Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging
Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging
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DOI：
10.1128/jvi.01559-06
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发表时间：
2006-11-01
影响因子：
5.4
通讯作者：
Khromykh, Alexander A.
中科院分区：
文献类型：
--
作者：
Pijlman, Gorben P.;Kondratieva, Natasha;Khromykh, Alexander A.
我们之前的研究使用昆津病毒（KUN）全长cDNA克隆的反式互补分析，这些克隆在NS 3基因中具有框内缺失，证明这些有缺陷的互补RNA无法包装到病毒颗粒中（W. J. Liu，P. L. Sedlak，N. Kondratieva和A. A. Khromykh，J. Virol. 76：10766-10775）。在这项研究中，我们的目的是确定是否这种要求NS 3在RNA包装是由二级RNA结构的NS 3基因或翻译的NS 3基因产物的重要作用。在KUN复制子RNA的NS 3编码区中，旨在破坏RNA二级结构而不影响氨基酸序列的三个计算机预测的稳定RNA结构的多个沉默突变不影响包装细胞系中的RNA复制和包装成病毒样颗粒，从而证明NS 3基因中预测的保守RNA结构在RNA复制和/或包装中不起作用。相反，在全长KUN RNA的NS 3编码区中的双移码突变，产生乱序NS 3蛋白但保留二级RNA结构，导致这些缺陷RNA在KUN复制子表达细胞中的互补实验中被包装成病毒颗粒的能力丧失。此外，基于产生大量互补复制型NS 3缺陷型复制子RNA的已建立稳定细胞系和用KUN病毒感染以提供结构蛋白的更稳健的互补包装系统也未能检测到含有包装的NS 3缺陷型复制子RNA的任何分泌的病毒样颗粒。这些结果现在已经牢固地确立了将基因组包装到病毒颗粒中的顺式翻译的KUN NS 3蛋白的要求。
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.