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Shahid I, Khan KM, Khan TM, Imran MS, Shahid N, Alnafoosi FN, Baig MR, Al-Worafi YM. Clinical Efficacy of LivPro® Herbal Medicine among Hepatitis C Patients Pakistan: Longitudinal Interventional Study. ARCHIVES OF PHARMACY PRACTICE 2021. [DOI: 10.51847/v5lmbrlqnq] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
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2
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Noreen S, Hussain I, Tariq MI, Ijaz B, Iqbal S, Qamar-ul-Zaman, Ashfaq UA, Husnain T. Portulaca oleracea L. as a Prospective Candidate Inhibitor of Hepatitis C Virus NS3 Serine Protease. Viral Immunol 2015; 28:282-9. [PMID: 25871297 DOI: 10.1089/vim.2014.0079] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a worldwide health problem affecting about 300 million individuals. HCV causes chronic liver disease, liver cirrhosis, hepatocellular carcinoma, and death. Many side effects are associated with the current treatment options. Natural products that can be used as anti-HCV drugs are thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for the screening of antiviral activity against HCV. The present work explores plants with anti-HCV potential, isolating possible lead compounds. Ten plants, used for medicinal purposes against different infections in rural areas of Pakistan, were collected. The cellular toxicity effects of methanolic extracts of the plants on the viability of Huh-7 cells were studied through the Trypan blue dye exclusion method. Following this, the anti-HCV potential of phytoextracts was assessed by infecting liver cells with HCV-3a-infected serum inoculum. Only the methanolic extract of Portulaca oleracea L. (PO) exhibited more than 70% inhibition. Four fractions were obtained through bioassay-guided extraction of PO. Subsequent inhibition of all organic extract fractions against NS3 serine protease was checked to track the specific target in the virus. The results showed that the PO methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. Hence, PO extract and its constituents either alone or with interferon could offer a future option to treat chronic HCV.
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Affiliation(s)
- Sobia Noreen
- 1Department of Chemistry, University of Sargodha, Sargodha, Pakistan
| | - Ishtiaq Hussain
- 1Department of Chemistry, University of Sargodha, Sargodha, Pakistan
| | | | - Bushra Ijaz
- 2Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan
| | - Shahid Iqbal
- 1Department of Chemistry, University of Sargodha, Sargodha, Pakistan
| | - Qamar-ul-Zaman
- 3Department of Pharmacy, University of Sargodha, Sargodha, Pakistan
| | - Usman Ali Ashfaq
- 4Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, Pakistan
| | - Tayyab Husnain
- 2Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan
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3
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Hartjen P, Höchst B, Heim D, von der Kammer H, Lucke J, Reinholz M, Baier A, Smeets R, Wege H, Borowski P, Schulze Zur Wiesch J. The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity. Cell Mol Biol Lett 2013; 18:447-58. [PMID: 23893289 PMCID: PMC6275940 DOI: 10.2478/s11658-013-0099-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2013] [Accepted: 07/17/2013] [Indexed: 11/20/2022] Open
Abstract
Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC₅₀-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC₅₀ in this assay, which compares well with our result for NS3h-D1316A (IC₅₀ = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.
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Affiliation(s)
- Philip Hartjen
- Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.
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4
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Subgenomic HCV RNA replication and its localization in the nucleus of the infected cells. Saudi J Biol Sci 2011; 18:213-8. [PMID: 23961127 DOI: 10.1016/j.sjbs.2011.01.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2010] [Revised: 01/30/2011] [Accepted: 01/30/2011] [Indexed: 11/22/2022] Open
Abstract
Cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. It is known that HCV contains proteins which interact with host cell proteins. To see whether a HCV RNA replicon can interact in the same way with host cell proteins, HCV RNA replicon was transfected in Huh7 cells. In most infected cells, HCV replicon is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own is positive for NS3. The presence of NS3 in the nuclei of Huh7 cells indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.
