Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry

Abstract

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLNdeficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond.Hepatitis C virus (HCV), a member of the family Flaviviridae, is the causative agent of classically defined non-A, non-B hepatitis and is highly prevalent, with approximately 3% of the worldwide population infected (48). HCV infection often results in a chronic, life-long infection that can have severe health consequences, including hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. There is no HCV vaccine available, and the currently employed interferon-based treatment is inadequate as it has severe side effects and is effective only in half of the major genotype-infected individuals (22,32). Specific anti-HCV inhibitors targeting the viral proteases and polymerase are currently being developed and will likely improve therapeutic options substantially. Undoubtedly, however, the emergence of viral resistance to such inhibitors will be a problem facing future HCV treatment options. As such, developing a spectrum of inhibitors targeting diverse steps in the virus life cycle, including HCV cell entry, is a priority for HCV research. Such inhibitors may be particularly useful following liver transplantation. Although HCV is the leading cause of liver transplants worldwide (10), the usefulness of such procedures is limited by subsequent universal graft reinfection and often accelerated disease progression (21). Even transiently inhibiting graft reinfection with HCV cell entry inhibitors could greatly improve the effectiveness of this procedure. Therefore, a greater understanding of HCV cell entry is required for the ...

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

“…Moreover, we show that murine OCLN can serve as a coreceptor for HCV, although with reduced efficiency compared to that of its human homologue, and that the loss in efficiency is attributable to the same two alanine residues previously reported to be critical for HCVpp entry (29). Lastly, our data suggest that OCLN not only enhances but also is essential for spread of HCV between adjacent cells (cell-to-cell route) and that different OCLN variants, including murine OCLN, support this route of viral spread.…”

“…1A). While changes in transmembrane segments have the potential to disrupt the topology of membrane proteins, L233S lies in the C-terminal half of the second extracellular loop of OCLN, previously reported to be critical for species selectivity and in close proximity to two residues (alanines 223 and 224) that are responsible for the more efficient coreceptor function of human OCLN rather than murine OCLN in HCVpp assays (29). The frequency of the L233S SNP, but not that of the A151V or V255E SNP, has been determined to be about 1% in African and Chinese HapMap panels (Table 1) (1).…”

“…OCLN, together with CD81, has been suggested to be a determinant of host restriction of HCV entry (16,31). In a detailed mutagenesis study using HCVpp and 786-O cells as a cellular background, the better coreceptor function of human OCLN has been pinpointed to two residues, alanines 223 and 224, in the second extracellular loop (29). Murine OCLN has two glycines in positions 223 and 224.…”

“…At the cell entry stage, it has been shown that human and murine SR-BI and CLDN1 can serve as HCV (co)receptors with about equal efficiency (8, 13). Conversely, CD81 and OCLN have been reported to confer species restriction since the murine homologues of these entry factors are significantly less efficient than their human counterparts (16,29,31). In both cases, the second of two extracellular loops has been identified as the critical region determining species restriction.…”

“…In both cases, the second of two extracellular loops has been identified as the critical region determining species restriction. In the case of OCLN, this characteristic has been pinpointed to two amino acid residues (29). However, a limitation is that while in the case of CD81, this notion has been substantiated in several studies, including ones using authentic cell culture-grown HCV (HCVcc) as a model system (4,16), data on the species specificity of OCLN stems purely from experiments with lentiviruses pseudotyped with HCV E1E2 (HCV pseudoparticles [HCVpp]), because a cell line that is OCLN negative yet permissive to HCV replication is not available.…”