Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome
Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome
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DOI：
10.1073/pnas.97.10.5516
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发表时间：
2000-05-09
影响因子：
11.1
通讯作者：
Enjuanes, L
中科院分区：
文献类型：
--
作者：
Almazán, F;González, JM;Enjuanes, L
编码具有生物学意义的大尺寸RNA分子的cDNA克隆的构建，如冠状病毒基因组，是生物学已知的最大的成熟RNA分子之一，一直受到细菌中这些cDNA的不稳定性的阻碍。在此，我们展示了两种策略的应用，克隆cdna到细菌人工染色体和细胞核表达RNA，通常在细胞质内产生，对于大RNA分子的工程是有用的。编码传染性冠状病毒RNA基因组的cDNA已被克隆为细菌人工染色体。获救的冠状病毒保留了整个序列中引入的所有遗传标记，并显示出标准的mRNA模式和合成病毒的抗原特征。cDNA在细胞核内转录，RNA转位到细胞质中。有趣的是，恢复的病毒基本上与原始病毒具有相同的序列，并且没有观察到剪接。该cDNA来源于一种减毒分离物，它只在猪的呼吸道中复制。在感染性cDNA的工程过程中，病毒的刺突基因被肠道分离物的刺突基因所取代。合成病毒在肠道内大量复制，具有完全的毒力，表明回收的冠状病毒的嗜性和毒力是可以修饰的。这一证明为利用这种传染性cDNA作为对1族冠状病毒易感的人、猪、犬和猫的疫苗开发载体提供了可能性。
The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved ail of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a Vector for Vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.