozzoli U, Vertemara J, De Gioia L, Clerici M, Sironi M. Evolutionary Analysis Provides Insight Into the Origin and Adaptation of HCV. Front Microbiol 2018; 9:854. [PMID: 29765366 PMCID: PMC5938362 DOI: 10.3389/fmicb.2018.00854] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Accepted: 04/13/2018] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) belongs to the Hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. HCV origin was previously dated in a range between ∼200 and 1000 years ago. Hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. The closest relatives of HCV were found in horses/donkeys (equine hepaciviruses, EHV). However, the origin of HCV as a human pathogen is still an unsolved puzzle. Using a selection-informed evolutionary model, we show that the common ancestor of extant HCV genotypes existed at least 3000 years ago (CI: 3192–5221 years ago), with the oldest genotypes being endemic to Asia. EHV originated around 1100 CE (CI: 291–1640 CE). These time estimates exclude that EHV transmission was mainly sustained by widespread veterinary practices and suggest that HCV originated from a single zoonotic event with subsequent diversification in human populations. We also describe a number of biologically important sites in the major HCV genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. HCV exploits several cell-surface molecules for cell entry, but only two of these (CD81 and OCLN) determine the species-specificity of infection. Herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at CD81 were only observed in Chiroptera. No evidence of selection was detected for OCLN in any mammalian order. These results shed light on the origin of HCV and provide a catalog of candidate genetic modulators of HCV phenotypic diversity.
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Affiliation(s)
- Diego Forni
- Bioinformatics Laboratory, Scientific Institute IRCCS E.Medea, Bosisio Parini, Italy
| | - Rachele Cagliani
- Bioinformatics Laboratory, Scientific Institute IRCCS E.Medea, Bosisio Parini, Italy
| | - Chiara Pontremoli
- Bioinformatics Laboratory, Scientific Institute IRCCS E.Medea, Bosisio Parini, Italy
| | - Uberto Pozzoli
- Bioinformatics Laboratory, Scientific Institute IRCCS E.Medea, Bosisio Parini, Italy
| | - Jacopo Vertemara
- Department of Biotechnology and Biosciences, University of Milan-Bicocca, Milan, Italy
| | - Luca De Gioia
- Department of Biotechnology and Biosciences, University of Milan-Bicocca, Milan, Italy
| | - Mario Clerici
- Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.,Don C. Gnocchi Foundation Onlus, IRCCS, Milan, Italy
| | - Manuela Sironi
- Bioinformatics Laboratory, Scientific Institute IRCCS E.Medea, Bosisio Parini, Italy
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19
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Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis. J Virol 2018; 92:JVI.02036-17. [PMID: 29444947 DOI: 10.1128/jvi.02036-17] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2017] [Accepted: 02/06/2018] [Indexed: 02/06/2023] Open
Abstract
Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression.
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20
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Abstract
Reproduction of RNA viruses is typically error-prone due to the infidelity of their replicative machinery and the usual lack of proofreading mechanisms. The error rates may be close to those that kill the virus. Consequently, populations of RNA viruses are represented by heterogeneous sets of genomes with various levels of fitness. This is especially consequential when viruses encounter various bottlenecks and new infections are initiated by a single or few deviating genomes. Nevertheless, RNA viruses are able to maintain their identity by conservation of major functional elements. This conservatism stems from genetic robustness or mutational tolerance, which is largely due to the functional degeneracy of many protein and RNA elements as well as to negative selection. Another relevant mechanism is the capacity to restore fitness after genetic damages, also based on replicative infidelity. Conversely, error-prone replication is a major tool that ensures viral evolvability. The potential for changes in debilitated genomes is much higher in small populations, because in the absence of stronger competitors low-fit genomes have a choice of various trajectories to wander along fitness landscapes. Thus, low-fit populations are inherently unstable, and it may be said that to run ahead it is useful to stumble. In this report, focusing on picornaviruses and also considering data from other RNA viruses, we review the biological relevance and mechanisms of various alterations of viral RNA genomes as well as pathways and mechanisms of rehabilitation after loss of fitness. The relationships among mutational robustness, resilience, and evolvability of viral RNA genomes are discussed.
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21
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Morozov VA, Lagaye S. Hepatitis C virus: Morphogenesis, infection and therapy. World J Hepatol 2018; 10:186-212. [PMID: 29527256 PMCID: PMC5838439 DOI: 10.4254/wjh.v10.i2.186] [Citation(s) in RCA: 87] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2017] [Revised: 01/11/2018] [Accepted: 02/07/2018] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a major cause of liver diseases including liver cirrhosis and hepatocellular carcinoma. Approximately 3% of the world population is infected with HCV. Thus, HCV infection is considered a public healthy challenge. It is worth mentioning, that the HCV prevalence is dependent on the countries with infection rates around 20% in high endemic countries. The review summarizes recent data on HCV molecular biology, the physiopathology of infection (immune-mediated liver damage, liver fibrosis and lipid metabolism), virus diagnostic and treatment. In addition, currently available in vitro, ex vivo and animal models to study the virus life cycle, virus pathogenesis and therapy are described. Understanding of both host and viral factors may in the future lead to creation of new approaches in generation of an efficient therapeutic vaccine.
