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Characterisation of the oxidation state of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 during glycoprotein biosynthesis and virus entry
No full textHepatitis C virus (HCV) is a major cause of chronic liver disease, including cirrhosis and hepatocellular carcinoma (HCC) and is a leading reason for liver transplantation in industrialised countries. The most recent estimate of global prevalence is 2-3% representing 130-170 million HCV-positive people [1]. HCV is an enveloped, positive strand RNA virus which comprises the hepacivirus genus within the Flaviviridae family. The virus displays two glycoproteins on its surface, E1 and E2, which are required to mediate entry into hepatocytes [2,3,4]. Cellular infection is dependent on receptors CD81, SR-BI, claudin-1, 6 or 9, and occludin [5,6,7,8,9,10]. Following attachment HCV is thought to be internalised via clathrin mediated endocytosis where the low pH of the endosome is required for fusion of the viral and cellular membranes [11]. Productive infection occurs following delivery of the viral nucleocapsid to the cytoplasm where RNA translation, replication and virus assembly takes place. This project investigated the significance of the redox state of HCV envelope glycoproteins E1 and E2 during glycoprotein biosynthesis and virus entry. It was found that virion incorporated forms of E1 and E2 contain at least one free cysteine residue each prior to cellular attachment. Alkylation of reduced sulfhydryl groups prior to cellular attachment abolished virus infectivity. However, it was found that cell culture derived HCV (HCVcc) became insensitive to thiol-reactive agents after binding to cells indicating that the thiol groups were no longer available for modification following receptor engagement. These data suggest that free sulfhydryl groups within E1 and/or E2 are required to mediate virus entry and that receptor engagement may induce a switch in glycoprotein redox state from reduced to oxidised. A highly conserved C226VPC thiol isomerisation motif was identified in E1 critical for formation of a functional E1E2 heterodimer. Mutation of Val227 to Ile or Leu allowed normal E1 and E2 biosynthesis and virion incorporation. However,mutant viruses demonstrated significantly reduced entry compared to wild type. These findings indicate that the C226VPC motif is required to function during virus entry. Domain organisation within E1 was examined by expression of E1 ectodomain residues in bacteria. A stable domain consisting of N-terminal amino acids 191-259 was identified and produced as a soluble monomer. When expressed in mammalian cells, ectodomain residues 191-329 were efficiently expressed and secreted from cells suggesting that when glycosylated these residues may fold independently from other HCV protein sequences. The findings from this study indicate that HCV functionality is highly dependent on careful regulation of the redox state of the viral envelope glycoproteins, possibly mediated by the E1 C226VPC motif
Functional interactions of structural and NS proteins of hepatitis c virus
No full textHepatitis C virus (HCV) is a small enveloped virus with a positive-sense single stranded RNA genome. Based on its molecular genetic characteristics, the virus has been classified into the Hepacivirus genus of the family Flaviviridae. HCV is one of the major causes of chronic hepatitis which can lead to liver cirrhosis and hepatocellular carcinoma. According to the recent WHO published data, 123 million individuals are infected with HCV (approximately 3% of the world’s population) throughout the world. Due to its highly variable nature, HCV is classified into six major genotypes. The HCV genome encodes a single polyprotein that is cleaved to yield at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The recently developed HCV cell culture system, based on the JFH1 strain of HCV, has provided an opportunity to study the role of the viral proteins in the complete HCV replication cycle in human hepatoma cells. How the viral proteins functionally interact during replication of HCV in cell culture is not completely understood. Passage of cell cultures transfected with HCV genomic RNA containing attenuating mutations allows for the selection of genomes with second site compensatory mutations that restore replication to wild type levels. Using this approach, the functional interactions of p7 and E2 with other viral proteins during HCV replication was investigated. A small protein of 63 amino acids, p7 is encoded at the junction of the structural and non-strucutural region. p7 is a highly hydrophobic, integral membrane protein and is classified in the viroporin family. In this thesis, it is shown that p7 is critical for production of viral particles and is implicated in a late step of particle assembly. Since the protein plays a critical role in the virus life cycle, chemical compounds that block p7 function are potential candidates for anti-viral therapy. In this thesis, a chimeric JFH1 genome that encodes the p7 protein of genotype (GT) 1b strain J4 was generated. The intergenotypic chimeric genome was nonviable in human hepatoma cells and infectious chimeric virions were only produced after cells harboring the chimeric genomes were passaged several times. To investigate the emergence of compensatory mutations