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Liefhebber JMP, Hensbergen PJ, Deelder AM, Spaan WJM, van Leeuwen HC. Characterization of hepatitis C virus NS3 modifications in the context of replication. J Gen Virol 2009; 91:1013-8. [PMID: 19923258 DOI: 10.1099/vir.0.016881-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV non-structural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.
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Affiliation(s)
- Jolanda M P Liefhebber
- Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
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Hartjen P, Medom BK, Reinholz M, Borowski P, Baier A. Regulation of the biochemical function of motif VI of HCV NTPase/helicase by the conserved Phe-loop. Biochimie 2009; 91:252-60. [DOI: 10.1016/j.biochi.2008.09.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2008] [Accepted: 09/26/2008] [Indexed: 11/29/2022]
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Abstract
Hepatitis C virus may cause hepatic and extrahepatic diseases. Extrahepatic manifestations range from disorders for which a significant association with viral infection is supported by epidemiologic and pathogenetic data, to anecdotal observations without clear proof of causality. This article describes the diagnosis and treatment of these diseases.
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9
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Viral NS3 helicase activity is inhibited by peptides reproducing the Arg-rich conserved motif of the enzyme (motif VI). Biochem Pharmacol 2008; 76:28-38. [PMID: 18479669 DOI: 10.1016/j.bcp.2008.03.018] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2008] [Revised: 03/28/2008] [Accepted: 03/31/2008] [Indexed: 11/23/2022]
Abstract
The NTPase/helicase of Flaviviridae viruses is one of the essential components of their replication complex. The enzyme is defined by the presence of seven highly conserved amino acid motifs. Random screening of numerous hepatitis C virus (HCV) derived peptides, revealed a basic amino acid stretch corresponding to motif VI of the HCV NTPase/helicase (amino acids 1487-1500 of the HCV polyprotein). This peptide inhibited the unwinding activity of the enzyme with an IC(50)=0.2 microM. Peptides corresponding to motif VI of HCV, West Nile virus (WNV) and Japanese encephalitis virus (JEV) were synthesized and tested as inhibitors of NTPase and unwinding reactions mediated by the viral enzymes. Peptides distinguished in regard to their length and structure. Between the peptides tested HCV(1487-1500) reproducing the sequence of motif VI was the most potent inhibitor of helicase activities of investigated enzymes. Other respective peptides were rather modest inhibitors. The examined peptides inhibited the Flaviviridae helicases in the following order of potency: HCV(1487-1500)>WNV(1959-1572)>JEV(1962-1975). Interestingly, the susceptibility of the helicase activity to the inhibition by the peptides was similar and in the row: HCV>WNV>JEV. The inhibition results from binding and blockade of the active site of the enzyme lyes beyond the NTP-binding and hydrolyzing site. The kinetic analyses indicated that the binding of the peptides do not interfere with the NTPase activity of the enzymes. The peptide may serve as effective and selective tool to reduce the virus propagation.
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Abstract
In recent years, the effects of hepatitis C virus (HCV) proteins on hepatocarcinogenesis have undergone intense investigations. The potentially oncogenic proteins include at least three HCV proteins: core (C) protein, NS3, and NS5A. Several authors indicated relationships between subcellular localization, concentration, a specific molecular form of the proteins (full length, truncated, phosphorylated), the presence of specific domains (the nuclear localization signal homologous to e.g. Bcl-2) and their effects on the mechanisms linked to oncogenesis. The involvement of all the proteins has been described as being in control of the cell cycle, through interactions with key proteins of the process (p53, p21, cyclins, proliferating cell nuclear antigen), transcription factors, proto-oncogenes, growth factors/cytokines and their receptors, and proteins linked to the apoptotic process. Untilnow, the involvement of the core protein of HCV in liver carcinogenesis is the most recognized. One of the most common proteins affected by HCV proteins is the p53 tumor-suppressor protein. The p21/WAF1 gene is a major target of p53, and the effect of HCV proteins on the gene is frequently considered in parallel. The results of studies on the effects of HCV proteins on the apoptotic process are controversial. This work summarizes the information collected thus far in the field of HCV molecular virology and principal intracellular signaling pathways in which HCV oncogenic proteins are involved.