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Affiliation(s)
- Vladimir Alexei Morozov
- Center for HIV and Retrovirology, Department of Infectious Diseases, Robert Koch Institute, Berlin 13353, Germany
| | - Sylvie Lagaye
- Department of Immunology, Institut Pasteur, INSERM U1223, Paris 75015, France
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22
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Kassela K, Karakasiliotis I, Charpantidis S, Koskinas J, Mylopoulou T, Mimidis K, Sarrazin C, Grammatikos G, Mavromara P. High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis. J Gen Virol 2017; 98:1713-1719. [PMID: 28708052 DOI: 10.1099/jgv.0.000851] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD <15) and 164 chronic HCV patients without cirrhosis. 28.7 % of HCV patients with cirrhosis were positive for anti-core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (P<0.05). These findings, together with the high prevalence of anti-core+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.
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Affiliation(s)
- Katerina Kassela
- Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece.,Laboratory of Molecular Virology, Hellenic Pasteur Institute, Athens, Greece
| | - Ioannis Karakasiliotis
- Laboratory of Medical Biology, Department of Medicine, Democritus University of Thrace, Alexandroupolis, Greece.,Laboratory of Molecular Virology, Hellenic Pasteur Institute, Athens, Greece
| | - Stefanos Charpantidis
- Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece
| | - John Koskinas
- Department of Internal Medicine, Medical School of Athens, Hippokration Hospital Athens, Greece
| | - Theodora Mylopoulou
- First Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece
| | - Konstantinos Mimidis
- First Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece
| | - Christoph Sarrazin
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany
| | - Georgios Grammatikos
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany
| | - Penelope Mavromara
- Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece.,Laboratory of Molecular Virology, Hellenic Pasteur Institute, Athens, Greece
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23
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Zhao SN, Liu LL, Lv ZP, Wang XH, Wang CH. Network analysis of HBV‑ and HCV‑induced hepatocellular carcinoma based on Random Forest and Monte Carlo cross‑validation. Mol Med Rep 2017; 16:2411-2416. [PMID: 28656273 DOI: 10.3892/mmr.2017.6861] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Accepted: 03/28/2017] [Indexed: 11/06/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer‑associated mortality worldwide. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are two common risk factors for HCC. The majority of patients with HCC present at an advanced stage and are refractory to therapy. It is important to identify a method for efficient diagnosis at early stage. In the present study gene expression profile data, generated from microarray data, were pretreated according to the annotation files. The genes were mapped to pathways of Ingenuity Pathways Analysis. Dysregulated pathways and dysregulated pathway pairs were identified and constructed into individual networks, and a main network was constructed from individual networks with several edges. Random Forest (RF) classification was introduced to calculate the area under the curve (AUC) value of this network. Subsequently, 50 runs of Monte Carlo cross‑validation were used to screen the optimal main network. The results indicated that a total of 4,929 genes were identified in the pathways and gene expression profile. By combining dysregulated pathways with Z<0.05 and dysregulated pathway pairs with Z<0.2, individual networks were constructed. The optimal main network with the highest AUC value was identified. In the HCV group, the network was identified with an AUC value of 0.98, including 41 pairs of pathways, and in the HBV group, the network was identified with an AUC value of 0.94, including eight pairs of pathways. In addition, four pairs were identified in both groups. In conclusion, the optimal networks of HCV and HBV groups were identified with the highest AUC values. The use of these networks is expected to assist in diagnosing patients effectively at an early stage.
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Affiliation(s)
- Shan-Na Zhao
- Department of Clinical Laboratory, Yantaishan Hospital, Yantai, Shandong 264008, P.R. China
| | - Ling-Ling Liu
- Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated Shandong University, Jinan, Shandong 250021, P.R. China
| | - Zhi-Ping Lv
- Department of Clinical Laboratory, Yantaishan Hospital, Yantai, Shandong 264008, P.R. China
| | - Xiao-Hua Wang
- Department of Clinical Laboratory, Yantaishan Hospital, Yantai, Shandong 264008, P.R. China
| | - Cheng-Hong Wang
- Department of Clinical Laboratory, Yantaishan Hospital, Yantai, Shandong 264008, P.R. China
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Computational Prediction of the Heterodimeric and Higher-Order Structure of gpE1/gpE2 Envelope Glycoproteins Encoded by Hepatitis C Virus. J Virol 2017; 91:JVI.02309-16. [PMID: 28148799 DOI: 10.1128/jvi.02309-16] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2016] [Accepted: 01/25/2017] [Indexed: 12/24/2022] Open
Abstract
Despite the recent success of newly developed direct-acting antivirals against hepatitis C, the disease continues to be a global health threat due to the lack of diagnosis of most carriers and the high cost of treatment. The heterodimer formed by glycoproteins E1 and E2 within the hepatitis C virus (HCV) lipid envelope is a potential vaccine candidate and antiviral target. While the structure of E1/E2 has not yet been resolved, partial crystal structures of the E1 and E2 ectodomains have been determined. The unresolved parts of the structure are within the realm of what can be modeled with current computational modeling tools. Furthermore, a variety of additional experimental data is available to support computational predictions of E1/E2 structure, such as data from antibody binding studies, cryo-electron microscopy (cryo-EM), mutational analyses, peptide binding analysis, linker-scanning mutagenesis, and nuclear magnetic resonance (NMR) studies. In accordance with these rich experimental data, we have built an in silico model of the full-length E1/E2 heterodimer. Our model supports that E1/E2 assembles into a trimer, which was previously suggested from a study by Falson and coworkers (P. Falson, B. Bartosch, K. Alsaleh, B. A. Tews, A. Loquet, Y. Ciczora, L. Riva, C. Montigny, C. Montpellier, G. Duverlie, E. I. Pecheur, M. le Maire, F. L. Cosset, J. Dubuisson, and F. Penin, J. Virol. 89:10333-10346, 2015, https://doi.org/10.1128/JVI.00991-15). Size exclusion chromatography and Western blotting data obtained by using purified recombinant E1/E2 support our hypothesis. Our model suggests that during virus assembly, the trimer of E1/E2 may be further assembled into a pentamer, with 12 pentamers comprising a single HCV virion. We anticipate that this new model will provide a useful framework for HCV envelope structure and the development of antiviral strategies.IMPORTANCE One hundred fifty million people have been estimated to be infected with hepatitis C virus, and many more are at risk for infection. A better understanding of the structure of the HCV envelope, which is responsible for attachment and fusion, could aid in the development of a vaccine and/or new treatments for this disease. We draw upon computational techniques to predict a full-length model of the E1/E2 heterodimer based on the partial crystal structures of the envelope glycoproteins E1 and E2. E1/E2 has been widely studied experimentally, and this provides valuable data, which has assisted us in our modeling. Our proposed structure is used to suggest the organization of the HCV envelope. We also present new experimental data from size exclusion chromatography that support our computational prediction of a trimeric oligomeric state of E1/E2.