in the viral proteins during cell passaging, the consensus sequences of the entire polyprotein coding regions of the wild type JFH1 and three chimeric viruses were determined. Sequence analysis revealed mutations in core, NS2, NS5A and NS5B. Reverse genetic analysis demonstrated that any one of the single mutations restored the infectivity of the defective chimeric genomes. These data suggest that there are critical genetic interactions between p7 with core, NS2, NS5A and NS5B. In addition, a stable physical interaction between p7 and NS2 is shown in a transient expression system. The HCV glycoproteins E1 and E2 are present on the surface of virions as a heterodimer that attach virions to host cell receptors and facilitate virus fusion and entry. HCV entry proceeds via attachment to glycosaminoglycans followed by binding to scavenger receptor type B class I, and the tetraspanin CD81. Recently, claudin-1 and occludin have emerged as additional receptors required for entry. E2 has a receptor binding domain (E2661RBD) that conatins three variable regions, hypervariable regions 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). In this thesis, HVR1 of E2 was deleted in the context of full-length replication comptetent HCV. Deletion of HVR1 increases CD81-binding ability of the mutant and also increases its susceptibility to neutralizing antibody MAb 24. The infectivity of the HVR1 deleted virions was attenuated approximately 10-fold prior to accumulation of compensatory mutations. Sequencing of cDNA obtained from reverted virions revealed mutations in E1 (I262L) and E2 (N415D). Reverse genetic studies revealed that I262L improved the infectivity of HVR1 deleted virions 2.5 fold while N415D restored infectivity to wild type levels. These data suggest that mutations within E1 or E2 can compensate for the reduction in infectivity observed for HVR1 deleted viruses. In summary, this thesis demonstrates the importance of functional interactions between HCV proteins during virus morphogenesis and infectivity
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Functions of hepatitis C virus glycoprotein E2 variable regions
No full textHCV chronically infects ~3% of the global human population, including 200,000 Australians, causing recurring, progressively worsening liver disease, and cirrhosis and hepatocellular carcinoma. Preventative vaccines are not available. However, the advent of direct acting antivirals for the treatment of HCV results in viral clearance in at least 90% of treated individuals. Hepatitis C virus encodes two structural glycoproteins; E1 and E2. Glycoproteins E1 and E2 associate covalently and non-covalently to form heterodimers that mediate HCV binding to cell surface receptors. The E2 ectodomain region spanning residues 384-661 constitutes the receptor binding domain (RBD; E2661) that includes three highly variable regions: hypervariable region 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). The HVR1 is known to elicit type-specific neutralizing antibodies (NAb). The HVR2 is located downstream of HVR1 within a region flanked by two cysteine residues (Cys-459 and Cys-486). HVR2 is not a direct target of the antibody response and the reason for variation within its sequence is unknown. Similar to HVR2, the igVR is flanked by two cysteine residues (Cys-569 and Cys-581). The igVR is relatively conserved within subtypes but the sequence and length of this region varies widely between genotypes. Patients infected with HCV develop NAb during their infection. The natural targets for NAb are the envelope glycoproteins E1 and E2 especially within the E2 RBD. The majority of NAb are directed to the binding sites within E2 for the major cell surface receptor CD81. In this study, the role of HVRs in modulating the exposure of both neutralizing and non-neutralizing epitopes within E2 has been examined. Examination of sequence evolution within HCV infected patients revealed that in addition to HVR1, both HVR2 and igVR are under selective pressure. In the case of the igVR, this contrasts to earlier reports suggesting that the igVR does not vary within genotypes. Mutations in the igVR were shown to directly modulate the exposure of non-neutralizing antibody epitopes in the patient that cleared their infection. In addition, the CD81 binding site was more occluded in E2 RBDs isolated during the chronic phase of the disease and was partly attributable to mutation in HVR2. In another approach, intragenotypic HVR2 and igVR replacement attenuate genotype 2 (G.2) HCVcc virus, whereas intergenotypic HVR2 and igVR chimeras between G.2a and G.1a results in non-infectious HCVcc. Passaging attenuated virus restored replication due to acquisition of adaptive mutations in the E2 transmembrane domain. With the aid of neutralizing antibodies, we also show that neutralizing epitope I encompassing 411-428 is more accessible when replacing HVR1 of G.1a to that of G.1b, but becomes occluded by replacing igVR. In contrast, neutralizing epitope III covering residues 512-529 is occluded by HVR1 and becomes inaccessible by exchanging igVR of G.1a to that of G.1b. The results show that HCV variable regions play a role in modulating the exposure of NAb epitopes and access to the CD81 binding site on the E2 RBD
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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