1,720,967 research outputs found
The predominant species of nonstructural protein 4B in hepatitis C virus-replicating cells is not palmitoylated
No full textHepatitis C virus (HCV) represents a significant global health burden. Viral replication is thought to occur in close association with remodelled host cell membranes with non-structural protein 4B (NS4B) being a key player in this process. NS4B is a poorly characterized integral membrane protein, which has been reported to be palmitoylated at its carboxy-terminal end. In order to extend this observation and to establish a functional role for NS4B palmitolylation, we sought to determine the status of this post-translational modification when the protein was expressed as part of a functional viral replicase. We performed direct metabolic labelling and polyethylene glycol-maleimide palmitoylation reporter assays on NS4B expressed in cells containing subgenomic replicons and infectious viral RNA. In a vaccinia virus-based expression system NS4B palmitoylation was detected in a genotype-dependent manner. However, in spite of the high sensitivity of the methods used, no NS4B palmitoylation was found in physiologically more relevant systems. Thus, NS4B palmitoylation is most likely dispensable for HCV RNA replicatio
Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors
No full textBaculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a ‘minigenome’ derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but {beta}-galactosidase activity was limited to 10–20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis {delta} virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis {delta} virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host–cell interactions
Translation termination reinitiation between open reading frame 1 (ORF1) and ORF2 enables capsid expression in a bovine norovirus without the need for production of viral subgenomic RNA
No full textA generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.<br/
A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A
No full textHyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection
Interplay between respiratory viruses and cilia in the airways
Full text linkThe airway epithelium is the first point of contact for inhaled pathogens. The role of epithelial cells in clearance, infection and colonisation of bacteria is established. The interactions of respiratory viruses and cilia is less understood, but viruses are known to target ciliated epithelial cells for entry, replication and dissemination. Furthermore, some respiratory viruses impair and/or enhance ciliary activity. This review examines what is known about the interactions between cilia and viral infection and how respiratory viruses effect cilia function with subsequent consequences for human health. We discuss the models which can be used to investigate the relationship between respiratory viruses and the host airway.</p
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
Increasing the rate of cleavage at the boundary between non-structural protein 4B and 5A inhibits replication of hepatitis C virus
No full textIn hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. While proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However it is not known whether slow cleavage is important for viral replication, or a consequence of restrictions on sequences which can be tolerated at the cleaved ends of non-structuralproteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary and their effect on replication and polyprotein processing examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codondegeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall the data indicate that slow cleavage of the NS4B5A boundary is important, and identify a previously unrecognised role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time
Real-world performance of a single-use, analyser-free, molecular point-of-care test for COVID-19 used in the emergency department: results of a prospective trial (ED-POC)
No full textBackground: a novel single-use, analyser-free, molecular point-of-care test for SARS-CoV-2 (Veros COVID-19 test, Sherlock Biosciences) could reduce time to results and improve patient care and flow in the emergency department (ED), but its performance in this setting is unknown. Methods: adults aged ≥18 years presenting to Southampton General Hospital (UK) with suspected COVID-19 were tested with the Veros COVID-19 test in addition to standard of care near-patient PCR. Measures of diagnostic accuracy were calculated for the Veros COVID-19 test stratified by Ct value. Discrepant results underwent viral culture. Findings: between Jan 16 and May 2, 2023, 400 patients were enrolled with a median (IQR) age of 60 (34−77) and 141 (35·3%) were SARS-CoV-2 positive by PCR. The Veros test gave valid results on the first test in 384 (96·0%), and sensitivity and specificity were 127/141 (90·1%, 95%CI 83·9–94·5) and 258/259 (99·6%, 95%CI 97·9–100) overall. For those with high or moderate viral load (Ct ≤30), sensitivity was 125/129 (96·9%, 95%CI 92·3–99·2). One (7·1%) of 14 PCR positive/Veros test negative samples was culture positive. Median (IQR) time from sample collection to result was 19 (18−20) mins with the Veros test versus 73 (59−92) mins with PCR (p < 0·0001). Interpretation: the Veros COVID-19 test generated results in near real-time, around 1 h sooner than rapid, near-patient, analyser-based PCR, and accuracy was excellent for samples with moderate and high viral loads. The Veros test represents a step-change in molecular diagnostics for infection and could significantly reduce time to results and improve patient management in EDs and other settings.</p
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
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