Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP, et al. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 2009;457:882-886. www.nature.com (Reprinted with permission.)
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp) or HCV produced in cell culture (HCVcc) (A.P., M.E. and C.M.R., unpublished observations), suggesting that there is a block at the level of entry. Here we show, using an iterative complementary DNA library screening approach, that human occludin (OCLN) is an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for the HCV-susceptibility of human cells, because its overexpression in uninfectable cells specifically enhanced HCVpp uptake, whereas its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors CD81 (ref. 4), scavenger receptor class B type I (SR-BI, also known as SCARB1) and claudin-1 (CLDN1). Although the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, both OCLN and CD81 must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process, and provides an important advance towards efforts to develop small animal models for HCV.
A major hurdle to the development of effective vaccines and antiviral therapies against hepatitis C virus (HCV) infection has been the lack of a suitable small-animal model. Although mice transgenic for HCV protein expression and humanized mouse models (human hepatocyte xenograft transplantation into severe combined immunodeficient/albumin–urokinase plasminogen activator mice) have shed light on numerous aspects of HCV pathogenesis and allowed validation of antiviral therapies (for a review, see Barth et al.1), their use is limited because they allow the study of only certain aspects of virus-host interactions and infection pathogenesis. If indeed a robust mouse model is to be developed, a complete understanding of host factors required for HCV entry and replication is required. Studies to date using recombinant HCV envelope proteins, retroviral pseudoparticles expressing HCV E1/E2 glycoproteins (HCVpp), and most recently cell culture–derived infectious HCV particles (HCVcc) have determined that the candidate HCV entry factors include the low-density lipoprotein receptor, glycosaminoglycans, the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction protein claudin-1 (CLDN1). Despite the identification and validation of CD81, SR-BI, and CLDN1 as essential HCV entry cofactors in various model systems, a number of human cell lines and all cell lines of nonprimate origin remained resistant to HCV entry, even after forced expression of these three human HCV entry factors. These observations suggested the presence of an additional entry factor. This crucial factor has now been identified by Ploss and colleagues2 as the tight junction protein occludin (OCLN). Expression of OCLN in combination with other requisite entry factors (CD81, SR-BI, and CLDN1) renders mouse cell lines susceptible to infection with HCVpp. This represents a major breakthrough in the struggle toward the identification of the factors that restrict HCV infection to the human liver (Fig. 1). In turn, the findings of this study bring closer the possibility of the development of an urgently needed small-animal model for the study of HCV infection in vivo.
Ploss and colleagues2 screened a retroviral complementary DNA (cDNA) expression library derived from the hepatocarcinoma cell line Huh-7.5 (these cells are highly permissive for HCV infection) against a mouse NIH3T3 embryonic fibroblast cell line that overexpressed human CD81, SR-BI, and CLDN1 (termed N3xF26) yet remained resistant to HCVpp infection. After transduction with the Huh-7.5–derived expression library, N3xF26 cells were exposed to HCVpp encoding a puromycin resistance gene. Puromycin-resistant subpopulations were selected and transfected with retroviral packaging plasmids to rescue the Huh-7.5–derived cDNA elements that bestowed upon N3xF26 cells increased susceptibility to HCVpp infection. Subsequent rounds of transduction and selection in this vein resulted in the emergence of subpopulations of cells that displayed increased susceptibility to HCVpp infection. Finally, an analysis of Huh-7.5–derived cDNA from clonal populations of these cells identified human OCLN as a common thread.
The requirement of OCLN for HCV entry in both human and mouse cell lines was demonstrated by either silencing of OCLN expression in HCV-permissive cells or overexpression of all key entry factors in cells resistant to HCV infection. A concurrent report by Liu et al.3 also confirms the requirement for OCLN in HCV entry with a small interfering RNA knockdown approach. Furthermore, murine cell lines that were refractory to HCV infection became permissive to HCVpp infection only after overexpression of all four entry factors.
Perhaps the most interesting findings of this study come from the analysis of which HCV entry factors dictate species-specific tropism. To address this question, the authors expressed various combinations of mouse and human variants of the four entry factors in Chinese hamster ovary cells and analyzed their susceptibility to HCVpp infection. Intriguingly, mouse variants of SR-BI and CLDN1 could participate in HCV entry equally as well as their human counterparts, whereas only human variants of OCLN and CD81 could enable HCV entry when coexpressed with either mouse or human variants of the remaining complementary HCV entry factors. These findings indicate that CD81 and OCLN represent the minimal human-specific entry factors, at least in the context of mouse and hamster cells. Furthermore, these exciting observations may be the prelude to the generation of a mouse model with which to study the HCV life cycle and host response in vivo. The most pertinent question that comes to mind is whether mice expressing human CD81 and OCLN in the liver will be susceptible to HCV infection. Although expression of all four entry factors in mouse cell lines renders them permissive to infection with HCVpp, these cells could not support HCVcc infection. This is not surprising given past reports of inefficient replication of HCV RNA in mouse cell lines.4, 5 Therefore, expression of human OCLN and CD81 in a mouse liver is unlikely to provide a mouse model for the study of genuine HCV infection in itself in the short term. The investigation of additional level(s) of species tropism restriction is being actively pursued by several laboratories, and the identification of OCLN with CD81 as the minimal human-specific HCV entry factors by Ploss et al.2 clearly provides the first important step for the development of a mouse model of HCV infection in the future.
Like CLDN1, OCLN is a major component of the tight junction complexes that border adjacent epithelial and endothelial cells and regulate paracellular transit of solutes and cell polarity (for a review, see Chiba et al.6). It appears to contribute to the regulation of a number of signaling pathways to and from tight junctions. Although it is feasible to envisage interactions between HCV and all four entry factors in a cell culture system, it is difficult to conceptualize interactions in the HCV-infected liver given that CLDN1 and OCLN are sequestered at the hepatocyte tight junction. Recent studies addressing this question suggest that expression of the HCV envelope glycoprotein7 or engagement of envelope glycoprotein E2 with CD818 can alter the subcellular localization of the tight junction proteins CLDN1 and OCLN in Huh7-derived cell lines. The significance of these observations for the HCV-infected liver is unknown but clearly deserves further investigations. The use of several uptake factors with distinct cell surface distributions is consistent with the hypothesis that HCV could follow a coordinated entry pathway similar to that of Coxsackievirus B.2
What are the clinical implications of these findings? HCV entry is a key target for host neutralizing antibodies and a promising target for antiviral therapies.9, 10 Although HCV clearance can be achieved by a combination treatment using pegylated interferon-alpha and ribavirin, a large number of patients have contraindications to one of the components, cannot tolerate the treatment, or do not respond at all. The introduction of novel antiviral substances targeting HCV protein processing and replication appears to improve the response to interferon-based therapies in randomized clinical trials. Nevertheless, the clinical development of these antivirals is also characterized by significant toxicity and viral resistance. Therefore, novel and complementary antiviral treatment strategies targeting different steps of the viral life cycle will be needed. The relevance of the HCV entry step as a therapeutic target is underlined by the successful clinical development of entry inhibitors for human immunodeficiency virus infection (for a review, see Este and Telenti11). Thus, the identification of OCLN as a novel essential and human-specific HCV entry factor not only significantly advances the understanding of the molecular mechanisms of HCV entry but may ultimately contribute to the development of novel therapeutic and preventive antiviral strategies.