Study of hepatitis C virus entry in genetically humanized mice

Abstract

Approximately 2% of the world's population is chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can culminate in end stage liver disease and liver cancer if the infection is untreated. Current therapy is only partially effective and a vaccine for HCV does not exist. Since the discovery of HCV as the etiologic agent causing hepatitis C several experimental tools have been developed which have improved our understanding of the viral life cycle and the interaction of HCV with human cells. However, it remains challenging to study HCV infection in its native liver environment given its narrow species tropism, limited to humans and chimpanzees. Mice can be rendered susceptible to HCV infection by transplanting human hepatocytes into immunocompromized liver injury strains. Such human liver chimeric mice are useful as a challenge model for human hepatotropic pathogens but their utility is hampered by their inability to mount functional immune responses and practical aspects including high costs, low throughput, and donor-to-donor variability. The barriers that restrict HCV species tropism are incompletely understood. We have previously shown that expression of human CD81 and human OCLN is required for HCV uptake into mouse cells. This led to the construction of a genetically humanized mouse model for HCV infection. Here, we provide a detailed protocol for the generation of these animals and highlight some of its applications for studying HCV biology and preclinical testing of drug and vaccine candidates.

Figure 1. Mechanism of studying HCV entry in vivo

(a) The four HCV entry factors CD81, scavenger receptor BI (SCARB1), claudin-1 (CLDN1) and occludin (OCLN) (b) are delivered to the hepatocytes of reporter mice via adenoviral delivery. 24–48 hours post intravenous adenovirus delivery, 2×10⁷ TCID₅₀ of a Cre recombinase (Cre)-expressing HCV genome are injected i.v. and entry is analyzed either by bioluminescence imaging, histology or flow cytometry 72 hours post injection of HCV-Cre.

Figure 2. Constructs used for the detection of HCV entry in vivo

(a) Genome structure of the adenoviral Ad5 constructs used to deliver the four HCV entry factors and (b) structure of the CD81, SCARB1, CLDN1 and OCLN expressing adenoviral constructs. (c) Structure of the Cre recombinase-expressing HCV genomes. Bicistronic vectors expressing Cre recombinase driven by the HCV IRES are followed by different intergenotypic chimeras of HCV genotypes 1 (Con1), 2 (Jc1), 4 (ED43), 6 (HK6a) or 7 (QC69) driven by an EMCV IRES.

Figure 3. Detection of HCV entry by flow cytometry

(a) Isolated hepatocytes following isolation by perfusion of the murine liver with Collagenase. (b) Gating scheme of murine hepatocytes following injection of fluorescently labeled entry factor-expressing adenovirus and HCV-Cre.