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Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

ABSTRACT: The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype.

SUBMITTER: Pentecost M

PROVIDER: S-EPMC4363627 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

Pentecost Mickey M Vashisht Ajay A AA Lester Talia T Voros Tim T Beaty Shannon M SM Park Arnold A Wang Yao E YE Yun Tatyana E TE Freiberg Alexander N AN Wohlschlegel James A JA Lee Benhur B

PLoS pathogens 20150317 3

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nucle ...[more]

PMID: 25782006

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Project description:Abnormalities in nuclear phenotype and chromosome structure are key features of cancer cells. Investigation of the protein determinants of nuclear subfractions in cancer may yield molecular insights into aberrant chromosome function and chromatin organization and in addition may yield biomarkers for early cancer detection. Here we evaluate a proteomics work flow for profiling protein constituents in subnuclear domains in colorectal cancer tissues and apply this work flow to a comparative analysis of the nuclear matrix fraction in colorectal adenoma and carcinoma tissue samples. First, we established the reproducibility of the entire work flow. In a reproducibility analysis of three nuclear matrix fractions independently isolated from the same colon tumor homogenate, 889 of 1,047 proteins (85%) were reproducibly identified at high confidence (minimally two peptides per protein at 99% confidence interval at the protein level) with an average coefficient of variance for the number of normalized spectral counts per protein of 30%. This indicates a good reproducibility of the entire work flow from biochemical isolation to nano-LC-MS/MS analysis. Second, using spectral counting combined with statistics, we identified proteins that are significantly enriched in the nuclear matrix fraction relative to two earlier fractions (the chromatin-binding and intermediate filament fractions) isolated from six colorectal tissue samples. The total data set contained 2,059 non-redundant proteins. Gene ontology mining and protein network analysis of nuclear matrix-enriched proteins revealed enrichment for proteins implicated in "RNA processing" and "mRNA metabolic process." Finally, an explorative comparison of the nuclear matrix proteome in colorectal adenoma and carcinoma tissues revealed many proteins previously implicated in oncogenesis as well as new candidates. A subset of these differentially expressed proteins also exhibited a corresponding change at the mRNA level. Together, the results show that subnuclear proteomics of tumor tissue is feasible and a promising avenue for exploring oncogenesis.

Dataset Information

Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

Publications

Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

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