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Affiliation(s)
- Aldona Kasprzak
- Department of Histology and Embryology, Medical University, Poznań, Poland
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11
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Zignego AL, Ferri C, Pileri SA, Caini P, Bianchi FB. Extrahepatic manifestations of Hepatitis C Virus infection: a general overview and guidelines for a clinical approach. Dig Liver Dis 2007; 39:2-17. [PMID: 16884964 DOI: 10.1016/j.dld.2006.06.008] [Citation(s) in RCA: 181] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/28/2005] [Revised: 06/03/2006] [Accepted: 06/06/2006] [Indexed: 02/08/2023]
Abstract
Hepatitis C Virus is associated with a wide series of extrahepatic manifestations. Based on available data the link between the virus and some of these extrahepatic diseases is only suggested and needs further confirmation. Hepatitis C Virus-related lymphoproliferative disorders, whose prototype is mixed cryoglobulinaemia, represent the most closely related extrahepatic manifestations of Hepatitis C Virus. Other Hepatitis C Virus-associated disorders include nephropathies, thyreopathies, sicca syndrome, idiopathic pulmonary fibrosis, porphyria cutanea tarda, lichen planus, diabetes, chronic polyarthritis, cardiopathy and atherosclerosis. A pathogenetic link between Hepatitis C Virus and some extrahepatic manifestations was confirmed by their responsiveness to antiviral therapy, which is now deemed the first therapeutic option to consider. By contrast, there are diseases where treatment with interferon was ineffective or dangerous. The aim of the present paper is to outline the most recent evidence concerning extrahepatic disorders that are possibly associated with Hepatitis C Virus infection. Special emphasis will be given to discussion of the most appropriate clinical approaches to be adopted in order to diagnose, treat (possibly prevent) and follow-up extrahepathic diseases in patients with Hepatitis C Virus infection.
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Affiliation(s)
- A L Zignego
- Department of Internal Medicine, Medical School, Center for Research, Transfer and High Education DENOthe, Center for the Study of Systemic Manifestations of Hepatitis Viruses MaSVE, University of Florence, Florence, Italy.
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12
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Li JW, Li K, Jiang J, Xu XL, Huang ZQ. Construction of eukaryotic expression plasmid containing HCV NS3 segment and protein expression in human HL-7702 hepatocytes. World J Gastroenterol 2006; 12:1038-42. [PMID: 16534843 PMCID: PMC4087894 DOI: 10.3748/wjg.v12.i7.1038] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct the eukaryotic expression plasmid containing HCV NS3 segment and to analyze the expression of NS3 protein in normal human hepatocyte HL-7702.
METHODS: We amplified HCV NS3 fragment from plasmid pBRTM/HCV 1-3011 containing the whole length of HCV genome, recombined it with expression vector pcDNA3.1(-) to form the eukaryotic expression vector pcDNA3.1(-)/NS3, and transfected human HL-7702 hepatocytes with the recombined plasmid by cationic polymers. The expressed HCV NS3 protein was detected and analyzed by immunohistochemical method and Western blot.
RESULTS: The amplified NS3 fragments had correct molecule weight and sequence. The successfully constructed eukaryotic expression plasmids were transfected to HL-7702 cells. The expressed NS3 proteins had correct molecular weight 70000.
CONCLUSION: Eukaryotic expression vector pcDNA3.1 (-)/NS3 containing NS3 segment of HCV can be constructed, the sequence of NS3 fragments is consistent with the template. Normal human HL-7702 hepatocytes can efficiently express specific HCV NS3 protein in vitro.
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Affiliation(s)
- Jun-Wu Li
- Microbiology and Immunology Department, Medical College, Jinan University, Guangzhou 510632, Guangdong Province, China.
