Rapid reversible osmoregulation of cytoplasmic biomolecular condensates of human interferon-α-induced antiviral MxA GTPase

Cellular and tissue-level edema is a common feature of acute viral infections such as covid-19, and of many hyponatremic hypoosmolar disorders. However, there is little understanding of the effects of cellular edema on antiviral effector mechanisms. We previously discovered that cytoplasmic human MxA, a major antiviral effector of Type I and III interferons against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes. In this study we investigated how hypoosmolar conditions, mimicking cellular edema, might affect the structure and antiviral function of MxA condensates. Cytoplasmic condensates of both IFN-α-induced endogenous MxA and of exogenously expressed GFP-MxA in human A549 lung and Huh7 hepatoma cells rapidly disassembled within 1-2 min when cells were exposed to hypotonic buffer (∼ 40-50 mOsm), and rapidly reassembled into new structures within 1-2 min of shifting of cells to isotonic culture medium (∼ 330 mOsm). MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. Condensate reassembly, whether induced by isotonic medium or occurring spontaneously under continued moderate hypotonicity, was preceded by “crowding” of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly. Overall, the data highlight the exquisite sensitivity of MxA condensates to rapid reversible osmoregulation. ### Competing Interest Statement The authors have declared no competing interest. * 2-DG : 2-deoxyglucose CLEM : correlated live-cell fluorescence and electron microscopy ELB : hypotonic “erythrocyte” lysis buffer EM : thin-section EM; FLUAV : influenza A virus FRAP : fluorescence recovery after photobleaching assay GAPDH : glycerandehyde 3-phosphate dehydrogenase LLPS : liquid-liquid phase separation MLO : membraneless organelle Mx : myxovirus resistance protein MuMx1 : murine Mx1 MxA or HuMxa : human MxA N protein : nucleocapsid protein P bodies : cytoplasmic processing bodies pi : post-infection PM : plasma membrane TEA : tetraethylammonium TGT-020 : N -1,3,4-thiadiazol-2-yl-3-pyridinecarboxamide VLD : vacuole-like dilation VSV : vesicular stomatitis virus