Effect of inserting paramyxovirus simian virus 5 gene junctions at the HN L gene junction: Analysis of accumulation of mRNAs transcribed from rescued viable viruses
Effect of inserting paramyxovirus simian virus 5 gene junctions at the HN L gene junction: Analysis of accumulation of mRNAs transcribed from rescued viable viruses
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DOI：
10.1128/jvi.73.8.6228-6234.1999
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发表时间：
1999-08-01
影响因子：
5.4
通讯作者：
Lamb, RA
中科院分区：
文献类型：
--
作者：
He, B;Lamb, RA
猴副流感病毒5(SV5)是副粘病毒科非节段性负义RNA病毒的原型。这些病毒的单链RNA基因组包含一系列由基因末端(GE)和基因起始(GS)序列隔开的基因间(IG)序列。病毒RNA聚合酶(VRNAP)复合体被认为在其3‘端进入基因组，mRNAs的合成被认为是以顺序和极性的停止-启动机制发生的，转录衰减主要发生在基因间隔区。为了研究GE、IG和GS序列在转录中的功能，我们构建了含有SV5全长基因组cDNA的质粒，其中位于血凝素神经氨酸酶(HN)和聚合酶(L)之间的基因连接序列(GE、IG和GS序列)被替换为来自其他基因连接的对应序列。通过使用反向遗传学，我们从每个cdna构建体中恢复了活病毒，尽管它们的生长特征各不相同。用定量核糖核酸酶保护实验对HN和L的mRNAs进行分析，结果表明，BN和L的mRNAs的比例在四倍范围内变化。基因连接序列的改变也允许对GS位点六聚体核苷酸位置的假设要求进行检查。具有转录起始点的传染性病毒的恢复发生在六聚体的第1、2、3、5和6位核苷酸位置，这表明这一要求是不严格的。
Simian parainfluenza virus 5 (SV5) is a prototype of the Paramyxoviridae family of nonsegmented negative-sense RNA viruses. The single-stranded RNA genomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences banked by gene-end(GE) and gene-start (GS) sequences. The viral RNA polymerase (vRNAP) complex is thought to enter the genome at its 3' end, and synthesis of mRNAs is thought to occur by a stop-start mechanism in a sequential and polar manner, with transcriptional attenuation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNAP, To investigate the functions of GE, IG, and GS sequences in transcription, we constructed plasmids containing cDNAs of the full-length SV5 genome in which the gene junction sequences (GE, IG, and GS sequences) located between the hemagglutinin-neuraminidase (HN) and the polymerase (L) genes were replaced with the counterpart sequences from other gene junctions. By using reverse genetics, we recovered viable viruses from each cDNA construct, although their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of BN to L mRNAs varied over a fourfold range. The alteration of the gene junction sequences also permitted examination of the hypothesized requirement for hexamer nucleotide position of the GS sites. The recovery of infectious viruses with transcription initiation sites that occurred at nucleotide positions 1, 2, 3, 5, and 6 of the hexamer suggest that the requirement is nonstringent.