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Hassan M, Ghozlan H, Abdel-Kader O. Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth. Virology 2005; 333:324-36. [PMID: 15721365 DOI: 10.1016/j.virol.2005.01.008] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2004] [Revised: 12/14/2004] [Accepted: 01/07/2005] [Indexed: 12/13/2022]
Abstract
Hepatitis C virus (HCV) non-structural protein 3 (NS3) has been shown to affect cellular functions and is thought to contribute to the development of HCV-related hepatocarcinogenesis. In this study, we delineated part of the mechanisms whereby NS3 protein stimulates cell growth in liver (HepG2) and non-liver (HeLa) cells. The expression of NS3 protein enhanced cell growth, c-jun NH(2)-terminal kinase (JNK) activation, DNA binding activities of the transcription factors AP-1 and ATF-2, and c-jun expression, but not the activation of extracellular signal-regulated kinase (ERK) or p38(MAPK). Whereas co-expression of NS3 with its cofactor NS4A inhibited NS3-mediated cell growth without to influence NS3-mediated JNK activation, or to affect the basal activities of ERK or p38(MAPK). Pre-treatment of NS3 protein-expressing cells with JNK inhibitor, SP600125, abolished activation of AP-1 and ATF-2 and inhibited c-jun expression and induced cell growth, suggesting that JNK activation is essential for the stimulation of NS3-mediated cell growth.
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Affiliation(s)
- Mohamed Hassan
- Institute of Pathology, Faculty of Medicine, University of Duesseldorf, Mooren Str.5, 40225 Duesseldorf, Germany
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He QQ, Cheng RX, Sun Y, Feng DY, Chen ZC, Zheng H. Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 C-terminal deleted protein. World J Gastroenterol 2003; 9:474-8. [PMID: 12632500 PMCID: PMC4621564 DOI: 10.3748/wjg.v9.i3.474] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effect of hepatitis C virus nonstructural protein 3 c-terminal deleted protein (HCV NS3-5’) on hepatocyte transformation and tumor development.
METHODS: QSG7701 cells were transfected with plasmid pRcHCNS3-5’ (expressing HCV NS3 c-terminal deleted protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological behavior of transfected cells was observed through cell proliferation assay, anchorage-independent growth and tumor development in nude mice. The expression of HCV NS3 and c-myc proteins in the induced tumor was evaluated by immunohistochemistry.
RESULTS: HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5’ and the positive signal was located in cytoplasm. Cell proliferation assay showed that the population doubling time in pRcHCNS3-5’ transfected cells was much shorter than that in pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning ratio of cells transfected with pRcHCNS3-5’, pRcCMV and non-transfected cells was 33%, 1.46%, 1.11%, respectively, the former one was higher than that in the rest two groups (P < 0.01). Tumor development was seen in nude mice inoculated with pRcHCNS3-5’ transfected cells after 15 days. HE staining showed its feature of hepatocarcinoma, and immunohistochemistry confirmed the expressions of HCV NS3 and c-myc proteins in tumor tissue. The positive control group inoculated with HepG2 also showed tumor development, while no tumor developed in the nude mice injected with pRcCMV and non-transfected cells after 40 days.
CONCLUSION: 1.HCV NS3 c-terminal deleted protein has transforming and oncogenic potential. 2. Human liver cell line QSG7701 may be used as a good model to study HCV NS3 pathogenesis.
