Baer G S, Dermody T S
Department of Microbiology and Immunology, and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Virol. 1997 Jul;71(7):4921-8. doi: 10.1128/JVI.71.7.4921-4928.1997.
Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.
从持续感染培养物中分离出的呼肠孤病毒（PI病毒）中选择的突变会影响病毒进入细胞的过程。与野生型（wt）病毒不同，PI病毒可以在氯化铵存在的情况下生长，氯化铵是一种弱碱，在病毒进入细胞期间会阻断细胞内体中病毒外衣壳蛋白的酸依赖性蛋白水解。在本研究中，我们表明E64（一种半胱氨酸蛋白酶抑制剂，如存在于内吞区室中的那些）通过抑制病毒解体来阻断wt呼肠孤病毒的生长。为了确定PI病毒是否能在内吞蛋白酶抑制剂存在的情况下生长，我们比较了用E64处理的细胞中wt和PI病毒的产量。在E64处理的细胞中，原型PI病毒L/C、PI 2A1和PI 3-1产生的产量比wt 1型朗（T1L）和3型迪林（T3D）病毒高得多。为了鉴定在E64存在下与PI病毒生长相关的病毒基因，我们测试了从T1L与每种原型PI病毒的独立杂交中分离出的重配病毒在E64处理的细胞中的生长情况。在E64存在下重配病毒的生长仅与编码病毒外衣壳蛋白sigma3的S4基因相关。这些结果表明，在持续感染期间选择的sigma3蛋白中的突变改变了其在病毒解体过程中对切割的敏感性。为了确定呼肠孤病毒解体过程中酸依赖性和蛋白酶依赖性步骤的时间关系，用wt菌株T1L或T3D感染细胞，并在吸附后的不同时间加入含有氯化铵或E64d（E64的膜通透性形式）的培养基。当在吸附后60分钟以上的时间加入任何一种抑制剂时，对氯化铵和E64抑制的敏感性都被消除。这些发现表明，酸依赖性和蛋白酶依赖性解体事件在呼肠孤病毒复制早期以相似的动力学发生，这表明这些事件发生在内吞途径的同一区室中。
