Herold J, Andino R
Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0414, USA.
J Virol. 2000 Jul;74(14):6394-400. doi: 10.1128/jvi.74.14.6394-6400.2000.
Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5' end, which are deleted during replication to generate authentic viral genomes. We removed those 5'-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5' ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5' end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.
脊髓灰质炎病毒感染性RNA可利用噬菌体DNA依赖性RNA聚合酶在体外合成。这些合成转录本在5'端含有几个额外的核苷酸,在复制过程中会被删除以产生正宗的病毒基因组。我们利用锤头状核酶去除那些5'端额外的核苷酸,以产生具有精确5'端的转录本。这些转录本在细胞培养中复制得更快,在复制前没有延迟;它们在非洲爪蟾卵母细胞和体外翻译-复制细胞提取物中也能更有效地复制。在这两个系统中,精确的5'端对于正链RNA的合成是必需的,但对于负链RNA的合成则不是必需的。