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Affiliation(s)
- Qiong-Qiong He
- Department of Pathology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan Province, China
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Borowski P, Schalinski S, Schmitz H. Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy. Antiviral Res 2002; 55:397-412. [PMID: 12206878 DOI: 10.1016/s0166-3542(02)00096-7] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The RNA nucleoside triphosphatase (NTPase)/helicases represent a large family of proteins that are detected in almost all biological systems where RNA plays a central role. The enzymes are capable of enzymatically unwinding duplex RNA structures by disrupting the hydrogen bonds that keep the two strands together. The strand separating activity is associated with hydrolysis of nucleoside triphosphate (NTP). Because of this, potential specific inhibitors of NTPase/helicases could act by one or more of the following mechanisms: (i) inhibition of NTPase activity by interference with NTP binding, (ii) inhibition of NTPase activity by an allosteric mechanism and (iii) inhibition of the coupling of NTP hydrolysis at the unwinding reaction. There are also other inhibitory mechanisms conceivable, which may involve a modulation of the interaction of the enzyme with its RNA substrate, for example, (iv) the competitive inhibition of RNA binding and (v) the inhibition of the unwinding by sterical blockade of the translocation of the NTPase/helicase along the polynucleotide chain. NTPase/helicase has also been identified in the viral genome of hepatitis C virus (HCV) which is a member of the Flaviviridae family. It is conceivable that the inhibition of the unwinding activity of the enzyme leads to the inhibition of virus replication and this may represent a novel antiviral strategy. This review updates the current spectrum of inhibitors targeting different mechanisms by which the NTPase and/or helicase activities of the HCV NTPase/helicase are inhibited. Consequently, some of the compounds might be important as antiviral agents against HCV.
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Affiliation(s)
- Peter Borowski
- Abteilung für Virologie, Bernhard-Nocht-Institut für Tropenmedizin, 20359 Hamburg, Germany.
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16
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Rho J, Choi S, Seong YR, Choi J, Im DS. The arginine-1493 residue in QRRGRTGR1493G motif IV of the hepatitis C virus NS3 helicase domain is essential for NS3 protein methylation by the protein arginine methyltransferase 1. J Virol 2001; 75:8031-44. [PMID: 11483748 PMCID: PMC115047 DOI: 10.1128/jvi.75.17.8031-8044.2001] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg(1493) in the QRR(1488)GRTGR(1493)G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg(1488) is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.
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Affiliation(s)
- J Rho
- Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejeon 305-333, Republic of Korea
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17
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Aoubala M, Holt J, Clegg RA, Rowlands DJ, Harris M. The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis. J Gen Virol 2001; 82:1637-1646. [PMID: 11413375 DOI: 10.1099/0022-1317-82-7-1637] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.
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Affiliation(s)
- Mustapha Aoubala
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
| | - John Holt
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
| | | | - David J Rowlands
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
| | - Mark Harris
- Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
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18
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Borowski P, Niebuhr A, Mueller O, Bretner M, Felczak K, Kulikowski T, Schmitz H. Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme. J Virol 2001; 75:3220-9. [PMID: 11238848 PMCID: PMC114115 DOI: 10.1128/jvi.75.7.3220-3229.2001] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.
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Affiliation(s)
- P Borowski
- Abteilung für Virologie, Bernhard-Nocht-Institut für Tropenmedizin, D-20359 Hamburg, Germany.
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Naganuma A, Nozaki A, Tanaka T, Sugiyama K, Takagi H, Mori M, Shimotohno K, Kato N. Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein. J Virol 2000; 74:8744-50. [PMID: 10954577 PMCID: PMC116387 DOI: 10.1128/jvi.74.18.8744-8750.2000] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2'-5'-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2'-5'-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2'-5'-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2'-5'-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2'-5'-OAS gene.
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Affiliation(s)
- A Naganuma
- Virology and Glycobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan
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Affiliation(s)
- R Bartenschlager
- Institute for Virology, Johannes-Gutenberg University Mainz, Obere Zahlbacher Strabetae 67, 55131 Mainz, Germany.
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Abstract
Infection with the hepatitis C virus (HCV) is the major cause of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in vitro, several of its key features have been elucidated in the past few years. The viral genome is a positive-sense, single-stranded, 9.6 kb long RNA molecule. The viral genome is translated into a single polyprotein of about 3000 amino acids. The viral polyprotein is proteolytically processed by the combination of cellular and viral proteinases in order to yield all the mature viral gene products. The genomic order of HCV has been shown to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C, E1 and E2 are the virion.structural proteins. The function of p7 is currently unknown. These proteins have been shown to arise from the viral polyprotein via proteolytic processing by the host signal peptidases. Generation of the mature nonstructural proteins, NS2 to NS5B, relies on the activity of viral proteinases. Cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine proteinase also contained within the N-terminal region of NS3. NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the proteinase. While no function has yet been attributed to NS4B, it has recently been suggested that NS5A is involved in mediating the resistance of the hepatitis C virus to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase.
