Abstract：
Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that βγ-crystallin fused aerolysin-like protein and trefoil factor complex (βγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. βγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, βγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the βγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that βγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the βγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of βγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that βγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.
摘要：
因为大多数动物病毒都有包膜，这些病毒通过与细胞膜融合进入细胞质开始入侵。然而,宿主细胞阻止此类病毒进入细胞质的策略尚不完全清楚。成孔毒素气溶素样蛋白（ALPs）存在于整个动物界，但它们的功能大多是未知的。在这项研究中,我们报道了βγ-晶状体蛋白融合的气溶素样蛋白和三叶因子复合物（βγ-CAT），来自青蛙Bombinamaxima的ALP和三叶因子复合物，通过干扰细胞质进入直接阻止包膜病毒入侵。βγ-CAT靶向HSV1型（HSV-1）包膜上的酸性鞘糖脂诱导孔形成，如蛋白质和钾和钙离子流出的低聚物形成所示。同时,βγ-CAT在脂质体上形成了直径为10nm的环状低聚物，并诱导了模拟病毒包膜的脂质体中的染料释放。出乎意料的是,透射电子显微镜分析显示,βγ-CAT处理的HSV-1明显与载体处理的HSV-1一样完整,表明βγ-CAT不溶解病毒包膜。然而,βγ-CAT处理的HSV-1进入HeLa细胞的细胞质完全受阻。在体内,局部应用βγ-CAT可减轻小鼠HSV-1角膜感染。总的来说，这些结果表明，βγ-CAT具有抵抗包膜病毒侵袭的能力，具有抗病毒作用的特点。我们的发现也将在很大程度上有助于说明动物ALP的推定抗病毒活性。
