He B, Paterson R G, Ward C D, Lamb R A
Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Virology. 1997 Oct 27;237(2):249-60. doi: 10.1006/viro.1997.8801.
A complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3' end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals.
副粘病毒SV5基因组（15246个核苷酸）的完整cDNA克隆由多个cDNA构建而成，使得T7 RNA聚合酶能够转录出反基因组RNA，并通过使用丁型肝炎病毒核酶切割产生正确的3'末端。将编码反基因组序列的质粒转染到先前感染了表达T7 RNA聚合酶的重组痘苗病毒的细胞中，同时转染在T7聚合酶启动子控制下表达病毒复制蛋白NP、P和L的辅助质粒。通过使用基因组RNA的RT-PCR和核苷酸测序，从引入DNA克隆的标签限制位点回收SV5，证明了从DNA中拯救RNA基因组。从DNA中拯救SV5不需要作为辅助质粒表达病毒V蛋白，这表明V蛋白对于初始复制不是必需的。还对SV5的感染性cDNA进行了操作，使其在HN和L基因之间引入的SV5转录起始和终止信号的控制下表达绿色荧光蛋白（GFP）。表达的GFP量因新引入的转录信号的性质而异。
