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Osmosensing by cytoplasmic biomolecular condensates of antiviral MxA GTPase

Cellular and tissue‐level edema is a common feature of acute viral infections, including covid‐19. However, there is little understanding of the effects of tissue edema on antiviral effector mechanisms. In as much as defects in IFN‐α function (such as due to presence of autoantibodies) are now implicated in enhanced severity of covid‐19 disease, we investigated how hypoosmolarity and cellular edema might affect the antiviral activity of IFN‐α‐induced human MxA GTPase, a major antiviral effector against several RNA and DNA viruses. We had previously discovered that cytoplasmic human MxA existed in the cytoplasm in phase‐separated membraneless biomolecular condensates with a gel‐like internal consistency. The vesicular stomatitis virus (VSV) nucleocapsid (N) protein associated with such MxA condensates in cells exhibiting an antiviral phenotype. Unexpectedly, MxA condensates displayed rapid and reversible osmosensing properties. Condensates of both IFN‐α‐induced endogenous MxA and of exogenous GFP‐MxA expressed in human A549 lung and Huh7 hepatoma cells rapidly disassembled within 1‐2 min when cells were exposed to hypotonic buffer (approx. 50 mOsm), and rapidly reassembled into new structures within 1‐2 min of shifting of cells to isotonic culture medium (approx. 330 mOsm). GFP‐MxA condensates in cells continuously exposed to culture medium in the range one‐fourth, one‐third or one‐half isotonicity first rapidly disassembled within 1‐2 min, and then, in most cells. spontaneously reassembled 7‐15 min later into new structures. The data are consistent with cytoplasmic “crowding” as the mechanism driving MxA condensate formation. In terms of antiviral function, one 5 min cycle of disassembly and reassembly of GFP‐MxA condensates at 1 or 2 hr after VSV infection did not affect MxA antiviral activity. Additionally, continuous exposure of GFP‐MxA expressing A549 lung cells to culture medium at one‐fourth or one‐third tonicity (thus triggering a cycle of disassembly and slower spontaneous reassembly in the continued presence of hypotonic culture medium) together with a VSV challenge, left antiviral activity largely unaffected. Such cultures showed an increase in number of cells with VSV N colocalized with the reassembled GFP‐MxA condensates. The data reveal a novel property of MxA biomolecular condensates ‐ rapid and reversible osmosensing. Importantly, antiviral activity was largely unaffected by tonicity‐driven disassembly/reassembly mimicking conditions of cellular and tissue‐level edema.