Human antiviral protein MxA forms novel metastable membrane-less cytoplasmic condensates exhibiting rapid reversible "crowding"-driven phase transitions

Phase-separated biomolecular condensatesof proteins and nucleic acids form functional membrane-less organelles in the mammalian cell cytoplasmand nucleus. We report that the interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA) forms membrane-less metastable condensatesin the cytoplasm. Light and electron microscopy studies revealed that transient expression of HA- or GFP-tagged MxA in Huh7, HEK293T or Cos7 cells, or exposure of Huh7 cells to IFN-α2a led to the appearance of MxA in the cytoplasmin membrane-less variably-sized spherical or irregular bodies, in filaments and even a reticulum. 1,6-Hexanedioltreatment led to rapid disassembly of these condensates; however, FRAP revealed a relative rigidity with a mobile fraction of only 0.24±0.02 within condensates. In vesicular stomatitis virus(VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with GFP-MxA condensates. Remarkably, the cytoplasmicGFP-MxA condensatesdisassembled within 1-3 min of exposure of cells to hypotonic medium (40-50 milliosmolar) and reassembled within 0.5-2 min of re-exposure of cells to isotonic medium (310-325 milliosmolar) through multiple cycles. Mechanistically, the extent of cytoplasmic"crowding" regulated this phase-separation process. GFP-MxA condensatesalso included the DNA sensor protein cyclic GMP-AMP synthase(cGAS), another protein known to be associated with liquid-like condensates. Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced relative inducibility of ISG54-luc by IFN-α, revealing a physical separation between condensate- and cytosol-based signaling pathways in the cytoplasm. Taken together, the data reveal a new aspect of the cell biology of MxA in the cell cytoplasm.