School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
J Virol. 2017 Nov 14;91(23). doi: 10.1128/JVI.01348-17. Print 2017 Dec 1.
Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion. The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.
Zika 病毒（ZIKV）是一种通过蚊子传播的黄病毒，可导致非洲和亚洲零星爆发轻微发热疾病。该病毒在过去十年中再次出现，引起了严重的人类疾病，包括小头畸形、先天畸形和格林-巴利综合征。虽然基因组和系统发育分析表明遗传进化可能导致 ZIKV 的毒力增强，但目前缺乏支持特定遗传变化在毒力中作用的实验证据。一个序列基序，VNDT，在包膜（E）蛋白中含有一个 N 连接的糖基化位点，是多态的；它在许多非洲分离株中不存在，但在最近爆发的所有分离株中都存在。在本研究中，我们研究了这个序列基序和 E 蛋白糖基化在 ZIKV 致病性中的作用。我们首先在一种新的线性载体中构建了 ZIKV 的稳定全长 cDNA 克隆，从中恢复了感染性病毒。从含有 VNDT 基序的感染性克隆中产生的重组 ZIKV 具有高度致病性，可导致小鼠模型死亡。相比之下，缺失 VNDT 基序或其中 N 连接的糖基化位点通过单个氨基酸取代发生突变的重组病毒高度衰减且非致死性。当皮下接种时，突变病毒在感染小鼠的大脑中复制不良，但在颅内接种时复制良好。我们的研究结果首次提供了证据表明 E 蛋白的 N 连接糖基化是 ZIKV 毒力和神经侵袭的重要决定因素。最近 Zika 病毒（ZIKV）在美洲的出现引起了全球公共卫生的严重关注。该病毒似乎获得了显著的致病性，导致严重的人类疾病，包括小头畸形和格林-巴利综合征。目前尚不清楚导致致病性 ZIKV 出现的因素，但已表明遗传变化有助于病毒传播。最近爆发的所有分离株的病毒包膜（E）蛋白中都含有一个 N 连接的糖基化位点，而许多非洲谱系病毒的分离株则缺乏该位点。为了阐明糖基化在 ZIKV 致病性中的功能意义，从具有或不具有 E 蛋白上糖基化的感染性克隆中生成了重组 ZIKV。缺乏糖基化的 ZIKV 引起小鼠模型死亡率的能力显著减弱，并且严重损害了其神经侵袭能力。我们的研究表明，E 蛋白的糖基化是导致 ZIKV 致病性的一个重要因素。