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Affiliation(s)
- R De Francesco
- I.R.B.M.-Istituto di Ricerche di Biologia Molecolare, Rome, Italy.
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Borowski P, Resch K, Schmitz H, Heiland M. A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC. Biol Chem 2000; 381:19-27. [PMID: 10722046 DOI: 10.1515/bc.2000.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.
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Affiliation(s)
- P Borowski
- Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany
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Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 1999; 242:55-84. [PMID: 10592656 DOI: 10.1007/978-3-642-59605-6_4] [Citation(s) in RCA: 239] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- K E Reed
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110-1093, USA
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24
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Abstract
Hepatitis C virus (HCV) NS3 is a multifunctional protein with both protease and helicase activities and has been shown to interact with host cell proteins. It is shown that NS3 is present in the hepatocytes from patients with chronic HCV infection by using anti-NS3 antisera. NS3 is detectable in approximately 4% of the hepatocytes from these patients. In most infected cells, NS3 is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own are positive for NS3. The presence of NS3 in the nuclei of hepatocytes in chronically infected patients indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.
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Affiliation(s)
- W Errington
- Department of Medicine, Imperial College School of Medicine, London, England
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25
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Borowski P, Kuehl R, Mueller O, Hwang LH, Schulze Zur Wiesch J, Schmitz H. Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus. EUROPEAN JOURNAL OF BIOCHEMISTRY 1999; 266:715-23. [PMID: 10583365 DOI: 10.1046/j.1432-1327.1999.00854.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.
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Affiliation(s)
- P Borowski
- Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
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Borowski P, Schulze zur Wiesch J, Resch K, Feucht H, Laufs R, Schmitz H. Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus. J Biol Chem 1999; 274:30722-8. [PMID: 10521461 DOI: 10.1074/jbc.274.43.30722] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.
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Affiliation(s)
- P Borowski
- Abteilung für Virologie, Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany
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Borowski P, Kühl R, Laufs R, Schulze zur Wiesch J, Heiland M. Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus. J Clin Virol 1999; 13:61-9. [PMID: 10405893 DOI: 10.1016/s1386-6532(99)00007-4] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND Chronic hepatitis resulting from the hepatitis C virus (HCV) infection leads to cirrhosis in at least half the infected patients and increases the risk of hepatocellular carcinoma. There are indications that this pathogenic effect may result from the disturbance of intracellular signal cascades caused by the interaction with viral antigens. Although a great amount of data has been accumulated about functional regions in HCV proteins, relatively little is known about their intracellular targets. Previously, we have demonstrated that the full-length non-structural protein 3 of HCV (NS3) (Borowski P, Heiland M, Feucht H, Laufs R. Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC. Evidences for action on the level of substrate and enzyme. Arch Virol 1999a; 144) and its NH2- and COOH-terminal truncated form (Borowski P, Heiland M, Oehlmann K, Becker B, Kornetzky L, Feucht HH, Laufs R. Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur J Biochem 1996;237:611-618) associate to stable complexes with core histones H2B and H4. The changes of the properties of histones as substrate for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) were found as a direct consequence of the interaction. OBJECTIVE In the present study we further these observations, localize the histone binding domain of NS3 and investigate the mechanisms by which NS3 affects the functions of the histones in vitro. STUDY DESIGN HCV protein exhibiting the mentioned histone binding activity was produced in a bacterial expression system, purified and binding to histones was biochemically characterized. The region of NS3 involved in the interaction with histones was defined by proteolytic fragmentation, microsequencing and a specific histone binding assay. Furthermore, a functional test to quantify the interaction of histones with DNA was established and the binding of DNA to histone as a function of NS3 concentration was analysed by means of graphical methods. RESULTS The investigated fragment of HCV polyprotein consisting of amino acid residues 1189-1525 (HCV-polyprotein-(1189-1525)) displayed significant histone binding activity. The binding occurred at a molar ratio 1:1 of histone to HCV-polyprotein-(1189-1525) and was mediated by a linear stretch of amino acids located between the residues 1343 and 1379 of the HCV polyprotein. To demonstrate that HCV-polyprotein-(1189-1525) affects the binding of DNA to histones we used two independent methods: overlay assay and binding assay on Sepharose beads. Graphic analysis of the binding kinetics revealed an uncompetitive type of inhibition. CONCLUSIONS Our results provide the first evidence that NS3 binds and affects the functions of core histones. The mechanism by which the NS3 interferes with the histone functions involves conformational changes of histone molecule.
