Impact of Intra- and Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

Abstract

Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN low subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN low cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.The hepatitis C virus (HCV) is a small enveloped viral pathogen of the Flaviviridae family with a single plus-strand RNA genome (26). About 130 million individuals worldwide are currently chronically HCV infected and thus at risk for the development of cirrhosis, end-stage liver disease, and hepatocellular carcinoma (34).HCV primarily infects and replicates in hepatocytes. All stages of its replication cycle are dependent on various hostencoded factors. Completion of the earliest stage of the replication cycle, HCV cell entry, requires at least four host factors on the hepatocyte surface. These include the tetraspanin CD81 (30), scavenger receptor class B type I (SR-BI) (32), and more recently described, the tight junction components claudin 1 (CLDN1) (13) and occludin (OCLN) (27,31). It is thought that the viral envelope glycoproteins E1 and E2 interact with these four (co)receptors sequentially and that these interactions are orchestrated by cell signaling processes (6,14,28). SR-BI may act early and the tight junction components may act later in this process (11,13,21,24,42). The exact mechanisms and sequence of the interactions remain to be determined, but the result is known to be the uptake of the virion or a virion-coreceptor complex into an endosomal compartment. Acidification then triggers fusion of the viral envelope with the endosomal membrane (20,24,40). This releases the nucleocapsid into the cytosol, completing the cell entry process.Only humans and chimpanzees are natural hosts of HCV. This is thought to be because HCV is unable to utilize other species' homologues of several esse...

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].…”

“…Although the quantified contribution of this cell-to-cell transmission and its modalities are still disputed, it can be evidenced in several systems. Usually, cell-to-cell transmission is evaluated between two co-cultured cell populations, one population of producer cells infected beforehand and one population of target cells that can be easily tracked due to a chemical [186], or geneticallyencoded fluorescent marker [101,102]. Infected events in the second populations are considered as indicator of cell-to-cell transmission if the cell-free infectivity is blocked.…”

“…Infected events in the second populations are considered as indicator of cell-to-cell transmission if the cell-free infectivity is blocked. This last condition can be 13 achieved by using saturating concentrations of neutralizing antibodies [171,186,187], particular virus mutants or target cells [101], or by culturing the cells under an agarose overlay that prevents Brownian motion of cell-free viruses [102,171]. The dual cell-based reporter system developed by Jones et al…”

“…[85] (see Table 1) is particularly amenable to investigate cell-to-cell transmission [85,102,188] as it allows at the same time distinguishing two different cell populations (producer and target cells) and tracking rare infection events at the single cell level. As an alternative to using two cell populations, counting the number of cells per infection focus after culture under an agarose overlay has been used as an estimate of cell-to-cell transmission efficiency [171,189].…”

Characterization of the inhibition of hepatitis C virus entry byIn vitro-generated and patient-derived oxidized low-density lipoprotein

“…Luciferase assay was performed as previously described. 32 Intra-and extracellular HCV core was measured by a commercially available enzyme-linked immunosorbent assay (ELISA) (Architect HCV Ag Assay; Abbott Laboratories, Abbott Park, IL).…”