Ding Qiang, von Schaewen Markus, Hrebikova Gabriela, Heller Brigitte, Sandmann Lisa, Plaas Mario, Ploss Alexander
Princeton University, Department of Molecular Biology, Lewis Thomas Laboratory, Princeton New Jersey, USA.
Hannover Medical School, Hannover, Germany.
J Virol. 2017 Jan 31;91(4). doi: 10.1128/JVI.01799-16. Print 2017 Feb 15.
Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo IMPORTANCE: At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients, but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize vaccine candidates more efficiently, small-animal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN), which differ between mice and humans, were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels, and support HCV uptake. This model is of considerable utility for studying viral entry in the three-dimensional context of the liver and to test approaches aimed at preventing HCV entry.
丙型肝炎病毒（HCV）在全球至少1.5亿人身上引发慢性感染。HCV的宿主范围狭窄，仅能有效感染人类和黑猩猩。目前对这种狭窄宿主范围的潜在机制尚不完全清楚。在病毒进入层面，两种关键宿主因子——四跨膜蛋白CD81和紧密连接蛋白闭合蛋白（OCLN）的人类和小鼠直系同源物之间的氨基酸序列差异，至少部分解释了HCV进入小鼠肝细胞的能力有限。我们之前已经表明，腺病毒介导或转基因过表达人类CD81和OCLN可促进HCV在体外和体内进入小鼠肝细胞。为了优化这些模型，我们构建了基因敲入小鼠，其中CD81和OCLN的第二个细胞外环被各自的人类序列取代，这些人类序列包含对HCV摄取至关重要的决定因素。我们证明，人源化的CD81和OCLN以组织适当的方式在生理水平表达。携带人源化等位基因的小鼠形成正常的紧密连接，且未表现出任何免疫异常，这表明它们与生理配体的相互作用是完整的。HCV进入因子基因敲入小鼠摄取HCV的效率与转基因或腺病毒表达HCV进入因子的小鼠相似，证明了该模型在体内研究HCV感染的实用性。重要性：至少1.5亿人慢性感染丙型肝炎病毒（HCV）。慢性丙型肝炎可导致进行性肝病和肝癌。新的抗病毒治疗方法可治愈大多数患者的HCV，但疫苗仍然难以实现。为了更好地理解导致肝衰竭和癌症的过程，并更有效地确定候选疫苗的优先级，需要小动物模型。在这里，我们描述了一种新的小鼠模型的特征，其中对HCV摄取至关重要的两个宿主因子（小鼠和人类之间存在差异的CD81和闭合蛋白（OCLN））的部分被人源化。我们证明，这种最小化人源化的小鼠发育正常，在生理水平表达修饰基因，并支持HCV摄取。该模型对于在肝脏的三维环境中研究病毒进入以及测试旨在预防HCV进入的方法具有相当大的实用性。
