Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation
Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation
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DOI：
10.1371/journal.ppat.1008428
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发表时间：
2020-03-01
期刊：
影响因子：
6.7
通讯作者：
Koike, Satoshi
中科院分区：
文献类型：
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作者：
Kobayashi, Kyousuke;Mizuta, Katsumi;Koike, Satoshi
作者概述病毒在从最初的复制站点传播到最终的目标站点的过程中，必须克服各种组织和细胞中的各种挫折。为了实现这一点，RNA病毒采用了一种策略，通过使用低保真RNA依赖的RNA聚合酶创建多样化的病毒种群来适应不同的环境。另一方面，当病毒在克隆细胞培养中繁殖时，就会发生体外适应。这些病毒可能会获得新的特性，或者失去它们在体内的一些特性。体外适应通常与衰减有关。因此，对在体外和体内复制的病毒施加的选择压力是相当不同的。目前还不清楚这种环境差异是如何影响病毒种群的。EV71临床分离株在培养细胞中复制能力很差。然而，经过几代之后，病毒适应了这种条件，并进行了有效的复制。在这项研究中，我们通过分析细胞培养适应病毒的种群动态，证明了附着受体的使用是EV71体外适应的主要选择压力。肠道病毒71型(EV71)是手足口病(HFMD)的病原体。然而，这种感染有时与严重的神经系统并发症有关。识别神经毒力决定因素对于了解EV71的发病机制很重要。评估EV71毒力的问题之一是其基因组序列在培养细胞复制过程中变化迅速。在培养细胞中导致EV71基因组快速突变的因素尚不清楚。在这里，我们使用从手足口病患者分离的EV71菌株说明了适应RD-A细胞的种群动态。我们发现，在人类清道夫受体B2转基因(HSCARB2 TG)小鼠中，VP1蛋白(VP1-145)第145位氨基酸残基(VP1-145)有一个由谷氨酸(E)到甘氨酸(G)或谷氨酰胺(Q)的可重复氨基酸替换，这与人清道夫受体B2转基因(HSCARB2 TG)小鼠的衰减有关。由于以前的报道表明VP1-145G和Q突变体通过与硫酸乙酰肝素(HS)结合而有效地感染培养细胞，我们假设HS在细胞表面的表达是这次选择的主要因素。支持这一假设的是，在RD-A细胞中，HS的缺失和hSCARB2的过表达阻止了VP1-145突变体的选择。此外，该突变促进了EV71衣壳不同位置的次级氨基酸替代，以增加其在培养细胞中的适合性。这些结果表明，附着受体，尤其是HS，是选择VP1-145突变体和随后的衣壳突变的重要因素。此外，我们还为EV71强毒株的分离和繁殖提供了一种有效的方法，并且具有最小的选择衰减压力。
Author summaryViruses must overcome various setbacks in a variety of tissues and cells during transmission from the initial replication site to the final target site. To achieve this, RNA viruses employ a strategy to adapt to different environments by creating a diverse viral population using low-fidelity RNA-dependent RNA polymerases. On the other hand, when the viruses are propagated in clonal cell cultures, in vitro adaptation occurs. The viruses may acquire new properties or lose some properties they had in vivo. In vitro adaptation is often associated with attenuation. Therefore, the selection pressures imposed on viruses replicating in vitro and in vivo are quite different. It is unclear how this environmental difference affects viral populations. Clinical isolates of EV71 replicate in cultured cells poorly. However, after a few passages, the viruses adapt to this condition and replicate efficiently. In this study, we demonstrate that attachment receptor usage is a major selection pressure for in vitro adaptation of EV71 by analyzing the population dynamics of cell culture-adapted viruses. This mechanism appears to be a major mode of attenuation.Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation.