title: 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.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Mickey
authors.initials: M
authors.lastname: Pentecost
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Ajay A
authors.initials: AA
authors.lastname: Vashisht
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Talia
authors.initials: T
authors.lastname: Lester
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Tim
authors.initials: T
authors.lastname: Voros
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Beaty
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Arnold
authors.initials: A
authors.lastname: Park
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Yao E
authors.initials: YE
authors.lastname: Wang
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors.forename: Tatyana E
authors.initials: TE
authors.lastname: Yun
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Freiberg
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America; Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Benhur
authors.initials: B
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D015151:Immunoblotting; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D019913:Nuclear Localization Signals / Q000378:metabolism; D018098:Paramyxovirinae / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D014162:Transfection; D025801:Ubiquitin; D014709:Vero Cells; D014763:Viral Matrix Proteins / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D025801:Ubiquitin; D014763:Viral Matrix Proteins