1,720,990 research outputs found
The C-terminal cytoplasmic tail of herpes simplex virus type 1 gE protein is phosphorylated in vivo and in vitro by cellular enzymes in the absence of other viral proteins
No full textHerpes simplex virus 1 glycoprotein E (gE-1) is highly phosphorylated in culture cells during infection. In this report, it is shown that phosphorylation is mediated by host enzymes in human cells stably transfected with gE, in the absence of other herpesvirus products. In contrast, a tailless gE product (C terminus deletion mutant) is not phosphorylated. By using an in vitro kinase assay combined with linker-insertion mutagenesis, it is shown that casein kinase II catalyses the phosphorylation of the C-terminal domain of the protein. Also, it is demonstrated that the serine residues at positions 476 and/or 477 in the cytoplasmic portion of the protein are the major accepters for the phosphate groups
Expression of hepatitis C virus envelope glycoproteins by herpes simplex virus type 1-based amplicon vectors
No full textHerpes simplex virus type 1 (HSV-1)-based amplicon vectors expressing hepatitis C virus (HCV) E1 and E2 glycoproteins were investigated. HSV-1 amplicon vectors carrying the E1E2p7- or E2p7-coding sequences of HCV type 1a under the control of the HSV-1 IE4 (α22/α47) promoter were constructed. Studies of infected HepG2, WRL 68 or Vero cells indicated that HSV-1-based amplicon vectors express high levels of HCV glycoproteins that are processed correctly. Immunofluorescence microscopy combined with immunoprecipitation and endoglycosidase treatment of cells infected with the HSV-1-based vectors expressing E1 and E2 showed that the two glycoproteins were retained in the endoplasmic reticulum and had the expected glycosylation patterns. Furthermore, although most of the E1 and E2 proteins formed disulfide-linked aggregates, significant amounts of monomeric forms of the two proteins were detected by SDS-PAGE under non-reducing conditions, suggesting the presence of non-covalently associated E1 and E2. Similar results were produced by a replication-competent recombinant HSV-1 vector expressing HCV E1 and E2. These results indicated that HSV-1-based amplicon vectors represent a useful expression system for the study of HCV glycoproteins
Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins
No full textWe report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384-611 or 384-711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK(-)gC(-), is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119-6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule
Escherichia coli expressed herpes simplex virus gG1 and gG2 proteins in ELISA and immunoblotting assays
No full textThe type 1 and type 2 glycoprotein G (gG1 and gG2) of herpes simplex virus (HSV) were expressed in Escherichia coli as fusion proteins with the maltose binding protein (MBP) using the pMAL-c2 expression vector. The MBP-gG1 fusion protein contains all but the four amino acids from the amino-terminus of gG1, whereas the MBP-gG2 fusion protein was missing the first 30 amino acids that comprise the signal peptide of the protein. The diagnostic value of these antigens was examined by two methods: (1) immunoblot assay based on MBP-gG1 and MBP-gG2 fusion proteins present in crude E. coli cell extracts and (2) enzyme-linked immunosorbent assay (ELISA) of immunoaffinity-purified recombinant MBP-gG1 and MBP-gG2 fusion proteins. Of 28 serum samples known to have antibody to HSV-1 (10 specimens positive for HSV-1 alone and 18 specimens positive for mixed antibody to HSV-1/HSV-2), 27 were reactive to the MBP-gG1 recombinant protein both in ELISA and in immunoblotting. In addition, of 20 serum samples known to have antibody to HSV-2 (2 specimens positive for HSV-2 alone and 18 samples positive for mixed antibody to HSV-1/HSV-2), 15 were found to be reactive to the MBP-gG2 recombinant protein by ELISA and 16 by immunoblotting. None of the 13 HSV-antibody-negative serum samples showed reactivity to the MBP-gG1 or MBP-gG2 antigens by either assay. Moreover, none of the serum samples known to have antibody to HSV-1 alone showed reactivity to the MBP-gG2 recombinant antigen. This study verified the potential application of the E. coli-expressed recombinant gG1 and gG2 proteins as diagnostic antigens and demonstrated the MBP fusion system to be a simple and effective method of producing adequate amounts of low-cost, easily purified gG antigens
Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: Protection studies against lethal viral infection in mice
No full textThe objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model. A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector. In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector. Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity. These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of value as a component of an HSV multi-subunit vaccine
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Variations on the Author
Full text link“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
Full text linkWe 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
Full text linkWe 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
- …
