1,720,974 research outputs found
RNA-stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3
No full textHepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviriridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/ NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRT-GRGRSGR, Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain
Mutational analysis of the hepatitis C virus RNA helicase
No full textThe carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. a serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed
Towards defining a minimal functional domain for NTPase and RNA helicase activities of the hepatitis C virus NS3 protein
No full textHepatitis C virus (HCV) possesses two separate enzymatic functions in the NS3 protein: a protease and an NTPase/RNA helicase. In order to determine the minimal domain for NTPase and RNA helicase activities of the HCV NS3 protein, serial deletion mutants were constructed. The NS3H protein, a fusion protein of 25 amino acids (aa) from an expression vector and the C-terminal 466 aa of the HCV NS3 protein, contains an NTPase/RNA helicase activity. We made deletion mutants of 10, 30, 50, 97, and 135 aa from the C-terminus and 16 and 32 aa from the N-terminus of the NS3H protein. The deleted protein lacking: 50 aa from the C-terminus still possessed both activities, while the protein lacking 97 aa from the C-terminus lost an RNA helicase activity. The mutant lacking 16 amino acids from the N-terminus retained the enzymatic activities and the N-terminal 32 aa deleted mutant lost an NTPase/RNA helicase activity. A combinational deletion mutant lacking 16 aa the N-terminus and 50 aa from the C-terminus retained the enzymatic activities. These results show that the functional domain of the HCV NTPase/RNA helicase is about 400 aa in length and maps between NS3 residues 1209 and 1608. (C) 1997 Elsevier Science B.V
Kaposis sarcoma-associated herpesvirus open reading frame (ORF) 50 transactivates K8 and ORF57 promoters via heterogeneous response elements
No full textKaposi's sarcoma (KS)-associated herpesvirus (KSHV) belongs to the human gammaherpesvirus family that undergoes both lytic and latent life cycles in host cells. Open reading frame (ORF) 50 is the most important protein in reactivation to lytic phase and functions as a strong transcriptional activator of the early and late genes of KSHV. Since transactivation of promoters by ORF50 is achieved via response elements, we have attempted to identify ORF50 response elements in K8 and ORF57 promoters of KSHV by transient transfection assays with deletion mutants. Our data reveal that specific regions within the K8 (74661-74760) and ORF57 (81851-81931) promoters contain ORF50 response elements, which are heterogeneous, unlike those of Epstein-Barr virus and Herpesvirus saimiri. We additionally identify an AP-1 binding site at the ORF57 promoter between 81882 and 81889, and show that AP-1 participates in ORF57 promoter activation by ORF50. Our findings collectively indicate that ORF50 activates various viral proteins through both direct binding and cellular transcriptional factor-mediated mechanisms
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
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