Template-switching of CMV-RNA at replication.

以前，我们已经表明，卫星RNA的多聚体存在于黄瓜花叶病毒（CMV）感染烟草叶片。本研究首先假设病毒感染植物体内合成了异源连接的RNA分子，如CMV卫星RNA与CMV基因组RNA连接，根据CMV基因组RNA和卫星RNA的核苷酸序列设计寡核苷酸引物，扩增头-尾异源连接分子中的连接区。从CMV感染的烟草叶片中提取总RNA合成cDNA，用引物进行PCR扩增。RT-PCR产物的序列分析表明，它们是CMV基因组RNA 1 -3之间的正义或负义首尾连接RNA，而不是正-负连接RNA。连接区序列分析表明，大多数分子缺少3'端序列，少数分子在5'端和3'端之间有插入。然而，从纯化的CMV制备物中没有检测到头-尾连接的RNA。我们还检测到正义或负义头-尾连接RNA从黄瓜绿色斑驳花叶病毒（CGMMV）感染的样品。这些数据表明，异源连接的重要RNA分子是由复制酶驱动的拷贝选择机制产生的。本研究成功地建立了检测CMV病毒RNA的直接试管RT-PCR方法，并构建了CMV感染性全长cDNA克隆，以证实病毒感染样品中的RNA是首尾相连的。

Previously we have showed that multimers of satellite RNA are present in cucumber mosaic virus (CMV) infecting tobacco leaves. First of all in this research we assumed that the hetero-joined RNA molecules were synthesized in virus-infected plants, for example CMV satellite RNA joined with CMV genomic RNA, oligonucleotide primers were designed on the basis of the nucleotide sequence of CMV genome- and satellite RNAs to amplify the junction region in head-to-tail hetero-joined molecules. The cDNA was synthesized from total RNA extracted from CMV-infecting tobacco leaves and amplified by PCR with the primers. Sequence analysis of RT-PCR products revealed that they were plus-sense or minus-sense head-to-tail joined RNAs between CMV genomic RNA1-3 but not plus-minus joined one. The junction sequences indicated that most molecules lacked sequence of 3' components and a few have insertion between 5' and 3' components. However, no head-to-tail joined RNA was detected from purified CMV preparation. We have also detected plus-sense or minus-sense head-to-tail joined RNAs from samples infected with cucumber green mottle mosaic virus (CGMMV). These data indicate that hetero-joined vital RNA molecules are generated by a replicase-driven copy-choice mechanism. In this research we have succeeded to develop the simple direct tube RT-PCR method that can detect virus RNA.Moreover, to confirm the head-to-tail joined RNAs from virus-infecting samples, the infectious full-length cDNA clone of CMV was constructed.