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Xiao W, Jiang LF, Deng XZ, Zhu DY, Pei JP, Xu ML, Li BJ, Wang CJ, Zhang JH, Zhang Q, Zhou ZX, Ding WL, Xu XD, Yue M. PD-1/PD-L1 signal pathway participates in HCV F protein-induced T cell dysfunction in chronic HCV infection. Immunol Res 2016; 64:412-23. [PMID: 26286967 DOI: 10.1007/s12026-015-8680-y] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Programmed cell death-1/programmed cell death-1 ligand 1 (PD-1/PD-L1) inhibitory signal pathway has been verified to be involved in the establishment of persistent viral infections. Blockade of PD-1/PD-L1 engagement to reinvigorate T cell activity is supposed to be a potential therapeutic scheme. Studies have verified the participation of PD-1/PD-L1 in hepatitis C virus (HCV) core protein-regulated immune response. To determine the roles of PD-1/PD-L1 signal pathway in HCV F protein-induced immunoreaction in chronic HCV infection, variations in T cells were examined. The results showed that PD-1 expression on CD8(+) and CD4(+) T cells was increased with HCV F stimulation in both chronic HCV patients and healthy controls, and could be reduced partly by PD-1/PD-L1 blocking. Additionally, by PD-1/PD-L1 blocking, HCV F-induced inhibition of T cell proliferation and promotion of cellular apoptosis were partly or even totally recovered. Furthermore, levels of both Th1 and Th2 cytokines were elevated in the presence of anti-PD-L1 antibody. All these results indicated that PD-1/PD-L1 signal pathway also participates in HCV F protein-induced immunoregulation. PD-1/PD-L1 blocking plays important roles in the restoration of effective functionality of the impaired T cells in chronic HCV patients.
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Affiliation(s)
- Wen Xiao
- School of Life Science and Technology, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, 210009, Jiangsu, China
| | - Long Feng Jiang
- Department of Infectious Diseases, The First Affiliated Hospital with Nanjing Medical University, Nanjing, 210002, China.
| | - Xiao Zhao Deng
- School of Life Science and Technology, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, 210009, Jiangsu, China.
- Huadong Research Institute for Medicine and Biotechnics, No. 293, Zhongshan East Road, Nanjing, 210002, China.
| | - Dan Yan Zhu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing, China
| | - Jia Ping Pei
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing, China
| | - Mao Lei Xu
- School of Life Science and Technology, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, 210009, Jiangsu, China
| | - Bing Jun Li
- Huadong Research Institute for Medicine and Biotechnics, No. 293, Zhongshan East Road, Nanjing, 210002, China
| | - Chang Jun Wang
- Huadong Research Institute for Medicine and Biotechnics, No. 293, Zhongshan East Road, Nanjing, 210002, China
| | - Jing Hai Zhang
- Huadong Research Institute for Medicine and Biotechnics, No. 293, Zhongshan East Road, Nanjing, 210002, China
| | - Qi Zhang
- Huadong Research Institute for Medicine and Biotechnics, No. 293, Zhongshan East Road, Nanjing, 210002, China
| | - Zhen Xian Zhou
- Department of Clinical Laboratory, Nanjing Second Hospital, Nanjing, China
| | - Wei Liang Ding
- Department of Clinical Laboratory, Yixing People's Hospital, Yixing, China
| | - Xiao Dong Xu
- Huadong Research Institute for Medicine and Biotechnics, No. 293, Zhongshan East Road, Nanjing, 210002, China
| | - Ming Yue
- School of Life Science and Technology, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, 210009, Jiangsu, China
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Tomei L, Altamura S, Paonessa G, De Francesco R, Migliaccio G. Review HCV Antiviral Resistance: The Impact of in vitro Studies on the Development of Antiviral Agents Targeting the Viral NS5B Polymerase. ACTA ACUST UNITED AC 2016; 16:225-45. [PMID: 16130521 DOI: 10.1177/095632020501600403] [Citation(s) in RCA: 49] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The high prevalence of the disease caused by hepatitis C virus (HCV) and the limited efficacy of interferon-based therapies have stimulated the search for safer and more effective drugs. The development of inhibitors of the HCV NS5B RNA polymerase represents a promising strategy for identifying novel anti-HCV therapeutics. However, the high genetic diversity, mutation rate and turnover of HCV are expected to favour the emergence of drug resistance, limiting the clinical usefulness of polymerase inhibitors. Thus, the characterization of the drug-resistance profile of these antiviral agents is considered crucial for identifying the inhibitors with a higher probability of clinical success. In the absence of an efficient in vitro infection system, HCV sub-genomic replicons have been used to study viral resistance to both nucleoside and non-nucleoside NS5B inhibitors. While these studies suggest that drug-resistant viruses are likely to evolve in vivo, they provide a wealth of information that should help in the identification of inhibitors with improved and distinct resistance profiles that might be used for combination therapy.
