RT Journal Article
SR Electronic
T1 Human antiviral protein MxA forms novel metastable membrane-less cytoplasmic condensates exhibiting rapid reversible “crowding”-driven phase transitions
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 568006
DO 10.1101/568006
A1 Deodate Davis
A1 Huijuan Yuan
A1 Feng-Xia Liang
A1 Yang-Ming Yang
A1 Jenna Westley
A1 Chris Petzold
A1 Kristen Dancel-Manning
A1 Yan Deng
A1 Joseph Sall
A1 Pravin B. Sehgal
YR 2019
UL http://biorxiv.org/content/early/2019/03/05/568006.abstract
AB Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles in the mammalian cell cytoplasm and nucleus. We report that the interferon (IFN)-inducible human “myxovirus resistance protein A” (MxA) forms membrane-less metastable condensates in 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 cytoplasm in membrane-less variably-sized spherical or irregular bodies, in filaments and even a reticulum. 1,6-Hexanediol treatment 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 cytoplasmic GFP-MxA condensates disassembled 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 condensates also 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.Importance The human interferon-inducible “myxovirus resistance protein A” (MxA), which displays antiviral activity against several RNA and DNA viruses, exists in the cytoplasm in phase-separated membrane-less metastable condensates of variably-sized spherical or irregular bodies, in filaments and even in a reticulum. MxA condensate formation appeared necessary but not sufficient for antiviral activity. 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 Mechanistically, these phase transitions were regulated by the extent of cytoplasmic “crowding.” Moreover, GFP-MxA condensates included the DNA sensor protein cyclic GMP-AMP synthase (cGAS). Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced inducibility of ISG54-luc by IFN-α, revealing a biological distinction between condensate- and cytosol-based signaling pathways. Since intracellular edema and ionic changes are hallmarks of cytopathic viral effects, the rapid hypotonicity-driven disassembly of MxA condensates may modulate MxA.function during virus infection.