Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV

Abstract

Type I and III interferons (IFNs) induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, while murine Mx1 forms nuclear bodies. While both HuMxA and MuMx1 are antiviral towards influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral towards vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (MLOs) ("biomolecular condensates"). In the present study we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1, 6-hexanediol (5% w/v), or to hypotonic buffer (40-50 mosM), consistent with properties of membraneless phase-separated condensates. FRAP assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ~18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity towards VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and, such cells showed antiviral activity towards VSV

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

“…All cultures were then immunostained for MxA. Panel C, IFN-α2-treated cultures (10 ng/ml, 2 days) were infected with VSV (moi >10 pfu/cell) (44, 46) for 5 hr, fixed and immunostained for MxA (green) and for VSV N (red). Arrows: cells showing condensates with both MxA and VSV N protein.…”

“…Cultures were fizxed in 4% PFA in isotonic PBS or one-third strength PBS (1 hr at 4 o C immunostained for VSV nucleocapsid (N) protein using an mAb provided by Dr. Douglas S. Lyles (mAb 10G4). N-protein immunofluorescence (in red) in GFP-positive (nuclear or cytoplasmic) and negative cells was quantitated on a per cell basis as summarized in Davis et al (44) and Sehgal et al (46) and expressed in arbitrary units (AU) as integrated intensity/cell.…”

“…(44); also see Kochs et al 2002 for very early data for La Crosse virus N protein in membraneless MxA structures (45) by electron microscopy]. Remarkably, condensates formed by exogenously expressed GFP-MxA in human Huh7 and Hep3B hepatoma cells showed rapid disassembly (within 1-2 min) in cells exposed to hypotonic medium, and rapid reassembly (within 1-2 min) into new condensate structures when cells were subsequently shifted to isotonic medium (44, 46). In the present study we investigated whether IFN-α-induced endogenous MxA structures in lung-derived cells showed osmosensing properties, the underlying mechanisms, and whether osmotic disassembly/reassembly of MxA condensates affected their antiviral activity (against VSV).…”

MxB inhibits long interspersed element type 1 retrotransposition

“…MLOs regulate diverse cellular function, such as protein turnover, mitosis mRNA storage and translation, virus replication, and antiviral activity. Murine Mx1 and Mx2 were observed in nuclear bodies and granular cytoplasmic structures [ 73 ]. Coincidentally, exogenously expressed as well as IFN induced human MxA protein also forms as membrane-less condensates in the cytoplasm, and interacts with and sequesters viral nucleocapsid proteins.…”

Advances in the phase separation-organized membraneless organelles in cells: a narrative review

“…A recent study by Stubbe and colleagues demonstrated that PML bodies were enriched around viral replication centers when viral DNA binding proteins were modified with SUMOs, thereby mediating the replication of the virus ( 50 ). In addition, Sehgal revealed that the murine cognate Mx1 could form nuclear condensates overlapping with PML nuclear bodies, further exerting a vital antiviral activity ( 51 ).…”