Buckley B, Bruening G
Department of Plant Pathology, University of California, Davis 95616.
Virology. 1990 Jul;177(1):298-304. doi: 10.1016/0042-6822(90)90483-8.
We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea (Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 micrograms/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.
我们构建了一个由宿主、烟草环斑病毒（TobRV）和卫星烟草环斑病毒RNA（sTobRV RNA）组成的三组分系统，用于研究宿主成分或TobRV基因产物对卫星RNA增殖周期的具体作用。通过电穿孔法将sTobRV和TobRV基因组RNA接种到豇豆（Vigna unguiculata）原生质体中。通过印迹杂交以及将[14C]尿苷掺入具有sTobRV RNA电泳迁移率的物质中，检测到sTobRV RNA有所增加。培养基中50微克/毫升的放线菌素D（Act D）可有效抑制未接种原生质体中的DNA依赖性RNA合成。在与sTobRV RNA和TobRV基因组RNA共接种后24或48小时向原生质体中添加Act D，对sTobRV RNA的积累影响不大，而在共接种前24小时添加则可阻止任何极性的sTobRV RNA的检测到的积累。我们的结果以及先前在感染组织提取物中发现的与衣壳化卫星RNA互补的RNA表明，一种RNA依赖性RNA聚合酶负责sTobRV RNA的合成。早期添加Act D时的强烈抑制作用意味着宿主因子在sTobRV RNA复制的早期阶段发挥作用。
