Mouse-Specific Residues of Claudin-1 Limit Hepatitis C Virus Genotype 2a Infection in a Human Hepatocyte Cell Line

Abstract

Recently, claudin-1 (CLDN1) was identified as a host protein essential for hepatitis C virus (HCV) infection.To evaluate CLDN1 function during virus entry, we searched for hepatocyte cell lines permissive for HCV RNA replication but with limiting endogenous CLDN1 expression, thus permitting receptor complementation assays. These criteria were met by the human hepatoblastoma cell line HuH6, which (i) displays low endogenous CLDN1 levels, (ii) efficiently replicates HCV RNA, and (iii) produces HCV particles with properties similar to those of particles generated in Huh-7.5 cells. Importantly, naïve cells are resistant to HCV genotype 2a infection unless CLDN1 is expressed. Interestingly, complementation of HCV entry by human, rat, or hamster CLDN1 was highly efficient, while mouse CLDN1 (mCLDN1) supported HCV genotype 2a infection with only moderate efficiency. These differences were observed irrespective of whether cells were infected with HCV pseudoparticles (HCVpp) or cell culture-derived HCV (HCVcc). Comparatively low entry function of mCLDN1 was observed in HuH6 but not 293T cells, suggesting that species-specific usage of CLDN1 is cell type dependent. Moreover, it was linked to three mouse-specific residues in the second extracellular loop (L152, I155) and the fourth transmembrane helix (V180) of the protein. These determinants could modulate the exposure or affinity of a putative viral binding site on CLDN1 or prevent optimal interaction of CLDN1 with other human cofactors, thus precluding highly efficient infection. HuH6 cells represent a valuable model for analysis of the complete HCV replication cycle in vitro and in particular for analysis of CLDN1 function in HCV cell entry.Hepatitis C virus (HCV) is a liver-tropic plus-strand RNA virus of the family Flaviviridae that has chronically infected about 130 million individuals worldwide. During long-term persistent virus replication, many patients develop significant liver disease which can lead to cirrhosis and hepatocellular carcinoma (54). Current treatment of chronic HCV infection consists of a combination of pegylated alpha interferon and ribavirin. However, this regimen is not curative for all treated patients and is associated with severe side effects (37). Therefore, an improved therapy is needed and numerous HCVspecific drugs targeting viral enzymes are currently being developed (47). These efforts have been slowed down by a lack of small-animal models permissive for HCV replication since HCV infects only humans and chimpanzees. Among small animals, only immunodeficient mice suffering from a transgene-induced disease of endogenous liver cells and repopulated with human primary hepatocytes are susceptible to HCV infection (39).The restricted tropism of HCV likely reflects very specific host factor requirements for entry, RNA replication, assembly, and release of virions. Although HCV RNA replication has been observed in nonhepatic human cells and even nonhuman cells, its efficiency is rather low (2,11,59,67). In addition, so far, efficient productio...

MicroRNA‐26‐5p functions as a new inhibitor of hepatoblastoma by repressing lin‐28 homolog B and aurora kinase a expression

“…Human hepatocyte cell line HC‐04, human hepatoblastoma cell line HepG2 (generously provided by Dr. Yuzuru Shiio and Dr. Hongbing Wang20, 21), and cell line HUH6 (a generous gift from Dr. Thomas Pietschmann22) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Hep293TT cells (a generous gift from Dr. Yuzuru Shiio) were cultured in Roswell Park Memorial Institute 1640 supplemented with 10% fetal calf serum, 25 mM 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid, and 1 mM sodium pyruvate 20…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Huh-7 Lunet, Huh-7.5 or Huh-7.5.1 cell lines) or genetically modified to encode a reporter system allowing easy infectivity assay or to permit receptor complementation studies. Importantly, the tools are now available to study individually the 4 main HCV receptors (CD81, SR-BI, Cldn1 and Ocln): cell lines expressing very low levels of one of the receptors and retroviral vectors to rescue the expression of this receptor or one of its homologs have been developed in several laboratories [44,101,102,104,106,116,165] (see Table 1). Thanks to HCVpp, it is also possible to extend these receptor-complementation studies in cell lines that poorly replicate HCV, since the readout of HCVpp infectivity is independent on HCV replication [113,116,119].…”

Cell Culture Systems for Hepatitis C Virus

“…Conversely, selection of Huh-Lunet cell clones essentially lacking CD81 expression permitted receptor complementation stud ies for this important entry factor and the analysis of HCV cell-tocell spread in the presence or absence of CD81. In a similar fashion, we have now HCV permissive cell lines available permitting re ceptor complementation assays for SR-BI (Dreux, Dao Thi et al 2009;Catanese, Ansuini et al 2010), CLDN1 (Haid, Windisch et al 2010), and OCLN (Ciesek, Westhaus et al 2011), thus greatly facil itating HCV cell entry studies with HCVcc. In parallel, a novel hu man hepatoma cell line, named LH86, was demonstrated to be per missive and susceptible to HCVcc and overexpression of CD81 and miR122 rendered HepG2 cells which were initially refractory to HCVcc infection fully permissive to HCV propagation (Narbus, Is raelow et al 2011).…”