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Berlin Brandenburg

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  • 1
    In: PLoS ONE, 2014, Vol.9(7)
    Description: Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se . The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).
    Keywords: Research Article ; Biology And Life Sciences
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: The Journal of Virology, 2011, Vol. 85(6), p.2565
    Description: The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.
    Keywords: Biology;
    ISSN: 0022-538X
    ISSN: 0022538X
    E-ISSN: 10985514
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  • 3
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(8), p.e69997
    Description: Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: PLoS ONE, July 15, 2014, Vol.9(7)
    Description: Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli [beta]-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide ([alpha]-peptide) is complementing the inactive mutant form [DELTA]M15 of [beta]-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).
    Keywords: Peptides – Chemical Properties ; Peptides – Health Aspects ; Cells (Biology) – Chemical Properties ; Cells (Biology) – Health Aspects ; Infection – Chemical Properties ; Infection – Health Aspects ; Enzymes – Chemical Properties ; Enzymes – Health Aspects
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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  • 5
    In: The Journal of Infectious Diseases, 2019, Vol. 219(5), pp.723-733
    Description: Respiratory syncytial virus (RSV) can infect neonatal and adult natural killer cells, thereby inducing a proinflammatory rather than a cytotoxic response. In subneutralizing concentrations, virus-specific antibodies can enhance infection. These findings provide novel insights into the potential mechanisms underlying severe RSV immunopathology.
    Keywords: Rsv ; Nk Cells ; Interferon - Γ ; Antibody ; Ade
    ISSN: 0022-1899
    E-ISSN: 1537-6613
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  • 6
    Language: English
    In: PLoS ONE, 01 January 2017, Vol.12(1), p.e0170877
    Description: Respiratory syncytial virus (RSV) is the leading cause for respiratory illness that requires hospitalization in infancy. High levels of maternal antibodies can protect against RSV infection. However, RSV-infected infants can suffer from severe disease symptoms even in the presence of high levels...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: Antiviral Research, August 2016, Vol.132, pp.1-5
    Description: Palivizumab efficiently blocks respiratory syncytial virus (RSV) infection . However, virus neutralization assays generally omit Fc region-mediated effects. We investigated the neutralization activity of RSV-specific monoclonal antibodies on cells with Fc receptors. Subneutralizing concentrations of antibodies resulted in antibody-dependent enhancement of RSV infection in monocytic cells. Contrary to antibodies targeting other epitopes, the neutralization by palivizumab was augmented in cells with Fc receptors. This unrecognized characteristic of palivizumab may be relevant for its performance .
    Keywords: Medicine ; Biology
    ISSN: 0166-3542
    E-ISSN: 1872-9096
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  • 8
    Language: English
    In: Cytokine, November 2015, Vol.76(1), pp.105-105
    Description: The pattern recognition receptor RIG-I is a pivotal sensor of viral infections. Its activation by 5′-triphosphorylated- or double-stranded-RNA leads to subsequent signaling via MAVS, TBK1 and IKK epsilon resulting in IRF3 nuclear translocation. Activated IRF3 induces transcription of type I and type III interferons and several interferon stimulated genes. Despite intensive investigations on the RIG-I signaling pathway, its regulatory network still remains largely elusive.To gain more insight into the complex regulation of this pathway a kinome-wide siRNA screen was performed. The primary screen revealed over 100 siRNAs that significantly altered the translocation of IRF3 to the nucleus upon RIG-I stimulation. The top 50 candidates were further analyzed in three independent validation screens based on IRF3-sensitive promoter reporter assays or Rift-valley-fever virus replication. Taking all three validation screens into account, 21 novel regulators of the RIG-I signaling pathway could be identified. Relevance of the identified hits in regulating the host-cell antiviral defense was demonstrated by analyzing cytokine profiles and the impact on Influenza A virus replication.In the course of this screen, DAPK1 was identified as an inhibitor of RIG-I mediated IRF3 activation. Extensive mapping experiments revealed a minimal construct, including the kinase domain, to be sufficient for inhibiting IRF3 reporter activation in over-expression experiments. Furthermore, interaction studies revealed binding of DAPK1 to ligand-activated RIG-I, suggesting that a DAPK1 mediated phosphorylation of RIG-I inhibits its activity. In fact, in an in vitro kinase assays we could demonstrate that RIG-I is a substrate of DAPK1.
    Keywords: Medicine ; Biology
    ISSN: 1043-4666
    E-ISSN: 1096-0023
    Source: ScienceDirect Journals (Elsevier)
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  • 9
    Language: English
    In: Journal of virology, August 2015, Vol.89(15), pp.8077-81
    Description: The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein, we created a recombinant PEDV with an artificial furin protease cleavage site N terminal of the putative fusion peptide (PEDV-SFCS). PEDV-SFCS exhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independently of trypsin, though to low titer.
    Keywords: Point Mutation ; Virus Internalization ; Coronavirus Infections -- Veterinary ; Furin -- Metabolism ; Porcine Epidemic Diarrhea Virus -- Genetics ; Spike Glycoprotein, Coronavirus -- Genetics ; Swine Diseases -- Enzymology ; Trypsin -- Metabolism
    ISSN: 0022538X
    E-ISSN: 1098-5514
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  • 10
    Language: English
    In: Journal of virology, July 2014, Vol.88(14), pp.7952-61
    Description: Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.
    Keywords: Virus Internalization ; Porcine Epidemic Diarrhea Virus -- Physiology ; Spike Glycoprotein, Coronavirus -- Metabolism ; Trypsin -- Metabolism
    E-ISSN: 1098-5514
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