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Affiliation(s)
- Licia Tomei
- Istituto di Ricerche di Biologia Molecolare P Angeletti, Pomezia-Roma, Italy
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Park SB, Seronello S, Mayer W, Ojcius DM. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I. PLoS One 2016; 11:e0158419. [PMID: 27404108 PMCID: PMC4942120 DOI: 10.1371/journal.pone.0158419] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Accepted: 06/15/2016] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.
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Affiliation(s)
- Seung Bum Park
- School of Natural Sciences, University of California Merced, Merced, California, United States of America
| | - Scott Seronello
- School of Natural Sciences, University of California Merced, Merced, California, United States of America
| | - Wasima Mayer
- School of Natural Sciences, University of California Merced, Merced, California, United States of America
| | - David M. Ojcius
- School of Natural Sciences, University of California Merced, Merced, California, United States of America
- University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, California, United States of America
- * E-mail:
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Rafik M, Bakr S, Soliman D, Mohammed N, Ragab D, ElHady WA, Samir N. Characterization of differential antibody production against hepatitis C virus in different HCV infection status. Virol J 2016; 13:116. [PMID: 27357382 PMCID: PMC4928299 DOI: 10.1186/s12985-016-0572-9] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 06/22/2016] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND The Centers for Disease Control and Prevention (CDC) issued an update on hepatitis C virus (HCV) testing approach, in which it omitted the use of recombinant immunoblot assay (RIBA) in the diagnostic algorithm and recommended that future studies are needed to evaluate the performance of HCV testing without RIBA. As Egypt has the highest prevalence of HCV worldwide, we aimed to evaluate the value of RIBA in HCV testing in a high prevalence population. Our objective was to clarify whether enzyme linked immunosorbent assay (ELISA) anti-HCV signal-to-cutoff (S/CO) ratios were able to discriminate true positive from false positive anti-HCV antibody status and to evaluate the role of RIBA in solving this problem which may lead to a redefined strategy for diagnosis of HCV infection. Our second objective was to elucidate the effects of different HCV peptides of both structural and non-structural proteins on the humoral immune response to HCV infection. METHODS The current study drew results from 167 individuals divided into three groups: Group I: included 77 HCV antibody positive (ELISA) high risk health care workers (HCW), Group II: included 56 presumably uninfected individuals who showed normal liver enzymes, negative HCV RNA and were asymptomatic. Their ELISA HCV antibody S/C ratio ranged from 0.9 to <5. Group III: included 34 patients enrolled from outpatient clinics of Ain Shams Hospital with persistent viral replication, elevated liver enzymes, and chronic HCV related liver disease. All study participants were assessed for the presence of anti-HCV antibodies by 3(rd) generation ELISA which was confirmed by RIBA. RESULTS Interpreting the results of both ELISA and RIBA together, false positive results were highly significantly increased in HCW when compared with the other two groups. Indeterminate and false negative results were only found in the presumably uninfected group. For differentiated antibody responses by RIBA, chronic HCV cases had the highest frequency of positive antibody response to core peptides while the presumably uninfected group had the lowest. Antibody response to E2 was found less frequently in chronic cases than Core 1, Core 2 and NS3. The specific antibody response to the different HCV peptides showed the same distribution of frequencies in both chronic HCV cases and the presumably uninfected individuals with the chronic cases having the highest frequencies. This distribution was different from the HCW. The most evident difference was the reaction towards NS3 which was the highest antibody producing peptide in chronic HCV and presumably uninfected individuals whereas in HCW Core1 was the highest. CONCLUSION The HCV antibody immunoblot assay (RIBA) is still necessary for the detection of false positive cases which can occur quite frequently in countries of high prevalence as Egypt. Indeterminate RIBA results indicate a waning antibody response in elderly individuals who recovered from previous or distant HCV infection.
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Affiliation(s)
- Mona Rafik
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Salwa Bakr
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Dina Soliman
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Nesrine Mohammed
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Dina Ragab
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt.
| | - Walid Abd ElHady
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Nancy Samir
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
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Zhu DY, Deng XZ, Jiang LF, Xiao W, Pei JP, Li BJ, Wang CJ, Zhang JH, Zhang Q, Zhou ZX, Ding WL, Xu XD, Yue M. Potential Role of Hepatitis C Virus Alternate Reading Frame Protein in Negative Regulation of T-Bet Gene Expression. Inflammation 2016; 38:1823-34. [PMID: 25894282 DOI: 10.1007/s10753-015-0160-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Hepatitis C virus (HCV) is a major cause of chronic liver disease and has led to cirrhosis or hepatocellular carcinoma in a majority of infected individuals. We have previously demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias in chronic hepatitis C (CHC) patients, and we aimed to explore the relative molecular mechanisms here. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA-3 expression levels were analyzed in peripheral blood mononuclear cells (PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of gamma interferon (IFN-γ) in sera, were decreased in anti-F protein antibody seropositive patients compared with anti-F protein antibody seronegative patients, whereas the levels of GATA-3 did not show difference between the two groups. Moreover, the decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV core/F protein stimulation with constant STAT1/6 expression. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV F protein, and the disruption of STAT phosphorylation may participate in this mediation.
