Multigene RNA vector based on coronavirus transcription
Multigene RNA vector based on coronavirus transcription
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DOI：
10.1128/jvi.77.18.9790-9798.2003
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发表时间：
2003-09-01
影响因子：
5.4
通讯作者：
Siddell, SG
中科院分区：
文献类型：
--
作者：
Thiel, V;Karl, N;Siddell, SG
冠状病毒基因组是已知最大的自主复制RNA，大小约为1000。30 kb。它们具有正极性，并被翻译以产生组装活性复制酶-转录酶复合物所需的病毒蛋白质。除了复制基因组RNA之外，这种复合物的一个关键特征是独特的转录过程，该过程导致合成一组嵌套的6至8个亚基因组mRNA。这些亚基因组mRNA以恒定但不等摩尔的量产生，并且通常每一种都被翻译以产生单一蛋白质。为了利用这些特点，我们已经开发了一种基于人冠状病毒229 E的多基因表达载体。我们已经构建了一个原型RNA载体，其含有人冠状病毒基因组的5'和3'末端、整个人冠状病毒复制酶基因和三个报告基因（即，氯霉素乙酰转移酶[CAT]基因、萤火虫荧光素酶[LUC]基因和绿色荧光蛋白[GFP]基因）。每个报告基因位于人冠状病毒转录相关序列的下游，这是合成单个亚基因组mRNA所必需的。将载体RNA和人冠状病毒核衣壳蛋白mRNA转染到BHK-21细胞中导致CAT、LUC和GFP报告蛋白的表达。序列分析证实了编码CAT、LUC和GFP的冠状病毒特异性mRNA的合成。此外，我们已经证明，基于人冠状病毒的载体RNA可以包装成病毒样颗粒，反过来，可以用于培养未成熟和成熟的人树突状细胞。总之，我们描述了一类新的真核多基因表达载体，其基于人冠状病毒229 E，并具有转染人树突状细胞的能力。
Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5' and 3' ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells.