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      Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts

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          ABSTRACT

          The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336–433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

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          Classical nuclear localization signals: definition, function, and interaction with importin alpha.

          The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.
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            Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.

            In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all cis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.
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              Importin alpha: a multipurpose nuclear-transport receptor.

              The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.
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                Author and article information

                Journal
                Virulence
                Virulence
                KVIR
                kvir20
                Virulence
                Taylor & Francis
                2150-5594
                2150-5608
                2018
                24 April 2018
                24 April 2018
                : 9
                : 1
                : 783-803
                Affiliations
                [a ]Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education, Guizhou University , Guiyang, China
                [b ]College of Animal Science, Guizhou University , Guiyang, China
                [c ]Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, Yangzhou University , Yangzhou, China
                Author notes
                CONTACT Zhiqiang Duan zqduan@ 123456gzu.edu.cn College of Animal Science, Guizhou University , Jiaxiu South Road, Huaxi District, Guiyang, Guizhou Province, 550025, China

                Supplemental data for this article can be accessed at http://doi.org/10.1080/21505594.2018.1449507.

                Article
                1449507
                10.1080/21505594.2018.1449507
                5955436
                29532715
                c6e93a29-6eda-444a-8e55-1c2e6ce27a23
                © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 November 2017
                : 28 February 2018
                Page count
                Figures: 10, Tables: 2, Equations: 0, References: 82, Pages: 21
                Funding
                Funded by: National Natural Science Foundation of China
                Award ID: 31502074
                Award ID: 31760732
                Funded by: Science and Technology Fund of Guizhou Province
                Award ID: QKH-2015-2054
                Funded by: Agricultural Research Project of Guizhou Province
                Award ID: QKHZC-2016-2588
                Funded by: Chinese Special Fund for Agro-scientific Research in the Public Interest
                Award ID: 201303033
                Funded by: Scientific Research Project of Guizhou University Talents Fund
                Award ID: GDRJHZ-2014-10
                This project was supported by National Natural Science Foundation of China (No. 31502074 and 31760732), the Science and Technology Fund of Guizhou Province (No. QKH-2015-2054), the Agricultural Research Project of Guizhou Province (No. QKHZC-2016-2588), the Chinese Special Fund for Agro-scientific Research in the Public Interest (No. 201303033) and the Scientific Research Project of Guizhou University Talents Fund (No. GDRJHZ-2014-10).
                Categories
                Research Paper

                Infectious disease & Microbiology
                newcastle disease virus,matrix protein,nuclear localization signal,nuclear import mechanism,chicken fibroblasts

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