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Affiliation(s)
- Dan Yan Zhu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing, China
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Piedade D, Azevedo-Pereira JM. MicroRNAs, HIV and HCV: a complex relation towards pathology. Rev Med Virol 2016; 26:197-215. [PMID: 27059433 DOI: 10.1002/rmv.1881] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Revised: 03/11/2016] [Accepted: 03/15/2016] [Indexed: 12/13/2022]
Abstract
MicroRNAs are small non-coding RNAs that modulate protein production by post-transcriptional gene regulation. They impose gene expression control by interfering with mRNA translation and stability in cell cytoplasm through a mechanism involving specific binding to mRNA based on base pair complementarity. Because of their intracellular replication cycle it is no surprise that viruses evolved in a way that allows them to use microRNAs to infect, replicate and persist in host cells. Several ways of interference between virus and host-cell microRNA machinery have been described. Most of the time, viruses drastically alter host-cell microRNA expression or synthesize their own microRNA to facilitate infection and pathogenesis. HIV and HCV are two prominent examples of this complex interplay revealing how fine-tuning of microRNA expression is crucial for controlling key host pathways that allow viral infection and replication, immune escape and persistence. In this review we delve into the mechanisms underlying cellular and viral-encoded microRNA functions in the context of HIV and HCV infections. We focus on which microRNAs are differently expressed and deregulated upon viral infection and how these alterations dictate the fate of virus and cell. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Diogo Piedade
- Host-Pathogen Interaction Unit, iMed.ULisboa, Faculdade de Farmácia, Universidade de Lisboa, Portugal
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31
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Khachatoorian R, French SW. Chaperones in hepatitis C virus infection. World J Hepatol 2016; 8:9-35. [PMID: 26783419 PMCID: PMC4705456 DOI: 10.4254/wjh.v8.i1.9] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2015] [Revised: 10/01/2015] [Accepted: 12/18/2015] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases: (1) binding and internalization; (2) cytoplasmic release and uncoating; (3) viral polyprotein translation and processing; (4) RNA genome replication; (5) encapsidation (packaging) and assembly; and (6) virus morphogenesis (maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses.
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Comparative Immunogenicity in Rabbits of the Polypeptides Encoded by the 5' Terminus of Hepatitis C Virus RNA. J Immunol Res 2015; 2015:762426. [PMID: 26609538 PMCID: PMC4644844 DOI: 10.1155/2015/762426] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Accepted: 09/29/2015] [Indexed: 12/26/2022] Open
Abstract
Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1–173, 1–152, and 147–191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147–191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147–191 reached 105; core aa 1–152, 5 × 105; core aa 1–173 and F-protein, 106. Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1–152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.
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Xiao W, Zhang Q, Deng XZ, Jiang LF, Zhu DY, Pei JP, Ge CY, Li BJ, Wang CJ, Zhang JH, Zhou ZX, Ding WL, Xu XD, Yue M. HCV F protein amplifies the predictions of IL-28B and CTLA-4 polymorphisms about the susceptibility and outcomes of HCV infection in Southeast China. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2015; 34:52-60. [PMID: 26079279 DOI: 10.1016/j.meegid.2015.06.016] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/07/2015] [Revised: 06/01/2015] [Accepted: 06/12/2015] [Indexed: 12/20/2022]
Abstract
Cytotoxic T lymphocyte associated antigen-4(CTLA-4) is an inhibitory receptor with great value in the progression of hepatitis C virus (HCV) infection related diseases. To determine the potential associations of IL-28B rs12979860 and CTLA-4 rs231775, rs3087243 and rs5742909 polymorphisms with the generation of HCV F protein, susceptibility and outcomes of HCV infection, a total of 375 healthy controls, 219 HCV spontaneous recovered patients and 600 chronic HCV patients from Southeast China were recruited and genotyped in this study. And the relative mRNA levels of CTLA-4 in T cells were detected. Logistic regression analysis showed that rs231775 A allele was associated with significantly higher rate of spontaneous viral clearance in anti-HCV F antibody negative patients (adjusted OR=0.512, P=0.008), but allele A was related to higher mRNA level of CTLA-4 with the generation of HCV F protein. And rs5742909 T allele added up to the risk of HCV infection chronicity significantly in patients with the presence of HCV F protein (adjusted OR=2.698, P=0.003). Also, the rs5742909 CC genotype, along with the presence of HCV F protein, indicated a significantly higher CTLA-4 level than that in anti-HCV F antibody negative patients. The AG+AA genotype of rs3087243 significantly increased the susceptibility to HCV infection in subjects over 56 years old (adjusted OR=1.595, P=0.011). Genotype-genotype interaction between IL-28B rs12979860 and CTLA-4 rs3087243 was found to be significantly associated with increased susceptibility to HCV infection (adjusted OR=1.509, P=0.005). Haplotype analysis in CTLA-4 also showed significant association with the generation of HCV F protein. All these results indicated the importance of IL-28B and CTLA-4 polymorphisms and their associations with HCV F protein in the risk and chronicity of HCV infection in Chinese Han population in Southeast China.
