Murine GFP-Mx1 forms phase-separated nuclear condensates and associates with cytoplasmic intermediate filaments: novel antiviral activity against vesicular stomatitis virus

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, some tethered to intermediate filaments. In contrast, murine Mx1 mainly forms nuclear bodies. Both HuMxA and MuMx1 are antiviral towards influenza A virus (FLUAV) (an orthomyxovirus). However, it has long been considered that HuMxA, but not MuMx1, was antiviral towards vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures in Huh7 hepatoma cells 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 Huh7 hepatoma and Mich-2H6 melanoma 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 MuMx1 in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic structures in 20-30% of transiently transfected Huh7 cells. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity towards VSV. Thus, murine GFP-Mx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 associated with cytoplasmic giantin-based intermediate filaments in a subset of Huh7 cells, and, such cells showed antiviral activity towards VSV.

Metastable biomolecular condensates of interferon-inducible antiviral Mx-family GTPases: A paradigm shift in the last three years

“…Human hepatoma Huh7 cells cultured in 35 mm plates were transiently transfected with expression vector for GFP-MxA (Panel A ) or HA-MxA (Panel B ). Two days later the cultures were fixed and imaged for GFP-MxA (Panel A , by fluorescence in green), HA-MxA (Panel B , by immunofluorescence using anti-HA mAb in green), and intermediate filaments (Panels A and B , using anti-giantin pAb in red; see Sehgal et al 2020b for validation). White arrows point to spherical GFP-MxA structures and streaks of HA-MxA adhering to intermediate filaments in Panels A and B respectively.…”

“…Human MxA forms cytoplasmic structures while murine Mx1 mainly forms nuclear bodies (table 1 ; figure 6 ; Haller and Kochs 2002 ; Kochs et al 2002 ; Engelhardt et al 2004 ; Haller et al 2015 ; Sehgal et al 2020b ). The molecular mechanisms of the Mx antiviral effects are incompletely understood; these effects include inhibition of early and late viral transcription and viral replication, as well as effects at the level of the transit of viral components through the nuclear pore (Haller and Kochs 2002 ; Verhelst et al 2013 ; Haller et al 2015 ; Steiner and Pavlovic 2020 ).…”

“… *See reviews Verhelst et al ( 2013 ), Haller et al ( 2015 , 2018 ) and Steiner and Pavlovic ( 2020 ) for detailed antiviral spectrum reported in the previous literature. Modified from Sehgal et al ( 2020b ). …”

“…Additionally, human MxA was antiviral towards FLUAV and vesicular stomatitis virus (VSV, a rhabdovirus whose entire life cycle is cytoplasmic), while human MxB was antiviral towards lentiviruses such as HIV but not FLUAV or VSV. In contrast to human MxA, the orthologous nuclear murine Mx1 was antiviral towards FLUAV (which has an obligatory nuclear step in its life cycle) but not against VSV, while, curiously, the cytoplasmic granular murine Mx2 (also an ortholog of human MxA) was antiviral towards VSV (Haller et al 2015 ; Sehgal et al 2020b ) (table 1 ). The GTPase activity was required for these antiviral activities (Dick et al 2015 ; Nigg and Pavlovic 2015 ; Haller et al 2015 , 2018 ).…”

“…The GTPase activity was required for these antiviral activities (Dick et al 2015 ; Nigg and Pavlovic 2015 ; Haller et al 2015 , 2018 ). However, despite extensive studies over the last six decades, the molecular mechanisms by which Mx proteins inhibit replication of different viruses in intact cells are incompletely understood (Haller et al 2015 , 2018 ; Sehgal et al 2020b ; Steiner and Pavlovic 2020 ). Surprisingly, even the understanding of the basic cell biology of Mx proteins needed a paradigm shift in the last three years.…”