Patton J T, Gallegos C O
Department of Microbiology and Immunology, University of Miami School of Medicine, Florida 33101.
Virology. 1988 Oct;166(2):358-65. doi: 10.1016/0042-6822(88)90506-5.
To understand the role of viral proteins in the replication of rotavirus RNA, we have characterized the structure of subviral particles (SVPs) that synthesize double-stranded RNA (DS RNA). Pulse-labeling of newly made RNA in infected cells showed that rotavirus DS RNA was synthesized either in single-shell (SS)-like particles or in precursor particles that rapidly mature into SS particles. Experiments using a cell-free system demonstrated that most replicase particles containing newly made DS RNA were of greater density in CsCl than single-shelled (SS) particles. However, this was partly due to the presence of single-stranded RNAs as the treatment of replicase particles with micrococcal nuclease reduced their density to between core particles and SS particles. Electrophoretic analysis indicated that replicase particles, purified by centrifugation on CsCl and glycerol gradients, were similar to SS particles, containing the structural proteins VP1, VP2, and VP6. Rotavirus replicase particles were also found to contain the nonstructural proteins NS34 and NS35 and possible host components. The presence of VP6 in enzymatically active replicase particles suggests that, like transcription, this protein may be required for rotavirus RNA replication.
为了解病毒蛋白在轮状病毒RNA复制中的作用，我们对合成双链RNA（DS RNA）的亚病毒颗粒（SVP）的结构进行了表征。对感染细胞中新合成RNA的脉冲标记显示，轮状病毒DS RNA是在单壳（SS）样颗粒或迅速成熟为SS颗粒的前体颗粒中合成的。使用无细胞系统进行的实验表明，大多数含有新合成DS RNA的复制酶颗粒在CsCl中的密度比单壳（SS）颗粒更高。然而，这部分是由于单链RNA的存在，因为用微球菌核酸酶处理复制酶颗粒会使其密度降低到核心颗粒和SS颗粒之间。电泳分析表明，通过在CsCl和甘油梯度上离心纯化的复制酶颗粒与SS颗粒相似，含有结构蛋白VP1、VP2和VP6。还发现轮状病毒复制酶颗粒含有非结构蛋白NS34和NS35以及可能的宿主成分。在具有酶活性的复制酶颗粒中存在VP6表明，与转录一样，这种蛋白质可能是轮状病毒RNA复制所必需的。