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Affiliation(s)
- Wen Xiao
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
| | - Qi Zhang
- Huadong Research Institute for Medicine and Biotechnics, Nanjing, China
| | - Xiao Zhao Deng
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China; Huadong Research Institute for Medicine and Biotechnics, Nanjing, China.
| | - Long Feng Jiang
- Department of Infectious Diseases, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China
| | - Dan Yan Zhu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing, China
| | - Jia Ping Pei
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, Nanjing, China
| | - Chi Yu Ge
- Jiangsu Food & Pharmaceutical Science College, Huaian, China
| | - Bing Jun Li
- Huadong Research Institute for Medicine and Biotechnics, Nanjing, China
| | - Chang Jun Wang
- Huadong Research Institute for Medicine and Biotechnics, Nanjing, China.
| | - Jing Hai Zhang
- Huadong Research Institute for Medicine and Biotechnics, Nanjing, China
| | - Zhen Xian Zhou
- Department of Clinical Laboratory, Nanjing Second Hospital, Nanjing, China
| | - Wei Liang Ding
- Department of Clinical Laboratory, Yixing People's Hospital, Yixing, China
| | - Xiao Dong Xu
- Huadong Research Institute for Medicine and Biotechnics, Nanjing, China
| | - Ming Yue
- School of Life Science and Technology, China Pharmaceutical University, Nanjing, China
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Immunization with Recombinant Adenoviral Vectors Expressing HCV Core or F Proteins Leads to T Cells with Reduced Effector Molecules Granzyme B and IFN-γ: A Potential New Strategy for Immune Evasion in HCV Infection. Viral Immunol 2015; 28:309-24. [DOI: 10.1089/vim.2015.0009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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Shehat MG, Bahey-El-Din M, Kassem MA, Farghaly FA, Abdul-Rahman MH, Fanaki NH. Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients. Arch Virol 2015; 160:1939-52. [PMID: 26036563 DOI: 10.1007/s00705-015-2465-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2014] [Accepted: 05/23/2015] [Indexed: 01/27/2023]
Abstract
HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.
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Affiliation(s)
- Michael G Shehat
- Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
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Hashempour T, Bamdad T, Bergamini A, Lavergne JP, Haj-Sheykholeslami A, Brakier-Gingras L, Ajorloo M, Merat S. F protein increases CD4+CD25+ T cell population in patients with chronic hepatitis C. Pathog Dis 2015; 73:ftv022. [PMID: 25862675 DOI: 10.1093/femspd/ftv022] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/17/2015] [Indexed: 01/28/2023] Open
Abstract
HCV is a global health problem with an estimated 230 million chronically infected people worldwide. It has been reported that a 17-kd protein translated from core-encoding genomic region can contribute to immune-mediated mechanisms associated with the development of the chronic disease. Also, Treg cells can be contributed to an inadequate response against the viruses, leading to chronic infection. Here we evaluated the ability of protein F to modulate the frequency of CD4+CD25+FoxP3+T and IL-10+T cells in patients with chronic HCV infection. F gene was amplified and cloned in the expression vector. The protein was purified and used for stimulation of PBMCs in the HCV chronic patients and the control groups. The frequency of CD4+CD25+FoxP3+ T cell-like populations and IL-10-producing CD4+CD25+ T cells was assessed in the HCV-infected patients and in the healthy controls by flow cytometry, which showed an increase of both CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells in the HCV-infected patients positive for anti-F antibody. Our results suggest the potential involvement of F and core antigens in increasing the frequency of CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells which may be associated with HCV-persistent infection.
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Affiliation(s)
- Tayebeh Hashempour
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, 14115-331 Tehran, Iran Digestive Disease Research Center, Shariati Hospital, Tehran University of Medical Sciences, 14117 Tehran, Iran
| | - Taravat Bamdad
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, 14115-331 Tehran, Iran
| | - Alberto Bergamini
- Department of Internal Medicine, University of Rome Tor Vergata, Via Montpellier 1,00133 Rome, Italy
| | - Jean Pierre Lavergne
- Laboratoire de Bioinformatique et RMN structurales, Institut de Biologie et chimie des protéines, UMR 5086 CNRS, Université Claude Bernard Lyon I
| | - Arghavan Haj-Sheykholeslami
- Digestive Disease Research Center, Shariati Hospital, Tehran University of Medical Sciences, 14117 Tehran, Iran
| | - Léa Brakier-Gingras
- Département de Biochimie et Médecine Moléculaire, Faculté de Médecine, Université de Montréal, Pavillon Roger-Gaudry, bureau E-519, C.P. 6128, Succ. Centre-ville, Montréal, Québec
| | - Mehdi Ajorloo
- Digestive Disease Research Center, Shariati Hospital, Tehran University of Medical Sciences, 14117 Tehran, Iran
| | - Shahin Merat
- Digestive Disease Research Center, Shariati Hospital, Tehran University of Medical Sciences, 14117 Tehran, Iran
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Zhu DY, Jiang LF, Deng XZ, Xiao W, Pei JP, Li BJ, Wang CJ, Zhang JH, Zhang Q, Zhou ZX, Ding WL, Xu XD, Yue M. TBX21 polymorphisms are associated with virus persistence in hepatitis C virus infection patients from a high-risk Chinese population. Eur J Clin Microbiol Infect Dis 2015; 34:1309-18. [PMID: 25759111 DOI: 10.1007/s10096-015-2337-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2014] [Accepted: 01/22/2015] [Indexed: 01/29/2023]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and the varied outcomes of the infection depend on both viral and host factors. We have demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias which is involved in virus persistence in chronic hepatitis C (CHC) patients. The purpose of this study was to test the hypothesis that genetic variants of TBX21 (T cell specific T-box transcription factor) were associated with the outcomes of HCV infection and F protein generation. Three single nucleotide polymorphisms (SNPs) (rs17250932, rs2074190, rs4794067) in the TBX21 gene were genotyped in a case-control study in a cohort of a high-risk group, including 354 healthy controls and 747 CHC patients (190 anti-F protein antibody seronegative patients and 557 anti-F protein antibody seropositive patients). Results showed that the rs4794067 C allele in the TBX21 promoter was significantly more common in CHC patients (OR = 1.335, 95% CI = 1.058-1.684, P = 0.015), exceptionally in anti-F protein seropositive patients (OR = 1.547, 95% CI = 1.140-2.101, P = 0.005), compared with healthy controls. And the risk effect was also significantly high in patients with HCV 1b genotype and mild fibrosis (P = 0.021, P = 0.010, respectively). Compared with the most frequent haplotype TAT, haplotype analysis showed that the distribution of TAC was significantly different between the chronic HCV carrier group and the healthy group, and so was the anti-F antibody seronegativity group and the anti-F antibody seronegativity group (all P < 0.001). Our results suggested that TBX21 variants may be involved in the etiology of this disease.
