Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes
Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes
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DOI：
10.1128/jvi.73.2.1535-1545.1999
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发表时间：
1999-02-01
影响因子：
5.4
通讯作者：
Enjuanes, L
中科院分区：
文献类型：
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作者：
Izeta, A;Smerdou, C;Enjuanes, L
已研究了涉及传染性胃肠炎病毒 (TGEV) RNA 的复制和包装的序列。先前描述了 9.7kb 的 TGEV 缺陷型干扰 RNA (DI-C) 的结构 (A. Mendez, C. Smerdou, A. Izeta, F. Gebamer, and L, Enjuanes, Virology 217:495-507, 1996)，并且将具有编码 DI-C RNA 的信息的 cDNA 克隆到 T7 启动子的控制下。分子克隆的 DI-C RNA 在转染辅助病毒感染的细胞后进行反式复制，并抑制亲代基因组的 20 倍复制。合成生成了 14 个 DI-C RNA 缺失突变体（TGEV 小基因组），并测试了它们的复制和包装能力。由辅助病毒复制并有效包装的最小迷你基因组 (M33) 为 3.3 kb。 2.1 kb (M21) 的小基因组也被复制，但它的包装效率比 M33 小基因组低得多，这表明它由于侧翼序列的改变而丢失了包含主要包装信号的序列或包装信号中所需的二级结构。 M21 小基因组的低包装和效率并不是由于最小尺寸限制。辅助病毒复制小基因组所必需的序列在 5' 和 3' 端分别减少至 1,348 nt 和 492 nt。 TGEV 衍生的 RNA 小基因组在两步扩增系统后成功表达，该系统将细胞核中 pol II 驱动的转录与细胞质中辅助病毒支持的复制耦合起来，没有任何明显的剪接。该系统和报告基因 P-葡萄糖醛酸酶 (GUS) 的使用允许在零代中检测小基因组，从而可以区分复制效率和包装能力。合成的小基因组已用于设计辅助依赖性表达系统，该系统可产生约 1.0 μg/10(6) 细胞的 GUS。
The sequences involved in the replication and packaging of transmissible gastroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV defective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mendez, C. Smerdou, A. Izeta, F. Gebamer, and L, Enjuanes, Virology 217: 495-507, 1996), and a cDNA with the information to encode DI-C RNA was cloned under the control of the T7 promoter. The molecularly cloned DI-C RNA was replicated in trans upon transfection of helper virus-infected cells and inhibited 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and tested for their ability to be replicated and packaged. The smallest minigenome (M33) that was replicated by the helper virus and efficiently packaged was 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was packaged with much lower efficiency than the M33 minigenome, suggesting that it had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging, efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for minigenome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5' and 3' ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that couples pol II-driven transcription in the nucleus to replication supported by helper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene P-glucuronidase (GUS) allowed minigenome detection at passage zero, making it possible to distinguish replication efficiency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 mu g/10(6) cells of GUS.