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

Pentecost M, Vashisht AA, Lester T, Voros T, Beaty SM, Park A, Wang YE, Yun TE, Freiberg AN, Wohlschlegel JA, Lee B - PLoS Pathog. (2015)

Bottom Line: However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread.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.

View Article: PubMed Central - PubMed

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.

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.

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Mutational analysis of the role of a putative NES and a lysine within the NLSbp in nuclear export of Paramyxovirinae matrix proteins.(A) Alignment of Paramyxovirinae M sequence motifs that correspond to NiV-M's leucine-rich NES and NLSbp, which contains a putative ubiquitinated lysine. Predicted critical residues are colored blue. Residues mutated in this study are also underlined in bold font. (B-G) Extended Focus (maximum intensity projection) views of 3D confocal micrographs of HeLa cells transfected with WT, or the indicated mutant GFP-tagged (B) NiV-M, (C) HeV-M, (D) SeV-M, (E) MuV-M, (F) NDV-M or (G) MeV-M. Cells were counterstained with DAPI to visualize nuclear DNA, blue, and fluorescent phalloidin to visualize the F-actin cytoskeleton, red. Scale bar 10 μm. (H) The amount of nuclear M fluorescence per cell was quantified from 3D reconstructed confocal micrographs. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, not significant by one-way ANOVA with Bonferroni adjustment for multiple comparisons.
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ppat.1004739.g002: Mutational analysis of the role of a putative NES and a lysine within the NLSbp in nuclear export of Paramyxovirinae matrix proteins.(A) Alignment of Paramyxovirinae M sequence motifs that correspond to NiV-M's leucine-rich NES and NLSbp, which contains a putative ubiquitinated lysine. Predicted critical residues are colored blue. Residues mutated in this study are also underlined in bold font. (B-G) Extended Focus (maximum intensity projection) views of 3D confocal micrographs of HeLa cells transfected with WT, or the indicated mutant GFP-tagged (B) NiV-M, (C) HeV-M, (D) SeV-M, (E) MuV-M, (F) NDV-M or (G) MeV-M. Cells were counterstained with DAPI to visualize nuclear DNA, blue, and fluorescent phalloidin to visualize the F-actin cytoskeleton, red. Scale bar 10 μm. (H) The amount of nuclear M fluorescence per cell was quantified from 3D reconstructed confocal micrographs. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, not significant by one-way ANOVA with Bonferroni adjustment for multiple comparisons.

Mentions: We have shown that the nuclear-export of NiV-M is regulated by a leucine-rich nuclear export signal (NES) as well as by the K258 lysine residue located within the second basic patch of the bipartite nuclear localization signal (NLSbp; Fig. 2A, blue residues) [39]. A K258A mutation partially disrupts the NLSbp and decreases nuclear localization of NiV-M, while a K258R mutation is unexpectedly retained in the nucleus despite having intact NES sequences and preservation of the positive charge necessary for NLSbp function. However, both mutants are impaired for ubiquitination [39]. Sequence alignment of M proteins indicates that a lysine is present in the homologously aligned position across the Paramyxovirinae genera. Thus, we hypothesized that this residue might be conserved for regulation of M ubiquitin-dependent nuclear export (Fig. 2A, bold and underlined blue residues). To interrogate our hypothesis, we mutated the NLSbp-lysine to an arginine in all M proteins studied and analyzed their subcellular localization by quantitative 3D confocal microcopy as described above (Fig. 2B-G, quantified in Fig. 2H). Since NDV-M contains another lysine adjacent to this position we mutated both (Fig. 2A, bold and underlined blue residues). A lysine to arginine mutation is expected to preserve the nuclear import function of the putative NLSbp, but prevents posttranslational modification at that position. As a comparison for nuclear retention, we also mutated the leucines that correspond to the NES of NiV-M within all M proteins (Fig. 2A, bold and underlined blue residues) [39].


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

Pentecost M, Vashisht AA, Lester T, Voros T, Beaty SM, Park A, Wang YE, Yun TE, Freiberg AN, Wohlschlegel JA, Lee B - PLoS Pathog. (2015)

Mutational analysis of the role of a putative NES and a lysine within the NLSbp in nuclear export of Paramyxovirinae matrix proteins.(A) Alignment of Paramyxovirinae M sequence motifs that correspond to NiV-M's leucine-rich NES and NLSbp, which contains a putative ubiquitinated lysine. Predicted critical residues are colored blue. Residues mutated in this study are also underlined in bold font. (B-G) Extended Focus (maximum intensity projection) views of 3D confocal micrographs of HeLa cells transfected with WT, or the indicated mutant GFP-tagged (B) NiV-M, (C) HeV-M, (D) SeV-M, (E) MuV-M, (F) NDV-M or (G) MeV-M. Cells were counterstained with DAPI to visualize nuclear DNA, blue, and fluorescent phalloidin to visualize the F-actin cytoskeleton, red. Scale bar 10 μm. (H) The amount of nuclear M fluorescence per cell was quantified from 3D reconstructed confocal micrographs. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, not significant by one-way ANOVA with Bonferroni adjustment for multiple comparisons.
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ppat.1004739.g002: Mutational analysis of the role of a putative NES and a lysine within the NLSbp in nuclear export of Paramyxovirinae matrix proteins.(A) Alignment of Paramyxovirinae M sequence motifs that correspond to NiV-M's leucine-rich NES and NLSbp, which contains a putative ubiquitinated lysine. Predicted critical residues are colored blue. Residues mutated in this study are also underlined in bold font. (B-G) Extended Focus (maximum intensity projection) views of 3D confocal micrographs of HeLa cells transfected with WT, or the indicated mutant GFP-tagged (B) NiV-M, (C) HeV-M, (D) SeV-M, (E) MuV-M, (F) NDV-M or (G) MeV-M. Cells were counterstained with DAPI to visualize nuclear DNA, blue, and fluorescent phalloidin to visualize the F-actin cytoskeleton, red. Scale bar 10 μm. (H) The amount of nuclear M fluorescence per cell was quantified from 3D reconstructed confocal micrographs. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS, not significant by one-way ANOVA with Bonferroni adjustment for multiple comparisons.
Mentions: We have shown that the nuclear-export of NiV-M is regulated by a leucine-rich nuclear export signal (NES) as well as by the K258 lysine residue located within the second basic patch of the bipartite nuclear localization signal (NLSbp; Fig. 2A, blue residues) [39]. A K258A mutation partially disrupts the NLSbp and decreases nuclear localization of NiV-M, while a K258R mutation is unexpectedly retained in the nucleus despite having intact NES sequences and preservation of the positive charge necessary for NLSbp function. However, both mutants are impaired for ubiquitination [39]. Sequence alignment of M proteins indicates that a lysine is present in the homologously aligned position across the Paramyxovirinae genera. Thus, we hypothesized that this residue might be conserved for regulation of M ubiquitin-dependent nuclear export (Fig. 2A, bold and underlined blue residues). To interrogate our hypothesis, we mutated the NLSbp-lysine to an arginine in all M proteins studied and analyzed their subcellular localization by quantitative 3D confocal microcopy as described above (Fig. 2B-G, quantified in Fig. 2H). Since NDV-M contains another lysine adjacent to this position we mutated both (Fig. 2A, bold and underlined blue residues). A lysine to arginine mutation is expected to preserve the nuclear import function of the putative NLSbp, but prevents posttranslational modification at that position. As a comparison for nuclear retention, we also mutated the leucines that correspond to the NES of NiV-M within all M proteins (Fig. 2A, bold and underlined blue residues) [39].

Bottom Line: However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread.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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus