Westaway Edwin G, Mackenzie Jason M, Khromykh Alexander A
Clinical Medical Virology Center-University of Queensland, Sir Albert Sakzewski Virus Research Center, Royal Children's Hospital, Herston, Brisbane, Australia.
Adv Virus Res. 2003;59:99-140. doi: 10.1016/s0065-3527(03)59004-2.
The Kunjin virus (KUNV) has provided a useful laboratory model for Flavivirus RNA replication. The synthesis of progeny RNA(+) strands occurs via asymmetric and semiconservative replication on a template of recycling double-stranded RNA (dsRna) or replicative form (RF). Kinetics of viral RNA synthesis indicated a cycle period of about 15 min during which, on average, a single nascent RNA (+) strand displaces the pre-existing RNA(+) strand in the replicative intermediate. Data on the composition of the replication complex (RC) in KUNV-infected cells were obtained from several sources, including analyses of the partially-purified still active RC, immunogold labeling of cryosections using monospecific antibodies to the nonstructural proteins and to the dsRNA, radioimmunoprecipitations of cell lysates using antibodies to dsRNA and to an RC-associated cell marker, and pull-down assays of cell lysates using fusion proteins GST-NS2A and GST-NS4A. These results yeilded a consensus composition of NS1, NS2A, NS3, NS4A, and NS5 strongly associated with the dsRNA template. The RC was located in induced membranes described as vesicle packets. The RNA-dependent RNA polymerase activity late in infection did not require continuing protein synthesis. Replication of genomic RNA was completely dependent on the presence of conserved complementary or cyclization sequences near the 5' and 3' ends. Assembly of the RC during translation in cis and the relationships, particularly those of NS1 and NS5 among the components, were deduced from an extensive set of complementation experiments in trans involving mutations/deletions in all the nonstructural proteins and use of KUN or alphahavirus replicons as helpers. The KUN replicon has found useful applications also as a noncytopathic vector for the continuing expression of foreign genes, delivered either as packaged RNA or as plasmid DNA.
库宁病毒（KUNV）为黄病毒RNA复制提供了一个有用的实验室模型。子代RNA（+）链的合成通过在循环双链RNA（dsRNA）或复制形式（RF）模板上的不对称和半保留复制进行。病毒RNA合成动力学表明，其周期约为15分钟，在此期间，平均而言，一条新生的RNA（+）链会取代复制中间体中预先存在的RNA（+）链。关于KUNV感染细胞中复制复合体（RC）组成的数据来自多个来源，包括对部分纯化的仍具活性的RC的分析、使用针对非结构蛋白和dsRNA的单特异性抗体对冷冻切片进行免疫金标记、使用针对dsRNA和RC相关细胞标志物的抗体对细胞裂解物进行放射免疫沉淀，以及使用融合蛋白GST-NS2A和GST-NS4A对细胞裂解物进行下拉试验。这些结果得出了与dsRNA模板紧密相关的NS1、NS2A、NS3、NS4A和NS5的一致组成。RC位于被描述为囊泡包的诱导膜中。感染后期的RNA依赖性RNA聚合酶活性不需要持续的蛋白质合成。基因组RNA的复制完全依赖于5'和3'末端附近保守的互补或环化序列的存在。通过一系列广泛的反式互补实验推断出RC在顺式翻译过程中的组装以及各组分之间的关系，特别是NS1和NS5之间的关系，这些实验涉及所有非结构蛋白中的突变/缺失，并使用KUN或甲病毒复制子作为辅助。KUN复制子还作为一种非细胞病变载体，用于持续表达外源基因，这些外源基因可以作为包装RNA或质粒DNA传递。
