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Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV

2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês

10.1074/jbc.ra120.015661

ISSN

1083-351X

Autores

Pravin B. Sehgal, Huijuan Yuan, Mia F. Scott, Yan Deng, Feng‐Xia Liang, Andrzej Maćkiewicz,

Tópico(s)

RNA modifications and cancer

Resumo

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV. Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV. Membraneless organelles (MLOs) in the cytoplasm and nucleus formed by liquid-liquid phase-separation (LLPS) biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions (1Mitrea D.M. Kriwacki R.W. Phase separation in biology, functional organization of a higher order.Cell Commun. Signal. 2016; 14 (26727894): 110.1186/s12964-015-0125-7Crossref PubMed Scopus (277) Google Scholar, 2Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18 (28225081): 285-29810.1038/nrm.2017.7Crossref PubMed Scopus (1348) Google Scholar, 3Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357 (28935776)eaaf438210.1126/science.aaf4382Crossref PubMed Scopus (925) Google Scholar, 4Alberti S. The wisdom of crowds: regulating cell function through condensed states of living matter.J. Cell Sci. 2017; 130 (28808090): 2789-279610.1242/jcs.200295Crossref PubMed Scopus (79) Google Scholar, 5Gomes E. Shorter J. The molecular language of membraneless organelles.J. Biol. Chem. 2019; 294 (30045872): 7115-712710.1074/jbc.TM118.001192Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Alberti S. Gladfelter A. Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176 (30682370): 419-43410.1016/j.cell.2018.12.035Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 7Bracha D. Walls M.T. Wei M.-T. Zhu L. Kurian M. Avalos J.L. Toettcher J.E. Brangwynne C.P. Mapping local and global liquid phase behavior in living cells using photo-oligomerized seeds.Cell. 2018; 175 (30500534): 1467-148010.1016/j.cell.2018.10.048Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Elbaum-Garfinkle S. Matter over mind: liquid phase-separation and neurodegeneration.J. Biol. Chem. 2019; 294 (30914480): 7160-716810.1074/jbc.REV118.001188Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 9Sehgal P.B. Westley J. Lerea K.M. DiSenso-Browne S. Etlinger J.D. Biomolecular condensates in cell biology and virology: phase-separated membraneless organelles (MLOs).Anal. Biochem. 2020; 597 (32194074)11369110.1016/j.ab.2020.113691Crossref PubMed Scopus (15) Google Scholar, 10Sehgal P.B. Lerea K.L. Biomolecular condensates and membraneless organelles (MLOs).in: Arthur Cooper Encyclopedia of Biological Chemistry. 3rd Ed. Elsevier, Amsterdam2020Google Scholar). These functions include cell differentiation, cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover, and mitosis (reviewed in Refs. 1Mitrea D.M. Kriwacki R.W. Phase separation in biology, functional organization of a higher order.Cell Commun. Signal. 2016; 14 (26727894): 110.1186/s12964-015-0125-7Crossref PubMed Scopus (277) Google Scholar, 2Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18 (28225081): 285-29810.1038/nrm.2017.7Crossref PubMed Scopus (1348) Google Scholar, 3Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357 (28935776)eaaf438210.1126/science.aaf4382Crossref PubMed Scopus (925) Google Scholar, 4Alberti S. The wisdom of crowds: regulating cell function through condensed states of living matter.J. Cell Sci. 2017; 130 (28808090): 2789-279610.1242/jcs.200295Crossref PubMed Scopus (79) Google Scholar, 5Gomes E. Shorter J. The molecular language of membraneless organelles.J. Biol. Chem. 2019; 294 (30045872): 7115-712710.1074/jbc.TM118.001192Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Alberti S. Gladfelter A. Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176 (30682370): 419-43410.1016/j.cell.2018.12.035Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 7Bracha D. Walls M.T. Wei M.-T. Zhu L. Kurian M. Avalos J.L. Toettcher J.E. Brangwynne C.P. Mapping local and global liquid phase behavior in living cells using photo-oligomerized seeds.Cell. 2018; 175 (30500534): 1467-148010.1016/j.cell.2018.10.048Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Elbaum-Garfinkle S. Matter over mind: liquid phase-separation and neurodegeneration.J. Biol. Chem. 2019; 294 (30914480): 7160-716810.1074/jbc.REV118.001188Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 9Sehgal P.B. Westley J. Lerea K.M. DiSenso-Browne S. Etlinger J.D. Biomolecular condensates in cell biology and virology: phase-separated membraneless organelles (MLOs).Anal. Biochem. 2020; 597 (32194074)11369110.1016/j.ab.2020.113691Crossref PubMed Scopus (15) Google Scholar, 10Sehgal P.B. Lerea K.L. Biomolecular condensates and membraneless organelles (MLOs).in: Arthur Cooper Encyclopedia of Biological Chemistry. 3rd Ed. Elsevier, Amsterdam2020Google Scholar). MLOs include the nucleolus, nucleoporin channels, nuclear speckles and paraspeckles, nuclear promyelocytic leukemia (PML) bodies, nuclear Cajal bodies, cytoplasmic processing bodies (P-bodies), germinal bodies, Balbiani bodies, Negri bodies, stress granules, translation-promoting TPA-inducible sequence (TIS) granules, and several more recent discoveries, such as condensates of synapsin, of the DNA sensor protein cyclic GMP-AMP synthase in the cytoplasm, and of active transcription-associated condensates in the nucleus (1Mitrea D.M. Kriwacki R.W. Phase separation in biology, functional organization of a higher order.Cell Commun. Signal. 2016; 14 (26727894): 110.1186/s12964-015-0125-7Crossref PubMed Scopus (277) Google Scholar, 2Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18 (28225081): 285-29810.1038/nrm.2017.7Crossref PubMed Scopus (1348) Google Scholar, 3Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357 (28935776)eaaf438210.1126/science.aaf4382Crossref PubMed Scopus (925) Google Scholar, 4Alberti S. The wisdom of crowds: regulating cell function through condensed states of living matter.J. Cell Sci. 2017; 130 (28808090): 2789-279610.1242/jcs.200295Crossref PubMed Scopus (79) Google Scholar, 5Gomes E. Shorter J. The molecular language of membraneless organelles.J. Biol. Chem. 2019; 294 (30045872): 7115-712710.1074/jbc.TM118.001192Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Alberti S. Gladfelter A. Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176 (30682370): 419-43410.1016/j.cell.2018.12.035Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 7Bracha D. Walls M.T. Wei M.-T. Zhu L. Kurian M. Avalos J.L. Toettcher J.E. Brangwynne C.P. Mapping local and global liquid phase behavior in living cells using photo-oligomerized seeds.Cell. 2018; 175 (30500534): 1467-148010.1016/j.cell.2018.10.048Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Elbaum-Garfinkle S. Matter over mind: liquid phase-separation and neurodegeneration.J. Biol. Chem. 2019; 294 (30914480): 7160-716810.1074/jbc.REV118.001188Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 9Sehgal P.B. Westley J. Lerea K.M. DiSenso-Browne S. Etlinger J.D. Biomolecular condensates in cell biology and virology: phase-separated membraneless organelles (MLOs).Anal. Biochem. 2020; 597 (32194074)11369110.1016/j.ab.2020.113691Crossref PubMed Scopus (15) Google Scholar, 10Sehgal P.B. Lerea K.L. Biomolecular condensates and membraneless organelles (MLOs).in: Arthur Cooper Encyclopedia of Biological Chemistry. 3rd Ed. Elsevier, Amsterdam2020Google Scholar). Overall, these condensates have liquid-like internal properties (as investigated using fluorescence recovery after photobleaching (FRAP) assays) and are metastable, changing to a gel or to filaments commensurate with the cytoplasmic environment (temperature, ionic conditions, physical deformation, or cytoplasmic "crowding"), and the incorporation of additional proteins, RNA or DNA molecules, or post-translational modifications (1Mitrea D.M. Kriwacki R.W. Phase separation in biology, functional organization of a higher order.Cell Commun. Signal. 2016; 14 (26727894): 110.1186/s12964-015-0125-7Crossref PubMed Scopus (277) Google Scholar, 2Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18 (28225081): 285-29810.1038/nrm.2017.7Crossref PubMed Scopus (1348) Google Scholar, 3Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357 (28935776)eaaf438210.1126/science.aaf4382Crossref PubMed Scopus (925) Google Scholar, 4Alberti S. The wisdom of crowds: regulating cell function through condensed states of living matter.J. Cell Sci. 2017; 130 (28808090): 2789-279610.1242/jcs.200295Crossref PubMed Scopus (79) Google Scholar, 5Gomes E. Shorter J. The molecular language of membraneless organelles.J. Biol. Chem. 2019; 294 (30045872): 7115-712710.1074/jbc.TM118.001192Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Alberti S. Gladfelter A. Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176 (30682370): 419-43410.1016/j.cell.2018.12.035Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 7Bracha D. Walls M.T. Wei M.-T. Zhu L. Kurian M. Avalos J.L. Toettcher J.E. Brangwynne C.P. Mapping local and global liquid phase behavior in living cells using photo-oligomerized seeds.Cell. 2018; 175 (30500534): 1467-148010.1016/j.cell.2018.10.048Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Elbaum-Garfinkle S. Matter over mind: liquid phase-separation and neurodegeneration.J. Biol. Chem. 2019; 294 (30914480): 7160-716810.1074/jbc.REV118.001188Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 9Sehgal P.B. Westley J. Lerea K.M. DiSenso-Browne S. Etlinger J.D. Biomolecular condensates in cell biology and virology: phase-separated membraneless organelles (MLOs).Anal. Biochem. 2020; 597 (32194074)11369110.1016/j.ab.2020.113691Crossref PubMed Scopus (15) Google Scholar, 10Sehgal P.B. Lerea K.L. Biomolecular condensates and membraneless organelles (MLOs).in: Arthur Cooper Encyclopedia of Biological Chemistry. 3rd Ed. Elsevier, Amsterdam2020Google Scholar). Indeed, specific DNA and RNA molecules participate in the assembly of many such cytoplasmic and nuclear condensates and in their functions (1Mitrea D.M. Kriwacki R.W. Phase separation in biology, functional organization of a higher order.Cell Commun. Signal. 2016; 14 (26727894): 110.1186/s12964-015-0125-7Crossref PubMed Scopus (277) Google Scholar, 2Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18 (28225081): 285-29810.1038/nrm.2017.7Crossref PubMed Scopus (1348) Google Scholar, 3Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357 (28935776)eaaf438210.1126/science.aaf4382Crossref PubMed Scopus (925) Google Scholar, 4Alberti S. The wisdom of crowds: regulating cell function through condensed states of living matter.J. Cell Sci. 2017; 130 (28808090): 2789-279610.1242/jcs.200295Crossref PubMed Scopus (79) Google Scholar, 5Gomes E. Shorter J. 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