Analysis of the Highly Diverse Gene Borders in Ebola Virus Reveals a Distinct Mechanism of Transcriptional Regulation
Analysis of the Highly Diverse Gene Borders in Ebola Virus Reveals a Distinct Mechanism of Transcriptional Regulation
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DOI：
10.1128/jvi.01863-14
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发表时间：
2014-11-01
影响因子：
5.4
通讯作者：
Muehlberger, Elke
中科院分区：
文献类型：
--
作者：
Brauburger, Kristina;Boehmann, Yannik;Muehlberger, Elke
埃博拉病毒（EBOV）属于非节段性负义RNA病毒。7个EBOV基因由可变基因边界分开，包括短（4或5个核苷酸）基因间区域（IR）、单个长（144个核苷酸）IR和基因重叠，其中相邻基因末端和起始信号共享5个保守核苷酸。基因重叠的独特结构和单个长IR的存在在所有丝状病毒中是保守的。在这里，我们试图确定EBOV基因边界在病毒转录过程中的影响。我们发现，通读mRNA的合成发生在EBOV感染的细胞中，而不管基因边界的结构，这表明基因重叠不促进基因末端信号的识别。然而，在VP 24基因处的两个连续的基因末端信号可能改善在VP 24-L基因边界处的终止，从而确保有效的L基因表达。我们进一步证明，长IR是不是必不可少的，但调节转录重新启动的长度依赖性，但序列独立的方式。双顺反子微型基因组和重组EBOV的突变分析表明，IR长度和再起始率之间没有直接的相关性，但表明特定的IR长度没有发现自然丝状病毒深刻抑制下游基因的表达。有趣的是，虽然截断的144个核苷酸长的IR到5个核苷酸并没有实质上影响EBOV转录，它导致了显着减少病毒growth.IMPORTANCEOur目前的理解EBOV转录调控是有限的，由于高遏制条件下研究这种高致病性病毒的要求。EBOV被认为与经过充分分析的原型非分段负义RNA病毒共享许多机制特征。基因组3'端的单个聚合酶进入位点决定了基因的转录主要受基因顺序和基因边界处发现的顺式作用信号控制。在这里，我们研究了结构独特的EBOV基因边界在病毒转录过程中的调节作用。我们的数据表明，EBOV中的转录调控是高度复杂的，与原型病毒不同，并进一步了解丝状病毒复制周期中最基本的过程。此外，我们对重组EBOV的研究结果表明，在病毒复制周期期间，在所有丝状病毒基因组中发现的长IR具有新的作用。
Ebola virus (EBOV) belongs to the group of nonsegmented negative-sense RNA viruses. The seven EBOV genes are separated by variable gene borders, including short (4- or 5-nucleotide) intergenic regions (IRs), a single long (144-nucleotide) IR, and gene overlaps, where the neighboring gene end and start signals share five conserved nucleotides. The unique structure of the gene overlaps and the presence of a single long IR are conserved among all filoviruses. Here, we sought to determine the impact of the EBOV gene borders during viral transcription. We show that readthrough mRNA synthesis occurs in EBOV-infected cells irrespective of the structure of the gene border, indicating that the gene overlaps do not promote recognition of the gene end signal. However, two consecutive gene end signals at the VP24 gene might improve termination at the VP24-L gene border, ensuring efficient L gene expression. We further demonstrate that the long IR is not essential for but regulates transcription reinitiation in a length-dependent but sequence-independent manner. Mutational analysis of bicistronic minigenomes and recombinant EBOVs showed no direct correlation between IR length and reinitiation rates but demonstrated that specific IR lengths not found naturally in filoviruses profoundly inhibit downstream gene expression. Intriguingly, although truncation of the 144-nucleotide-long IR to 5 nucleotides did not substantially affect EBOV transcription, it led to a significant reduction of viral growth.IMPORTANCEOur current understanding of EBOV transcription regulation is limited due to the requirement for high-containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative-sense RNA viruses. A single polymerase entry site at the 3' end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from that in prototype viruses and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle.