Golden Joseph W, Bahe Jessica A, Lucas William T, Nibert Max L, Schiff Leslie A
Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Biol Chem. 2004 Mar 5;279(10):8547-57. doi: 10.1074/jbc.M309758200. Epub 2003 Dec 11.
In murine fibroblasts, efficient proteolysis of reovirus outer capsid protein sigma3 during cell entry by virions requires the acid-dependent lysosomal cysteine protease cathepsin L. The importance of cathepsin L for infection of other cell types is unknown. Here we report that the acid-independent lysosomal cysteine protease cathepsin S mediates outer capsid processing in macrophage-like P388D cells. P388D cells supported infection by virions of strain Lang, but not strain c43. Genetic studies revealed that this difference is determined by S4, the viral gene segment that encodes sigma3. c43-derived subvirion particles that lack sigma3 replicated normally in P388D cells, suggesting that the difference in infectivity of Lang and c43 virions is at the level of sigma3 processing. Infection of P388D cells with Lang virions was inhibited by the broad spectrum cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane but not by NH(4)Cl, which raises the endocytic pH and thereby inhibits acid-dependent proteases such as cathepsins L and B. Outer capsid processing and infection of P388D cells with Lang virions were also inhibited by a cathepsin S-specific inhibitor. Furthermore, in the presence of NH(4)Cl, cell lines engineered to express cathepsin S supported infection by Lang, but not c43, virions. Our results thus indicate that differences in susceptibility to cathepsin S-mediated sigma3 processing are responsible for strain differences in reovirus infection of macrophage-like P388D cells and other cathepsin S-expressing cells. Additionally, our data suggest that the acid dependence of reovirus infections of most other cell types may reflect the low pH requirement for the activities of most other lysosomal proteases rather, than some other acid-dependent aspect of cell entry.
在鼠成纤维细胞中，病毒粒子在细胞进入过程中对呼肠孤病毒外衣壳蛋白σ3进行有效蛋白水解需要酸依赖性溶酶体半胱氨酸蛋白酶组织蛋白酶L。组织蛋白酶L对其他细胞类型感染的重要性尚不清楚。在此我们报告，酸非依赖性溶酶体半胱氨酸蛋白酶组织蛋白酶S介导巨噬细胞样P388D细胞中外衣壳的加工。P388D细胞支持Lang株病毒粒子的感染，但不支持c43株。遗传学研究表明，这种差异由编码σ3的病毒基因片段S4决定。缺乏σ3的c43衍生亚病毒粒子在P388D细胞中正常复制，这表明Lang株和c43株病毒粒子感染性的差异在于σ3加工水平。广谱半胱氨酸蛋白酶抑制剂反式环氧琥珀酰-L-亮氨酰胺基-（4-胍基）丁烷可抑制Lang株病毒粒子对P388D细胞的感染，但NH4Cl不能抑制，NH4Cl可提高内吞pH值，从而抑制酸依赖性蛋白酶如组织蛋白酶L和B。组织蛋白酶S特异性抑制剂也可抑制Lang株病毒粒子对P388D细胞的外衣壳加工和感染。此外，在存在NH4Cl的情况下，经基因工程改造以表达组织蛋白酶S的细胞系支持Lang株而非c43株病毒粒子的感染。因此，我们的结果表明，对组织蛋白酶S介导的σ3加工敏感性的差异是呼肠孤病毒感染巨噬细胞样P388D细胞和其他表达组织蛋白酶S的细胞时毒株差异的原因。此外，我们的数据表明，大多数其他细胞类型的呼肠孤病毒感染的酸依赖性可能反映了对大多数其他溶酶体蛋白酶活性的低pH要求，而不是细胞进入的其他一些酸依赖性方面。
