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TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein

ABSTRACT: Nuclear localization of cytoplasmic RNA virus proteins mediated by intrinsic nuclear localization signal (NLS) plays essential roles in successful virus replication. We previously reported that NLS mutation in the matrix (M) protein obviously attenuates the replication and pathogenicity of Newcastle disease virus (NDV), but the attenuated replication mechanism of NDV remains unclear. In this study, we showed that M/NLS mutation not only disrupted M’s nucleocytoplasmic trafficking characteristic but also impaired viral RNA synthesis and transcription. Using TMT-based quantitative proteomics analysis of BSR-T7/5 cells infected with the parental NDV rSS1GFP and the mutant NDV rSS1GFP-M/NLSm harboring M/NLS mutation, we found that rSS1GFP infection stimulated much greater quantities and more expression level changes of differentially expressed proteins involved in host cell transcription, ribosomal structure, posttranslational modification, and intracellular trafficking than rSS1GFP-M/NLSm infection. Further in-depth analysis revealed that early nuclear localization of M protein inhibited cell transcription and participated in reducing cellular protein synthesis, posttranscriptional modification and transport; whereas later cytoplasmic localization of M protein promoted viral protein synthesis and benefited for virus assembly and budding. Importantly, we first demonstrated that later cytoplasmic localization of M protein effected the inhibition of TIFA expression in a dose-dependent manner, and inhibiting TIFA expression was beneficial to NDV replication by down-regulating TIFA/TRAF6/NF-κB-mediated production of cytokines. Our findings suggest that precocious cytoplasmic localization of M protein caused by M/NLS mutation disrupts these important biological processes, and thereby causes the attenuated replication of NDV, demonstrating that NDV replication is closely related to the nucleocytoplasmic trafficking of M protein.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Cricetidae

SUBMITTER: zhiqiang duan

LAB HEAD: Zhiqiang Duan

PROVIDER: PXD018098 | Pride | 2020-05-22

REPOSITORIES: Pride

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TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein.

Duan Zhiqiang Z Yuan Chao C Han Yifan Y Zhou Lei L Zhao Jiafu J Ruan Yong Y Chen Jiaqi J Ni Mengmeng M Ji Xinqin X

Virulence 20201201 1

Nuclear localization of cytoplasmic RNA virus proteins mediated by intrinsic nuclear localization signal (NLS) plays essential roles in successful virus replication. We previously reported that NLS mutation in the matrix (M) protein obviously attenuates the replication and pathogenicity of Newcastle disease virus (NDV), but the attenuated replication mechanism remains unclear. In this study, we showed that M/NLS mutation not only disrupted M's nucleocytoplasmic trafficking characteristic but als ...[more]

PMID: 32420802

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Project description:The NS1 protein of influenza A virus (IAV) is a multifunctional virulence factor. Mouse adaptive mutations in the NS1 protein of the human isolate A/Hong Kong/1/1968(H3N2) (HK) have been previously reported to increase virulence, viral fitness, and interferon antagonism, but differ in binding to post-transcriptional processing factor CPSF30. Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. HK-wt virus NS1 partitioned equivalently between the cytoplasm and nucleus in human cells but was defective in cytoplasmic localization in mouse cells. The adaptive mutations either increased the proportion or abundance of NS1 in the cytoplasm, and/or the nucleus. NS1 mutations that increased cytoplasmic distribution identified a putative second nuclear export signal (NES) spanning aa positions 98-106 LSEDWFMLM, (mutation sites in bold); with the strongest effect seen for mutation M106I. The putative NES in the NS3 protein was associated with cytoplasmic localization. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low gene regulation (HGR or LGR) phenotypes that mapped to the amino-terminal and the carboxy-terminal regions respectively. The HGR and LGR mutations were predominantly down regulating versus up regulating respectively. The greatest effect on host gene expression in the HGR group correlated with the ability of the NS1 protein to bind CPSF30. To our knowledge this is the first report of roles of adaptive NS1 mutations that affect intracellular localization and regulation of host gene expression. biological triplicates of mock infected mouse M1 cells; cells infected with A/HK/1/1968(H3N2) wt and NS1 mtuatn with mutaions, D2N, V23A, L98S, L98S + M106I, F103L, M106V, M106V + M124I, D125G, V180A, V226I, M106I, and R227K. Cells were infected at a multiplicty of infection of 2 and cels were incubated for 8 hr at 37 C for 8 hrs before RNA extraction and analysis relative to mock PBS infected cells.

Dataset Information

TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein

Publications

TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein.

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