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Affiliation(s)
- D Y Zhu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical University, No. 293, Zhongshan East Road, Nanjing, 210002, China
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Expression of the novel hepatitis C virus core+1/ARF protein in the context of JFH1-based replicons. J Virol 2015; 89:5164-70. [PMID: 25694591 DOI: 10.1128/jvi.02351-14] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2014] [Accepted: 02/09/2015] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.
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Afzal MS, Alsaleh K, Farhat R, Belouzard S, Danneels A, Descamps V, Duverlie G, Wychowski C, Zaidi NUSS, Dubuisson J, Rouillé Y. Regulation of core expression during the hepatitis C virus life cycle. J Gen Virol 2015; 96:311-321. [DOI: 10.1099/vir.0.070433-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Affiliation(s)
- Muhammad Sohail Afzal
- Atta ur Rahman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), Islamabad, Pakistan
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Khaled Alsaleh
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Rayan Farhat
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Sandrine Belouzard
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Adeline Danneels
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Véronique Descamps
- EA4294, Unité de Virologie Clinique et Fondamentale, CHU d’Amiens, University of Picardie Jules Verne, Amiens, France
| | - Gilles Duverlie
- EA4294, Unité de Virologie Clinique et Fondamentale, CHU d’Amiens, University of Picardie Jules Verne, Amiens, France
| | - Czeslaw Wychowski
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Najam us Sahar Sadaf Zaidi
- Atta ur Rahman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), Islamabad, Pakistan
| | - Jean Dubuisson
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
| | - Yves Rouillé
- Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France
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Determination of the proteins encoded by BmBDV VD1-ORF4 and their interacting proteins in BmBDV-infected midguts. Curr Microbiol 2015; 70:623-9. [PMID: 25561406 DOI: 10.1007/s00284-014-0765-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2014] [Accepted: 11/14/2014] [Indexed: 12/23/2022]
Abstract
Bombyx mori bidensovirus (BmBDV) VD1-ORF4 consists of 3,318 nucleotides, which codes for a predicted protein with molecular weight of about 127 kDa. However, the authentic proteins encoded by VD1-ORF4 in silkworm midguts infected with BmBDV and their interacting proteins are still unclear. In this study, Western blot analysis revealed that a 127-kDa protein was confirmed to be translated from the VD1-ORF4 transcript using polyclonal antibodies and monoclonal antibodies against VD1-ORF4 deduced amino acid. Moreover, four smaller proteins with molecular weight of about 70, 60, 53, and 42 kDa were also examined in the infected midguts. Transient expression assay indicated that the expression amount of VD1-ORF4 fused with egfp was at least 30-fold lower than that of egfp gene, and immunofluorescence staining result indicated that these proteins encoded by VD1-ORF4 were located in both the cytoplasm and nucleus. Co-immunoprecipitation result showed that Aminopeptidase and Heat shock protein 90 can be captured by these proteins encoded by VD1-ORF4. In conclusion, multiple proteins were produced from the transcripts of VD1-ORF4 gene by an uncertain expression strategy, which may play important roles in viral replication and assembly.
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Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77. J Virol 2014; 89:811-23. [PMID: 25355880 DOI: 10.1128/jvi.02877-14] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
UNLABELLED The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. IMPORTANCE Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.
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Razzaq Z, Malik A. Viral load is associated with abnormal serum levels of micronutrients and glutathione and glutathione-dependent enzymes in genotype 3 HCV patients. BBA CLINICAL 2014; 2:72-8. [PMID: 26674880 PMCID: PMC4633942 DOI: 10.1016/j.bbacli.2014.09.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/25/2014] [Revised: 09/12/2014] [Accepted: 09/24/2014] [Indexed: 01/03/2023]
Abstract
Background Oxidative stress in hepatitis C patients has been linked to hepatitis C virus. We verified this assumption in HCV genotype 3 patients by detecting the relationship between viral load and certain specific oxidative stress markers like Cu, Mn, Fe, Se, Zn and glutathione and glutathione-dependent enzymes. Method Subjects (n = 200, average age 24 years) with quantitative HCV RNA polymerase chain reaction-proven genotype 3 hepatitis C were simultaneously evaluated. Cu, Mn, Fe, Se and Zn serum levels were by using atomic absorption spectrophotometer. Internationally accepted methods were used for viral load quantification of glutathione, GR and Gpx serum levels. Result There was a significant correlation between HCV viral load and studied parameters. With the increase of viral load from mild group (200,000–1,000,000 copies/ml) to severe group (5,000,000–25,000,000 copies/ml) the serum levels of Cu, Mn, Zn, and Fe and glutathione reductase were found to be abnormally high. However, in severe viral load group serum concentration of Se and glutathione was less than the healthy controls. Conclusion As a significant correlation was detected between the study parameters in genotype 3 HCV patients, it is concluded that the studied micronutrients and glutathione and glutathione-dependent enzymes are the biomolecular targets of HCV to induce oxidative stress. General significance Constant monitoring and regulation of the recommended biomolecular targets of HCV can improve the plight of more than 170 million patients suffering from hepatitis C virus around the globe.
