Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3.
Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3.
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拯救人类副流感病毒 3 型基因组 RNA 的合成类似物。
DOI：
10.1006/viro.1993.1486
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发表时间：
1993
期刊：
影响因子：
3.7
通讯作者：
Banerjee,AK
中科院分区：
文献类型：
--
作者：
De,BP;Banerjee,AK
一个简单的系统，允许外源基因的表达和包装人类副流感病毒3型（HPIV-3）已经描述。首先，构建cDNA编码HPIV-3基因组RNA的内部缺失版本。将病毒基因替换为细菌氯霉素乙酰转移酶（CAT）报告基因的阴性拷贝。用T7 RNA聚合酶体外转录合成了一个870个核苷酸的RNA，该RNA含有HPIV-3基因组的反义编码区、转录调控序列和3′和5′端外显子序列。当导入感染HPIV-3的细胞时，该RNA被扩增，报告基因被表达，通过细胞提取物中的CAT活性来测量。然后将合成的RNA包装成感染性病毒粒子。在亲本尾部5 ‘端添加两个额外的核苷酸使CAT活性降低了90%以上，这表明在病毒复制周期中需要完整的5 ’调控结构域。有趣的是，在3 ‘端添加一个额外的核苷酸完全消除了CAT活性，这表明精确的3 ’端在这个过程中是至关重要的。
A simple system that allows expression and packaging of a foreign gene by human parainfluenza virus type 3 (HPIV-3) has been described. First, a cDNA was constructed to encode an internally deleted version of HPIV-3 genome RNA. The viral genes were replaced with a negative sense copy of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene.In vitrorun-off transcription with T7 RNA polymerase synthesized an 870 nucleotide RNA that contained the antisense coding region of the CAT gene flanked by the transcription regulatory sequences and the 3′ and 5′ end extracistronic sequences of the HPIV-3 genome. When introduced into cells that are infected with HPIV-3, this RNA was amplified and the reporter gene was expressed, as measured by the CAT activity in the cell extract. Furthermore, the synthetic RNA was packaged into infectious virions. The addition of two extra nucleotides at the 5′ end of the parental trailer region decreased the CAT activity by more than 90%, suggesting a requirement for the intact 5′-regulatory domain in the viral replicative cycle. Interestingly, the addition of one extra nucleotide to the 3′ end totally abolished the CAT activity indicating that an exact 3′ terminus is critical in this process.