Human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause in vitro caspase-independent cell death, accompanied by previously undescribed cytoplasmic vacuolization with abnormal fusion of various types of endosomal-lysosomal organelles. Yeasts are unicellular eukaryotes widely used as a biological model to study fundamental cellular processes. In particular, yeast models of cell death are useful due to the similarity of regulated cell death mechanisms between yeast and humans. This, together with the functional similarity of yeast vacuoles and the lysosomal/endosomal compartment of mammalian cells, suggests yeasts to be a promising model to study molecular mechanisms of 3Cpro action. Here, we expressed 3Cpro and its mutant inactivated form (3Cmut) in Saccharomyces cerevisiae using pYES2 vector. It was shown that expression of 3Cpro, but not 3Cmut, caused cells to lose the ability to proliferate, but maintained viability, thus indicating the enzyme exerts a cytostatic effect. In addition, using vacuolar dyes and light and electron microscopy, it was found that 3Cpro causes vacuole distortion and fragmentation, accompanied by the abnormal vacuolar staining. In summary, the effects of the 3Cpro on yeast cells resemble the response of mammalian cells. Therefore, the experimental system based on S. cerevisiae can be used for further studies of 3Cpro action.

最近显示，人类甲型肝炎病毒3C蛋白酶（3Cpro）会导致体外不依赖caspase的细胞死亡，并伴有以前未描述的胞浆空泡化以及各种类型的内体-溶酶体细胞器的异常融合。酵母是单细胞真核生物，被广泛用作研究基本细胞过程的生物学模型。尤其是，由于酵母与人类之间调控的细胞死亡机制相似，因此细胞死亡的酵母模型非常有用。这与酵母液泡的功能相似性和哺乳动物细胞的溶酶体/内体区室一起，表明酵母菌是研究3Cpro作用分子机制的有前途的模型。在这里，我们在酿酒酵母中表达了3Cpro及其突变体失活形式（3Cmut）。使用pYES2向量。已经表明3Cpro而不是3Cmut的表达引起细胞丧失增殖能力，但是保持活力，因此表明该酶发挥了细胞抑制作用。此外，使用液泡染料和光镜和电子显微镜观察，发现3Cpro引起液泡变形和断裂，并伴有液泡异常染色。总之，3Cpro对酵母细胞的作用类似于哺乳动物细胞的反应。因此，基于酿酒酵母的实验系统可用于3Cpro作用的进一步研究。