Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells
Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells
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通过适应 Huh7.5.1 细胞分离出耐热丙型肝炎病毒 JFH1 衍生变体
DOI：
10.1099/jgv.0.001117
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发表时间：
2018
影响因子：
3.8
通讯作者：
Fukasawa Masayoshi
中科院分区：
文献类型：
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作者：
Shirasago Yoshitaka;Fukazawa Hidesuke;Aizaki Hideki;Suzuki Tetsuro;Suzuki Takeru;Sugiyama Kazuo;Wakita Takaji;Hanada Kentaro;Abe Ryo;Fukasawa Masayoshi
丙型肝炎病毒（HCV）在培养细胞中的感染和增殖主要使用感染性临床克隆JFH 1进行研究。然而，其传染性不足以使感染容易被检测到。在这项研究中，我们试图分离HCV-JFH 1适应人肝癌细胞Huh7.5.1的变异。通过在Huh 7.5.1细胞中连续传代野生型HCV-JFH 1，我们获得了能够诱导严重细胞病变效应的变体，其感染性比野生型HCV-JFH 1高约700倍。此外，当高容许性的Huh7.5.1-8细胞被该变体感染时，病毒颗粒以> 1011拷贝ml−1产生，使该变体成为最有效的HCV生产系统之一。在变异基因组中发现了两个适应性突变：核心蛋白区的a1994 c（K74 T）和E2蛋白区的t3014 c（I414 T）。这两种突变都有助于增强感染性，它们的组合在这方面显示出协同效应。对携带K74 T、I414 T和K74 T/I414 T突变的重组病毒的检查显示，这些突变对病毒进入后的步骤没有影响（基因组复制、颗粒组装和排出），但导致病毒感染变得不太依赖于I型B类清道夫受体，感染性颗粒改变为更宽和更低的密度范围，并增强了感染性病毒的热稳定性。因此，这种具有更高和稳定感染性的Huh7.5.1适应的HCV-JFH 1变体应该是研究HCV生命周期背后的分子机制和抗病毒筛选的有价值的工具。
Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1–8 cells were infected with this variant, viral particles were produced at >1011copies ml−1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.