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  1. Article ; Online: Protocol for assessing translational regulation in mammalian cell lines by OP-Puro labeling

    Jack Chun-Chieh Hsu / Joanna B. Pawlak / Maudry Laurent-Rolle / Peter Cresswell

    STAR Protocols, Vol 3, Iss 3, Pp 101654- (2022)

    2022  

    Abstract: Summary: Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin ...

    Abstract Summary: Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry.For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
    Keywords Cell biology ; Molecular biology ; Gene expression ; Molecular/Chemical probes ; Science (General) ; Q1-390
    Language English
    Publishing date 2022-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: CMPK2 restricts Zika virus replication by inhibiting viral translation.

    Joanna B Pawlak / Jack Chun-Chieh Hsu / Hongjie Xia / Patrick Han / Hee-Won Suh / Tyler L Grove / Juliet Morrison / Pei-Yong Shi / Peter Cresswell / Maudry Laurent-Rolle

    PLoS Pathogens, Vol 19, Iss 4, p e

    2023  Volume 1011286

    Abstract: Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can ... ...

    Abstract Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.
    Keywords Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2023-04-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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