Tapparel C, Maurice D, Roux L
Department of Genetics and Microbiology, University of Geneva Medical School, Centre Medical Universitaire, Switzerland.
J Virol. 1998 Apr;72(4):3117-28. doi: 10.1128/JVI.72.4.3117-3128.1998.
The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of approximately 15 kb, contains six transcription units flanked at the 3' and 5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3' end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.
副粘病毒基因组是一种约15kb的不分节段、负链、单链RNA，包含六个转录单元，在3'和5'末端侧翼有一段短的（约50至60个核苷酸）顺反子外序列，称为正链和负链前导区。这些位于基因组和反基因组3'端的前导模板区已被证明含有控制RNA复制活性的关键信号。它们是否足以促进复制仍有待探讨。通过使用一系列在启动子区域携带嵌套缺失的仙台病毒缺陷干扰RNA，本文表明，对于基因组和反基因组启动子，需要96个核苷酸的3'端RNA序列才能进行复制。活性和非活性启动子的序列比较导致鉴定出一组三个核苷酸六聚体（核苷酸79至84、85至90和91至96），其包含重复基序RXXYXX [以5'-3'正链显示]。将每个六聚体依次突变为其互补序列证实了它们的关键作用。这三个六聚体是必需的，并且它们的相对位置很重要，因为将它们移位6个核苷酸会破坏启动子功能。在这三个六聚体中携带简并核苷酸的RNA被用作复制模板。它们导致专门从核苷酸79至96选择携带基本基序(GNNNNN)3的活跃复制RNA种类。这些结果清楚地表明，除了先前确定为控制仙台病毒复制活性的核苷酸1至31区域外，另一个元件，最宽跨度为核苷酸79至96，似乎也是必需的。因此，副粘病毒复制启动子并不局限于前导模板区，弹状病毒似乎是这种情况。
