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Interaction of rotavirus nonstructural protein NSP5 with the viral replication complex
Get PDFRotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms,
where synthesis of the11 dsRNA genome segments and their packaging in new viral
particles take place. RNA replication is mediated by several viral proteins, of which
VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein,
were shown to be sufficient to provide replicase activity in vitro. In vivo, however,
viral replication complexes also contain the nonstructural proteins NSP2 and NSP5,
which were shown to be essential for replication, to interact with each other and to
form viroplasm-like structures (VLS) when coexpressed in uninfected cells.
In order to gain a better understanding of the intermediates formed during viral
replication, this work focused on the interactions of NSP5 with VP1, VP2 and NSP2.
We constructed a tagged form of VP1 and by coimmunoprecipitation experiments
we demonstrated that VP1 and NSP5 interact in virus-infected cells as well as in the
absence of other viral proteins or viral RNA in cotransfected cells. Using deletion
mutants of NSP5 or different fragments of NSP5 fused to EGFP, we identified the 48
C-terminal amino acids as the region essential for interaction with VP1. On the other
hand, removal of the C-terminal 15 amino acids from tagged VP1 resulted in a less
efficient coimmunoprecipitation with NSP5, suggesting an involvement of the Cterminus
of VP1. Interaction of NSP5 with VP2 was investigated by coexpression of
the two proteins in uninfected cells, which resulted in a strong hyperphosphorylation
of NSP5 and in the formation of VLS, that we named VLS(VP2i) to distinguish them
from those induced by NSP2, here designated as VLS(NSP2i). VLS(VP2i) were shown
to assemble independently of the phosphorylation degree of NSP5 and to recruit the
viroplasm-resident proteins NSP2, VP1, VP2 and VP6 (the protein forming the middle
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layer of the virion). Attempts to coimmunoprecipitate NSP5 and VP2 failed both from
infected and cotransfected cells.
Tagged VP1 was found to localize in VLS (both VP2i and NSP2i) and in viroplasms,
and to be able to replace wild-type VP1 structurally by being incorporated into
progeny viral particles. Coexpression of different combinations of tagged VP1,
NSP5, NSP2 and VP2 showed that the interaction of VP1 with NSP5 is not affected
by the other viral proteins and is stronger than the interaction with NSP2. In addition,
an inhibitory effect of VP1 on the levels of NSP5 hyperphosphorylation induced by
both NSP2 and VP2 was observed.
Altogether, these data confirmed an important role for NSP5 in replication, related
with the interactions with the two structural proteins essentially involved in viral
genome synthesis, and suggested that NSP5 plays a key role in architectural
assembly of viroplasms and in recruitment of the other viroplasmic proteins
Role of viral nonstructural proteins in rotavirus replication
No full textStudies on the molecular biology of rotavirus, the major etiologic agent of gastroenteritis in infants and young children worldwide, have so far led to a large but not exhaustive knowledge of the mechanisms by which rotavirus replicates in the host cell. While the role of rotavirus structural proteins in the replication cycle is well defined, the functions of nonstructural proteins remain poorly understood. Recent experiments of RNA interference have clearly indicated the phases of the replication cycle for which the nonstructural proteins are essentially required. In addition, biochemical studies of their interactions with other viral proteins, together with immunofluorescence experiments on cells expressing recombinant proteins in different combinations, are providing new indications of their functions. This article contains a critical collection of the most recent achievements and the current hypotheses about the roles of nonstructural proteins in virus replication
<it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system
Get PDFAbstract Background Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused to a 15 amino acid long Biotin Acceptor Peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin to the single lysine residue of the BAP sequence. However, this system is mainly based on the contemporaneous use of two different plasmids or on induction of expression of two proteins through an IRES-driven mechanism. Results We developed a single bigenic plasmid that contains two independent transcriptional units for the co-expression of both the protein tagged with BAP and an engineered version of the BirA enzyme. Upstream of the cDNA encoding BirA, a signal secretion leader sequence was added to allow translocation of the enzyme to the secretory pathway. Three different recombinant antibodies in the scFv format, a membrane bound and secretory truncated IgE Fc fragment and a soluble version of the human IgE high affinity receptor were shown to be efficiently biotinylated and to maintain their binding properties in immunofluorescence microscopy, flow cytometry and ELISA assays. Conclusion The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific in vivo biotinylation of target molecules tagged with a short acceptor peptide. These molecules could be easily obtained from supernatants or extracts of mammalian cells and used for a wide range of biological applications.</p
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
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
Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2
No full textRotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and
packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2,
the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however,
viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be
essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when
coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during
viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated
a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of
other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies
or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize
in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by
being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation
of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments
of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region
essential for interaction with VP1
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
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