Departments of Cell Biology and Anatomy, New York Medical College, Valhalla, New York, USA; Department of Medicine, New York Medical College, Valhalla, New York, USA.
Departments of Cell Biology and Anatomy, New York Medical College, Valhalla, New York, USA.
J Biol Chem. 2020 Dec 25;295(52):18023-18035. doi: 10.1074/jbc.RA120.015661. Epub 2020 Oct 19.
Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms structures, whereas murine Mx1 forms bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("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. Fluorescence recovery after photobleaching (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 toward 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 toward VSV.
I 型和 III 型干扰素诱导人细胞中“粘液病毒抗性蛋白” MxA 和鼠细胞中其同源物 Mx1 的表达。人 MxA 形成 结构，而鼠 Mx1 形成 体。尽管人 MxA 和鼠 Mx1 对甲型流感病毒 (FLUAV)（正粘病毒）均具有抗病毒作用，但只有人 MxA 被认为对水疱性口炎病毒 (VSV)（弹状病毒）具有抗病毒作用。我们之前报道细胞质中人 GFP-MxA 结构是相分离的无膜细胞器（“生物分子凝聚物”）。在本研究中，我们研究了核鼠 Mx1 结构是否也代表相分离的生物分子凝聚物。瞬时表达鼠 GFP-Mx1 于人 Huh7 肝癌细胞、人 Mich-2H6 黑色素瘤细胞和鼠 NIH 3T3 细胞中导致 Mx1 核体的出现。这些 GFP-MuMx1 核体在用 1,6-己二醇（5%，w/v）或低渗缓冲液（40-50mosm）处理细胞时迅速解体，与无膜相分离凝聚物的特性一致。荧光恢复后光漂白（FRAP）测定显示，GFP-MuMx1 核体在光漂白后显示出缓慢的部分恢复（移动分数：约 18%），提示凝胶样一致性。令人惊讶的是，GFP-MuMx1 在 Huh7 细胞中的表达也导致在 20-30%的转染细胞中出现新型细胞质巨丝蛋白为基础的中间丝网和细胞质体中的 GFP-MuMx1。值得注意的是，具有细胞质鼠 GFP-MuMx1 丝的 Huh7 细胞，但不是只有核体的细胞，显示出对 VSV 的抗病毒活性。因此，GFP-MuMx1 核体包含相分离的凝聚物。出乎意料的是，GFP-MuMx1 在 Huh7 细胞中也与细胞质巨丝蛋白为基础的中间丝相关联，并且此类细胞对 VSV 具有抗病毒活性。
