Fulton Benjamin O, Sachs David, Schwarz Megan C, Palese Peter, Evans Matthew J
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
J Virol. 2017 Jul 12;91(15). doi: 10.1128/JVI.00698-17. Print 2017 Aug 1.
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. 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.
影响寨卡病毒（ZIKV）进化的分子限制因素尚未得到充分理解。为了研究ZIKV的遗传灵活性，我们使用转座子诱变在ZIKV MR766基因组中各处添加15个核苷酸的插入片段，随后对存活的突变体进行深度测序。很少有ZIKV插入突变体能够复制，这可能反映出基因组存在高度的功能限制。NS1基因在筛选的不同阶段表现出不同的突变耐受性。这一结果可能确定了NS1蛋白在病毒生命周期不同阶段所必需的区域。ZIKV结构基因表现出最高程度的插入耐受性。尽管包膜（E）蛋白表现出特别的灵活性，但E蛋白高度保守的包膜结构域II（EDII）融合环对转座子插入不耐受。融合环也是针对其他黄病毒产生的泛黄病毒抗体的靶标，这些抗体可中和多种登革病毒和ZIKV分离株。在EDII融合环表位内鉴定出的遗传限制可能解释了这些区域在ZIKV以及多种黄病毒中的序列和抗原保守性。因此，我们的结果为可能影响该病毒进化的ZIKV遗传限制提供了见解。寨卡病毒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理解影响病毒进化的因素很重要。我们使用全基因组转座子诱变筛选来确定复制病毒中哪些位置可以容忍突变。我们发现参与RNA复制的遗传区域大多对突变不耐受。编码结构蛋白的基因对突变更具耐受性。尽管在这些区域观察到了灵活性，但我们发现被广泛反应性抗体结合的表位受到遗传限制。这一发现可能解释了这些表位在黄病毒之间的遗传保守性。
