Optimised cell systems for the investigation of hepatitis C virus E1E2 glycoproteins
Optimised cell systems for the investigation of hepatitis C virus E1E2 glycoproteins
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用于研究丙型肝炎病毒 E1E2 糖蛋白的优化细胞系统
DOI：
10.1101/2020.06.18.159442
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Kalemera M
中科院分区：
文献类型：
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作者：
Kalemera M
由于各种实验系统的开发，包括细胞培养熟练的 HCV、HCV 假颗粒系统和可溶性包膜糖蛋白，在理解和治疗丙型肝炎病毒 (HCV) 方面取得了长足的进步。 HCV 伪颗粒 (HCVpp) 系统是广泛用于细胞进入研究、新型进入抑制剂筛选、评估临床观察到的 E1 和 E2 糖蛋白表型以及最相关的表征患者接种疫苗和自然感染时诱导的中和抗体宽度的平台。尽管如此，一些源自患者的克隆产生的假颗粒要么不具有感染性，要么表现出感染性太低，无法进行有意义的表型分析。控制任何特定克隆是否产生传染性假粒子的机制尚不清楚。在这里，我们表明，在生产者 HEK 293T 细胞中，CD81（一种 HCV 受体和 E2 的同源结合伴侣）的内源表达对大多数菌株恢复的 HCVpp 的感染性有害。许多 HCVpp 克隆在经过 CRISPR/Cas9 改造以消除 CD81 表达 (293TCD81KO) 的 293T 细胞中产生时，表现出感染性增强或感染性得到恢复。 293TCD81KO细胞中产生的克隆在抗原性上与亲代细胞中产生的匹配对应物非常相似，并且似乎遵循公认的HCV进入途径。 CD81 的缺失并没有明显增加可溶性 E2 (sE2) 的恢复滴度。然而，我们确实出乎意料地发现，293T 细胞和 Freestyle 293-F (293-F) 细胞中产生的单体 sE2 表现出重要的差异。我们发现 293-F 产生的 sE2 主要含有复合型聚糖，而 293T 产生的 sE2 显示复合型聚糖和高甘露糖或混合型聚糖的异质混合物。此外，293T细胞产生的sE2具有优异的抗原性；表现出与构象抗体和 CD81 大细胞外环的结合增加。总之，这项工作描述了生产 HCVpp 的最佳细胞系，并揭示了 293T 和 293-F 细胞中产生的 sE2 的抗原性不同。我们的研究结果对 E1E2 的功能研究和候选免疫原的生产具有重要意义。
Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KOcells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.