Viral load, micronutrients, antioxidants are key factors involved in oxidative stress. We investigated viral load GSH, GPx, GR, Cu, Mn, Zn, Se, and Fe in genotype 3 HCV patients. Abnormal levels of GSH, GPx, Gr and micronutrients are linked with the progression of hepatitis C. These parameters are associated to immune response, mitochondrial damage and inflammation.
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Ren Q, Au HHT, Wang QS, Lee S, Jan E. Structural determinants of an internal ribosome entry site that direct translational reading frame selection. Nucleic Acids Res 2014; 42:9366-82. [PMID: 25038250 PMCID: PMC4132737 DOI: 10.1093/nar/gku622] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome.
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Affiliation(s)
- Qian Ren
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Hilda H T Au
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Qing S Wang
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Seonghoon Lee
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Eric Jan
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
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Choi J, Corder NLB, Koduru B, Wang Y. Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma. Free Radic Biol Med 2014; 72:267-84. [PMID: 24816297 PMCID: PMC4099059 DOI: 10.1016/j.freeradbiomed.2014.04.020] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/01/2014] [Revised: 04/16/2014] [Accepted: 04/18/2014] [Indexed: 02/08/2023]
Abstract
Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H2O2 formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor β. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC.
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Affiliation(s)
- Jinah Choi
- School of Natural Sciences, University of California at Merced, Merced, CA 95343, USA.
| | - Nicole L B Corder
- School of Natural Sciences, University of California at Merced, Merced, CA 95343, USA
| | - Bhargav Koduru
- School of Natural Sciences, University of California at Merced, Merced, CA 95343, USA
| | - Yiyan Wang
- School of Natural Sciences, University of California at Merced, Merced, CA 95343, USA
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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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Romero-López C, Berzal-Herranz A. Structure-function relationship in viral RNA genomes: The case of hepatitis C virus. World J Med Genet 2014; 4:6-18. [DOI: 10.5496/wjmg.v4.i2.6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Revised: 01/23/2014] [Accepted: 04/03/2014] [Indexed: 02/06/2023] Open
Abstract
The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus (HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5’-untranslatable regions (5’UTRs) and 3’UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5’ terminus of the viral genome and regulated by distal functional RNA domains placed at the 3’ end. Subsequent RNA replication strongly depends on the 3’UTR folding and is also influenced by the 5’ end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNA-RNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.
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Fan X, Xue B, Dolan PT, LaCount DJ, Kurgan L, Uversky VN. The intrinsic disorder status of the human hepatitis C virus proteome. MOLECULAR BIOSYSTEMS 2014; 10:1345-63. [PMID: 24752801 DOI: 10.1039/c4mb00027g] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Many viral proteins or their biologically important regions are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions do not possess unique structures and possess functions that complement the functional repertoire of "normal" ordered proteins and domains, with many protein functional classes being heavily dependent on the intrinsic disorder. Viruses commonly use these highly flexible regions to invade the host organisms and to hijack various host systems. These disordered regions also help viruses in adapting to their hostile habitats and to manage their economic usage of genetic material. In this article, we focus on the structural peculiarities of proteins from human hepatitis C virus (HCV) and use a wide spectrum of bioinformatics techniques to evaluate the abundance of intrinsic disorder in the completed proteomes of several human HCV genotypes, to analyze the peculiarities of disorder distribution within the individual HCV proteins, and to establish potential roles of the structural disorder in functions of ten HCV proteins. We show that the intrinsic disorder or increased flexibility is not only abundant in these proteins, but is also absolutely necessary for their functions, playing a crucial role in the proteolytic processing of the HCV polyprotein, the maturation of the individual HCV proteins, and being related to the posttranslational modifications of these proteins and their interactions with DNA, RNA, and various host proteins.
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Affiliation(s)
- Xiao Fan
- Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta AB T6G 2V4, Canada.
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XU XIAODONG, YU XIAOJIE, DENG XIAOZHAO, YUE MING, ZHANG JINHAI, ZHU DANYAN, ZHOU ZHENXIAN, ZHAI XIANGJUN, XU KE, ZHANG YUN. Hepatitis C virus alternate reading frame protein decreases interferon-α secretion in peripheral blood mononuclear cells. Mol Med Rep 2014; 9:730-736. [DOI: https:/doi.org/10.3892/mmr.2013.1816] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2023] Open
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Samrat SK, Li W, Singh S, Kumar R, Agrawal B. Alternate reading frame protein (F protein) of hepatitis C virus: paradoxical effects of activation and apoptosis on human dendritic cells lead to stimulation of T cells. PLoS One 2014; 9:e86567. [PMID: 24475147 PMCID: PMC3903568 DOI: 10.1371/journal.pone.0086567] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2013] [Accepted: 12/11/2013] [Indexed: 12/24/2022] Open
Abstract
Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans.
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Affiliation(s)
- Subodh Kumar Samrat
- Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Wen Li
- Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Shakti Singh
- Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Rakesh Kumar
- Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Babita Agrawal
- Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
- * E-mail:
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