Strategy for systematic assembly of large RNA and DNA genomes: Transmissible gastroenteritis virus model
Strategy for systematic assembly of large RNA and DNA genomes: Transmissible gastroenteritis virus model
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DOI：
10.1128/jvi.74.22.10600-10611.2000
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发表时间：
2000-11-01
影响因子：
5.4
通讯作者：
Baric, RS
中科院分区：
文献类型：
--
作者：
Yount, B;Curtis, KM;Baric, RS
开发了一种系统方法来组装大型 RNA 和 DNA 病毒的功能性全长基因组。冠状病毒含有自然界中最大的单链正极性RNA基因组。类似于 30 kb 的基因组，加上基因组不稳定区域，阻碍了全长感染性 cDNA 构建体的开发。我们组装了传染性胃肠炎病毒（TGEV）的全长感染性构建体，TGEV是猪的一种重要病原体。使用一种新颖的方法，分离出跨越整个 TGEV 基因组的 6 个相邻的 cDNA 亚克隆。每个克隆都设计有独特的侧翼互连连接，这些连接确定了仅与相邻 cDNA 亚克隆的精确系统组装，从而产生长度类似于 28.5 kb 的完整 TGEV cDNA 构建体。来自全长 TGEV 构建体的转录本具有感染性，并且子代病毒颗粒在允许的宿主细胞中连续传代。在感染期间和整个传代过程中，病毒抗原的产生和亚基因组 mRNA 的合成是明显的。来自感染性构建体的噬菌斑纯化病毒可有效复制，并在许可的宿主细胞中表现出相似的噬菌斑形态。分子克隆病毒和野生型病毒的宿主范围表型在猪和猫来源的细胞中相似。对重组病毒独特的互连连接点进行测序，最终证明了被设计到组件克隆中的标记突变和限制性位点。 TGEV 的拉长感染性构建体将允许对冠状病毒基因组进行精确的遗传修饰。我们设计的用于生成 TGEV 感染性 cDNA 构建体的方法理论上可用于精确重建长度接近数百万个碱基对的微生物或真核生物基因组。
A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The similar to 30 kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of similar to 28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Pull-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.