Pijlman Gorben P, Kondratieva Natasha, Khromykh Alexander A
School of Molecular and Microbial Sciences, University of Queensland, MBS Bldg. 76, Cooper Rd., St. Lucia, 4072 QLD, Australia.
J Virol. 2006 Nov;80(22):11255-64. doi: 10.1128/JVI.01559-06. Epub 2006 Sep 13.
Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.
我们之前利用对NS3基因存在读框内缺失的库京病毒（KUN）全长cDNA克隆进行反式互补分析的研究表明，这些有缺陷的互补RNA无法被包装进病毒颗粒（W. J. 刘、P. L. 塞德拉克、N. 孔德拉季耶娃和A. A. 赫罗梅赫，《病毒学杂志》76:10766 - 10775）。在本研究中，我们旨在确定RNA包装过程中对NS3的这种需求是由NS3基因的二级RNA结构决定的，还是由翻译后的NS3基因产物的关键作用决定的。针对破坏KUN复制子RNA的NS3编码区中三个计算机预测的稳定RNA结构而进行的多个沉默突变，旨在在不影响氨基酸序列的情况下破坏RNA二级结构，但这并未影响在包装细胞系中RNA的复制以及其被包装进病毒样颗粒的过程，从而表明NS3基因中预测的保守RNA结构在RNA复制和/或包装过程中不起作用。相比之下，全长KUN RNA的NS3编码区中的双移码突变，产生了混乱的NS3蛋白但保留了二级RNA结构，在KUN复制子表达细胞的互补实验中，导致这些有缺陷的RNA失去了被包装进病毒颗粒的能力。此外，基于已建立的稳定细胞系构建的更强有力的互补包装系统，该细胞系能产生大量互补的、复制的NS3缺陷复制子RNA，并感染KUN病毒以提供结构蛋白，但同样未能检测到任何含有被包装的NS3缺陷复制子RNA的分泌型病毒样颗粒。这些结果现已确凿地证实，顺式翻译的KUN NS3蛋白对于基因组包装进病毒颗粒是必需的。
