Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Abstract

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, smallanimal 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.KEYWORDS animal models, hepatitis C virus, viral entry, viral hepatitis H epatitis C virus (HCV) is a positive-sense, single-stranded RNA virus belonging to the Flaviviridae family, genus Hepacivirus (1). HCV progresses to persistent infection in 70 to 80% of those individuals who become acutely infected (2). Chronic carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) if they remain untreated. Over a few years, very potent directly acting antivirals (DAAs) which can cure

Tight Junction Proteins and the Biology of Hepatobiliary Disease

“…OCLN interacts with HCV surface glycoprotein E2 via its extracellular loop 2 (ECL2) [115]. Of note, transgenic expression of human OCLN enables HCV infection of non-permissive species like mice [116][117][118]. However, the exact mechanism and localization of OCLN-HCV interaction is not fully understood.…”

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

“…Indeed, expression of one or several of these host factors can confer cell susceptibility to infection by HCV [21][22][23]. While none of those factors individually confers tissue tropism to HCV, CD81 and OCLN are responsible for the human species-specific tropism of HCV [22,24,25]. In addition to these four essential entry factors, additional host factors play a role in HCV attachment (attachment/binding factors) and internalization/fusion (co-factors).…”

Hepatitis C Virus Entry: Protein Interactions and Fusion Determinants Governing Productive Hepatocyte Invasion

“…More recently, 3D organoid culture systems confirmed the role and spatiotemporal requirements of the major entry factors, namely, SR-BI, CD81, CLDN1, and OCLN, in more physiological environments (Baktash et al 2018). Finally, sophisticated mouse models using human xenotransplant mice, adenoviral overexpression of human factors, or genetic humanization of mice confirmed the role of these four HCV entry factors Meuleman et al , 2012Bissig et al 2010;Dorner et al 2011;Lacek et al 2012;Ding et al 2017). The lack of simple in vivo systems to study HCV infection and the sheer number of involved factors has provoked some skepticism as to which reported entry factors have physiological function.…”

“…The lack of simple in vivo systems to study HCV infection and the sheer number of involved factors has provoked some skepticism as to which reported entry factors have physiological function. Importantly, congruent evidence from in vivo models, primary hepatocyte cultures, and stem cellderived hepatocyte cultures clearly argues for the fact that HCV in vitro models can reliably mimic many parts of the HCV entry process into human hepatocytes (Fournier et al 1998;Codran et al 2006;Roelandt et al 2012;Belouzard et al 2017;Ding et al 2017). We will specify in detail below which level of evidence exists for the most important entry factors and cofactors.…”