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Affiliation(s)
- P Borowski
- Bernhard-Nocht-Institut für Tropenmedizin, Abt. Virologie, Hamburg, Germany
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28
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Bartenschlager R. The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy. J Viral Hepat 1999; 6:165-81. [PMID: 10607229 DOI: 10.1046/j.1365-2893.1999.00152.x] [Citation(s) in RCA: 105] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
The hepatitis C virus (HCV) is a major causative agent of transfusion-acquired and sporadic non-A, non-B hepatitis worldwide. Infections most often persist and lead, in approximately 50% of all patients, to chronic liver disease. As is characteristic for a member of the family Flaviviridae, HCV has a plus-strand RNA genome encoding a polyprotein, which is cleaved co- and post-translationally into at least 10 different products. These cleavages are mediated, among others, by a virally encoded chymotrypsin-like serine proteinase located in the N-terminal domain of non-structural protein 3 (NS3). Activity of this enzyme requires NS4A, a 54-residue polyprotein cleavage product, to form a stable complex with the NS3 domain. This review will describe the biochemical properties of the NS3/4A proteinase, its X-ray crystal structure and current attempts towards development of efficient inhibitors.
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Affiliation(s)
- R Bartenschlager
- Institute for Virology, Johannes-Gutenberg University Mainz, Mainz, Germany
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29
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30
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Di Pasquale G, Stacey SN. Adeno-associated virus Rep78 protein interacts with protein kinase A and its homolog PRKX and inhibits CREB-dependent transcriptional activation. J Virol 1998; 72:7916-25. [PMID: 9733829 PMCID: PMC110121 DOI: 10.1128/jvi.72.10.7916-7925.1998] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Adeno-associated virus (AAV) is a human parvovirus of the genus Dependovirus. AAV replication is largely restricted to cells which are coinfected with a helper virus. In the absence of a helper virus, the AAV genome can integrate into a specific chromosomal site where it remains latent until reactivated by superinfection of the host cell with an appropriate helper virus. Replication functions of AAV have been mapped to the Rep68 and Rep78 gene products. Rep proteins demonstrate DNA binding, endonuclease, and helicase activities and are involved in regulation of transcription from both AAV and heterologous promoters. AAV has been associated with suppression of oncogenicity in a range of viral and nonviral tumors. In this study we sought to identify and study cellular protein targets of AAV Rep, in order to develop a better understanding of the various activities of Rep. We used the yeast two-hybrid system to identify HeLa cell proteins that interact with AAV type 2 Rep78. We isolated several strongly interacting clones which were subsequently identified as PRKX (previously named PKX1), a recently described homolog of the protein kinase A (PKA) catalytic subunit (PKAc). The interaction was confirmed in vitro by using pMal-Rep pull-down assays. The region of Rep78 which interacts was mapped to a C-terminal zinc finger-like domain; Rep68, which lacks this domain, did not interact with PRKX. PRKX demonstrated autophosphorylation and kinase activity towards histone H1 and a PKA oligopeptide target. Autophosphorylation was inhibited by interaction with Rep78. In transfection assays, a PRKX expression vector was shown to be capable of activating CREB-dependent transcription. This activation was suppressed by Rep78 but not by Rep68. Since PRKX is a close homolog of PKAc, we investigated whether Rep78 could interact directly with PKAc. pMal-Rep78 was found to associate with purified PKAc and inhibited its kinase activity. Cotransfection experiments demonstrated that Rep78 could block the activation of CREB by a PKAc expression vector. These experiments suggest that AAV may perturb normal cyclic AMP response pathways in infected cells.
