Ruedas John B, Ladner Jason T, Ettinger Chelsea R, Gummuluru Suryaram, Palacios Gustavo, Connor John H
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA
National Emerging Infectious Diseases Laboratories, Boston, Massachusetts, USA.
J Virol. 2017 Jul 12;91(15). doi: 10.1128/JVI.00392-17. Print 2017 Aug 1.
Ebolaviruses have a surface glycoprotein (GP) that is required for virus attachment and entry into cells. Mutations affecting GP functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species , as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.
埃博拉病毒具有一种表面糖蛋白（GP），它是病毒附着和进入细胞所必需的。影响GP功能的突变可改变病毒的生长特性。我们构建了一种编码埃博拉病毒马科纳变种GP的重组水疱性口炎病毒（rVSV-MAK-GP），并观察到在某些细胞系的组织培养传代过程中，马科纳GP基因出现了T544I突变。当VSV-MAK-GP在Vero E6、Vero和BS-C-1细胞上生长时，T544I突变在两代内出现，但在Huh7和HepG2细胞上传代时则未出现。该突变导致病毒生长动力学显著增加，并在Vero E6细胞上相对于野生型rVSV-MAK-GP赋予了强大的生长优势。对从西非患者收集的完整病毒基因组的分析表明，埃博拉病毒临床样本中未发现该突变。然而，我们观察到在各种埃博拉马科纳分离株在Vero E6细胞上连续传代过程中出现了T544I。三个独立的分离株显示，在未传代的病毒或仅传代一次的病毒中无法检测到的T544I，在单次传代内频率增加到大于60%，这与其作为一种组织培养适应性变化一致。有趣的是，在任何苏丹、本迪布焦或塔伊森林埃博拉病毒序列中均未发现T544I。此外，当我们对编码来自这些埃博拉病毒的GP的重组VSV进行连续传代时，T544I并未出现。本报告提供了实验证据，表明自发突变T544I是某些细胞系中的一种组织培养适应性变化，并且它可能是该病毒种所特有的。就死亡率和死亡人数而言，埃博拉病毒（扎伊尔）种是所有埃博拉病毒中致死性最高的种。了解埃博拉病毒表面糖蛋白如何发挥功能以促进进入细胞是一个深入研究的领域。最近，三个研究小组独立鉴定出埃博拉糖蛋白中的一种多态性（I544），其增强了病毒进入，但他们对于其对发病机制的影响结论并不一致。我们在此的发现揭示了这种多态性的起源，并提供了实验证据表明它是组织培养条件特有的自发突变（T544I）的结果，这表明它在发病机制中没有作用。我们进一步表明，这种突变可能是该病毒种所特有的，因为它在苏丹、本迪布焦和塔伊森林埃博拉病毒中不发生。了解这种突变背后的机制可以深入了解培养条件和埃博拉病毒糖蛋白之间存在的功能差异。
