Khromykh A A, Varnavski A N, Westaway E G
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia.
J Virol. 1998 Jul;72(7):5967-77. doi: 10.1128/JVI.72.7.5967-5977.1998.
Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its amplification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an approximately 35-nm diameter, compared with approximately 50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in trans, and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.
通过对BHK-21细胞进行连续两次电穿孔的方法来包装库京病毒（KUN）复制子RNA，首先用电穿孔导入KUN复制子RNA C20DXrep（prME和大部分C区已缺失），约24小时后再导入表达KUN结构蛋白的重组辛德毕斯病毒（SFV）复制子RNA。通过在新感染的BHK-21细胞中对其进行扩增和表达，并用KUN特异性探针进行Northern印迹检测以及用抗NS3抗体进行免疫荧光分析，证明了包装颗粒中存在KUN复制子RNA。当同时电穿孔导入C20DXrep RNA和重组SFV RNA时，未产生感染性颗粒。当第二次电穿孔使用单个表达KUN连续prME基因和KUN C基因且受两个独立的26S亚基因组启动子控制的SFV复制子RNA（SFV-prME-C107）时，获得的感染性颗粒滴度比分别使用两个表达KUN C基因（SFV-C107）和prME基因（SFV-prME）的不同SFV复制子RNA时高10倍。没有表达KUN结构蛋白的SFV复制子RNA被包装到分泌颗粒中。通过超速离心从依次用C20DXrep和SFV-prME-C107 RNA转染的细胞培养液中沉淀得到的感染性颗粒，与抗KUN E蛋白单克隆抗体预孵育后可被中和。用抗E抗体对双重转染细胞的培养液进行放射免疫沉淀分析表明，免疫沉淀颗粒中存在C、prM/M和E蛋白。逆转录-PCR分析表明，免疫沉淀颗粒中也含有KUN特异性RNA。在5%至25%的蔗糖密度梯度中，包装的复制子颗粒比KUN病毒粒子沉降得更慢，并且呈均匀的球形，直径约为35纳米，而KUN病毒粒子的直径约为50纳米。本研究结果首次证明了黄病毒RNA的反式包装，并且排除了几乎整个结构区域在包装中的作用。所开发的包装系统的可能应用包括确定黄病毒RNA中的包装信号以及参与RNA包装、病毒粒子组装和分泌的结构蛋白中的氨基酸基序。此外，它可以促进基于包装表达异源基因的非细胞病变性黄病毒复制子的非感染性疫苗递送系统的开发。