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Affiliation(s)
- G Di Pasquale
- Cancer Research Campaign, Section of Molecular Genetics, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 4BX, United Kingdom
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31
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Borowski P, Heiland M, Kornetzky L, Medem S, Laufs R. Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C. Biochem J 1998; 331 ( Pt 2):649-57. [PMID: 9531509 PMCID: PMC1219400 DOI: 10.1042/bj3310649] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.
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Affiliation(s)
- P Borowski
- Institut für Medizinishe Mikrobiologie und Immunologie, Universitätskrankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Federal Republic of Germany
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Gwack Y, Kim DW, Han JH, Choe J. DNA helicase activity of the hepatitis C virus nonstructural protein 3. EUROPEAN JOURNAL OF BIOCHEMISTRY 1997; 250:47-54. [PMID: 9431989 DOI: 10.1111/j.1432-1033.1997.00047.x] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA x DNA and RNA x RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3' to 5'. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall order of inhibitory capacity was: adenosine 5'-O-(3-thiotriphosphate) > adenylyl-imidodiphosphate and adenylyl-(beta,gamma-methylene)-diphosphate > AMP. DNA helicase activity was inhibited strongly by ssDNA and ssRNA, but was little affected by dsDNA. The ATPase activity was stimulated greatly by ssDNA and ssRNA, but not by dsDNA. The NS3 protein could unwind up to 500 base pairs of duplex DNA. The possible multifunctional nature of the NS3 protein is discussed and compared with that of Simian virus 40 large T antigen.
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Affiliation(s)
- Y Gwack
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon
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Borowski P, Oehlmann K, Heiland M, Laufs R. Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase. J Virol 1997; 71:2838-43. [PMID: 9060639 PMCID: PMC191408 DOI: 10.1128/jvi.71.4.2838-2843.1997] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV.
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Affiliation(s)
- P Borowski
- Institut für Medizinische Mikrobiologie und Immunologie, Universitätskrankenhaus Eppendorf, Hamburg, Germany
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Leitz T, Müller WA. Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming. Dev Biol 1987; 8:26. [PMID: 21247464 PMCID: PMC3032714 DOI: 10.1186/1743-422x-8-26] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2010] [Accepted: 01/19/2011] [Indexed: 12/29/2022]
Abstract
Background Hepatitis C is a major health problem causes liver cirrhosis, hepatocellular carcinoma and death. The current treatment of standard interferon in combination with ribavirin, has limited benefits due to emergence of resistant mutations during long-term treatment, adverse side effects and high cost. Hence, there is a need for the development of more effective, less toxic antiviral agents. Results The present study was designed to search anti-HCV plants from different areas of Pakistan. Ten medicinal plants were collected and tested for anti-HCV activity by infecting the liver cells with HCV 3a innoculum. Methanol and chloroform extracts of Solanum nigrum (SN) seeds exhibited 37% and more than 50% inhibition of HCV respectively at non toxic concentration. Moreover, antiviral effect of SN seeds extract was also analyzed against HCV NS3 protease by transfecting HCV NS3 protease plasmid into liver cells. The results demonstrated that chloroform extract of SN decreased the expression or function of HCV NS3 protease in a dose- dependent manner and GAPDH remained constant. Conclusion These results suggest that SN extract contains potential antiviral agents against HCV and combination of SN extract with interferon will be better option to treat chronic HCV.
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