Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection.
Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection.
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DOI：
10.1371/journal.ppat.1002826
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Tapparel C
中科院分区：
文献类型：
--
作者：
Cordey S;Petty TJ;Schibler M;Martinez Y;Gerlach D;van Belle S;Turin L;Zdobnov E;Kaiser L;Tapparel C
肠病毒71 （EV71）是毒性最强的肠道病毒之一，但增强其在人类中传播能力的特定分子特征仍不清楚。我们根据感染的不同部位分析了EV71病毒在免疫功能低下的播散性疾病宿主中的基因组特征。对直接从呼吸、胃肠道、神经系统和血液标本中测序的5个全长基因组进行比较，发现在5天内发生了3个核苷酸变化：位于BC环（L97R）内的VP1的非保守氨基酸变化，该区域被认为是免疫原性位点，可能对脊髓灰质炎病毒宿主适应很重要；蛋白质2B （A38V）中保守的氨基酸取代；以及蛋白3D （L175）的沉默突变。使用BrCr（谱系A）和临床菌株（谱系C）背景构建感染性克隆，其中包含一个或两个非同义突变。体外细胞趋向性和竞争实验显示，BC环内VP197 Leu对Arg的替代在神经母细胞瘤起源的SH-SY5Y细胞中具有复制优势。有趣的是，这种突变在体外经常与神经母细胞瘤细胞VP1 EF环中的第二种非保守突变（E167G或E167A）相关。这些EV71 VP1变异体建立了比较模型，以确定取代如何影响VP1结构和/或与宿主细胞的相互作用，并表明，虽然没有观察到显著的结构变化，但取代可能会改变与宿主细胞受体的相互作用。综上所述，我们的研究结果表明，EV71的VP1 BC环区在独立于EV71谱系的细胞嗜性中起着关键作用，因此，可能有助于免疫功能不全患者的传播和神经嗜性。人类肠道病毒71型（EV71）一直是主要手足口病暴发的原因，特别是在亚太地区。EV71感染也可传播到中枢神经系统并导致脑膜脑炎。尽管进行了密集的流行病学筛查，并在动物模型中进行了实验，但导致嗜神经性疾病的病毒因素仍然不明确。我们在此描述了对来自具有播散性疾病的免疫功能低下宿主的不同感染位点的EV71全基因组的分析。我们的数据强调了EV71 VP1蛋白内的一个关键氨基酸变化，这可能在自然感染期间导致传播和嗜神经性。通过不同EV71谱系的体外反向遗传实验和计算机模拟，证实了这一假设。据我们所知，这项研究首次提供了EV71在感染过程中在单个人类宿主内进化和传播的全基因组分析，并强调了病毒基因组关键区域突变的出现如何可能导致新的表型和神经毒力。
Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient. Human enterovirus-71 (EV71) has been the cause of major hand-foot-and-mouth disease outbreaks, particularly in the Asia-Pacific region. EV71 infection can also disseminate to the central nervous system and result in meningo-encephalitis. Despite intensive epidemiological screening, as well as experimentation in animal models, viral factors contributing to neurotropism remain ill-defined. We describe here the analysis of the full-length genomes of EV71 from different infection sites in an immunocompromised host with disseminated disease. Our data highlight a critical amino acid change within the EV71 VP1 protein that could potentially lead to dissemination and neurotropism during natural infections. This hypothesis was confirmed in vitro through reverse genetic experiments in different EV71 lineages and by in silico modelling. To our knowledge, this study provides the first genome-wide analysis of EV71 evolution and dissemination within a single human host over the course of an infection, and highlights how the emergence of mutations at critical regions of the viral genome can potentially lead to new phenotypes and neurovirulence.
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