Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion
Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion
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DOI：
10.1128/jvi.00698-17
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发表时间：
2017-08-01
影响因子：
5.4
通讯作者：
Evans, Matthew J.
中科院分区：
文献类型：
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作者：
Fulton, Benjamin O.;Sachs, David;Evans, Matthew J.
寨卡病毒（ZIKV）是寨卡病毒的一种。为了研究ZIKV遗传灵活性，我们使用转座子诱变在整个ZIKV MR 766基因组中添加15个核苷酸的插入，随后对活突变体进行深度测序。几乎没有ZIKV插入突变体复制，这可能反映了基因组上高度的功能限制。NS1基因在筛选的不同阶段表现出不同的突变耐受性。这一结果可以定义病毒生命周期不同阶段所需的NS1蛋白区域。ZIKV结构基因显示出最高程度的插入耐受性。虽然包膜蛋白（E）表现出特殊的灵活性，高度保守的包膜结构域II（EDII）融合环的E蛋白是不容忍转座子插入。融合环也是针对其他黄病毒产生的泛黄病毒抗体的靶标，并中和广泛的登革病毒和ZIKV分离株。在EDII融合环中的表位内鉴定的遗传限制可能解释了ZIKV中和多种黄病毒中这些区域的序列和抗原保守性。因此，我们的研究结果提供了对ZIKV的遗传限制的见解，可能会影响这种病毒的进化。重要性ZIKV最近成为一种重要的人类病原体。确定寨卡病毒的遗传限制对于了解影响病毒进化的因素非常重要。我们使用全基因组转座子诱变筛选来确定在复制病毒中哪些突变是可以容忍的。我们发现，参与RNA复制的基因区域大多不能容忍突变。编码结构蛋白的基因更容易发生突变。尽管在这些区域观察到的灵活性，我们发现，广泛反应性抗体结合的表位是遗传限制。这一发现可以解释这些表位的遗传保守性黄病毒。
The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus.IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.