Human Antiviral Protein MxA Forms Novel Metastable Membrane-Less Cytoplasmic Condensates Exhibiting Rapid Reversible Tonicity-Driven Phase Transitions
Cell Biology and Anatomy
Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g. stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membrane-less metastable (shape-changing) condensates in the cytoplasm. In our studies we used the same cell lines and methods as used by previous investigators but concluded that wt MxA formed variably-sized spherical or irregular bodies, filaments and even a reticulum distinct from ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of GFP-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24+/-0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase respectively in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-alpha-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature, and provide evidence for membrane-less MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation, and further report that MxA forms membrane-less metastable (shape-changing) condensates in the cytoplasm consisting of variably-sized spherical or irregular bodies, filaments and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.
Davis, D., Yuan, H., Liang, F., Yang, Y., Westley, J., Petzold, C., Dancel-Manning, K., Deng, Y., Sall, J., & Sehgal, P. (2019). Human Antiviral Protein MxA Forms Novel Metastable Membrane-Less Cytoplasmic Condensates Exhibiting Rapid Reversible Tonicity-Driven Phase Transitions. Journal of Virology, 93 (22), e01014-19. https://doi.org/10.1128/JVI.01014-19