| |
Hepatitis C : Identification Of A Protein That Inhibits The Virus
|
| |
| |
ScienceDaily (Apr. 14, 2008) - Scientists in the Laboratoire Hepatite C of the Institut de Biologie de Lille in collaboration with INSERM Unit 602 and a laboratory at Stanford University have provided evidence of a protein, called EWI-2wint, that inhibits the hepatitis C virus at an early stage of its infective cycle. This research suggests possible new perspectives for the development of therapies to block the virus before it enters a cell.
HCV uses at least three receptors to enter and infect a hepatocyte. One of these receptors is the CD81 protein, which has the particular characteristic of associating with numerous other proteins. It was by studying these CD81 partner proteins that the researchers identified the EWI-2wint protein, which prevents the recognition of CD81 by the hepatitis C virus and inhibits it at a very early stage in its infective cycle. This protein is present in other types of cells, which could explain why they are not infected by HCV. Discovery of the role of EWI-2wint in hepatocytes has demonstrated the complexity of the mechanisms of entry of HCV into its target cells, and opens the way to new therapeutic approaches.
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001866
The CD81 Partner EWI-2wint Inhibits Hepatitis C Virus Entry
Vera Rocha-Perugini1#, Claire Montpellier1#, David Delgrange1, Czeslaw Wychowski1, Francois Helle1, Andre Pillez1, Herve Drobecq1, Francois Le Naour3, Stephanie Charrin3, Shoshana Levy2, Eric Rubinstein3, Jean Dubuisson1*, Laurence Cocquerel1,2*
1 Institut de Biologie de Lille (UMR8161), CNRS, Universites de Lille I et Lille II, Institut Pasteur de Lille, Lille, France2 Division of Oncology, Department of Medicine, Stanford University Medical Center, Stanford, California, United States of America3 INSERM-U602, Institut Andre-Lwoff, Universite Paris XI, Hopital Paul Brousse, Villejuif, France
Abstract
Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.
Introduction
Hepatitis C virus (HCV) infection is a global public health problem affecting over 130 million individuals worldwide; its symptoms including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma [1]. Unfortunately, no vaccine is currently available to prevent new infections and the current treatments are not fully efficient [2]. Clearly, new therapeutic strategies are urgently required.
Over the past decade, due to the lack of a cell culture system supporting production of infectious virus particles, several surrogate models have been developed to facilitate analysis of the HCV life cycle. Among these models, pseudoparticles (HCVpp), consisting of native HCV envelope glycoproteins assembled onto retroviral core particles [3], [4] have been useful in investigating the HCV entry process. More recently, however, production of infectious HCV particles in cell culture (HCVcc) has finally become possible [5], [6], [7]. This powerful system is based on the transfection of the human hepatoma cell line Huh-7 with the cloned JFH1 genome that replicates and produces infectious particles.
HCV encodes two envelope glycoproteins, E1 and E2, that interact to form a noncovalent E1E2 heterodimer [8] which is present at the surface of HCV particles [6], and is therefore the obvious candidate ligand for cellular receptor(s). Although the early steps of viral entry have yet to be elucidated, several cell-surface expressed molecules have been proposed as entry factors for HCV (reviewed in [9]). Among these molecules, the tetraspanin CD81 and the scavenger receptor class B type I (SR-BI) have been shown to play major roles in HCV entry. However, co-expression of these two molecules in non-hepatic cell lines does not lead to HCV entry [10], suggesting that additional molecule(s) are involved in control of HCV entry. Recently, the tight-junction components Claudins (CLDN-1, CLDN-6, CLDN-9) have been identified as additional key factors for HCV infection [11], [12]. Interestingly, CLDN-1 is the first entry factor shown to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Nevertheless, although CLDN-1 subcellular distribution seems to modulate HCV permissivity [13], some human cell lines expressing CD81, SR-BI and CLDN-1 remain resistant to HCV entry suggesting that one or more human-specific HCV entry factor(s) remain to be discovered [11].
CD81 belongs to the tetraspanin family. Members of this family organize and regroup their associated transmembrane proteins and are involved in various functions such as cell morphology, motility, fusion and signalling [14], [15]. A major characteristic of tetraspanins is their ability to interact with each other and with other transmembrane proteins, thus building membrane multi-molecular complexes, collectively referred to as the tetraspanin web [16], [17]. Within this network of interactions, tetraspanins form primary complexes with a limited number of proteins termed tetraspanin partners. These primary interactions are direct, highly specific and occur at high stoichiometry. Two major partners have been identified for CD81, EWI-F (also called CD9P-1, FPRP or CD315) and EWI-2 (also called PGRL, IgSF8 or CD316) [18], [19], [20], [21], [22], which may provide a link between the tetraspanin web and the actin cytoskeleton by interacting with Ezrin, an Ezrin-Radixin-Moesin (ERM) protein [23]. Although its function is still unclear, EWI-2 seems to participate in the regulation of cellular functions such as aggregation, spreading, motility and migration [24], [25], [26].
In this work, we identified a cleavage product of EWI-2, which associates with CD81 and inhibits its interaction with the HCV envelope glycoproteins. Most importantly, this molecule, that we called EWI-2wint (EWI-2 without its N-terminus), has an inhibitory effect on HCV entry, highlighting a potential new mechanism for the regulation of cellular invasion by this pathogen.
EWI-2wint inhibits HCV infection
We then sought to determine whether EWI-2wint is able to modulate HCV infection of Huh-7 target cells. Originally we used cell culture produced HCV particles (HCVcc) [5], [6], [7] to infect Huh-7 cells expressing EWI-2FLAGfur, HAEWI-2FLAG, pcDNA3.1 or naive Huh-7 cells. In the absence of a reporter gene in HCVcc, infection levels were evaluated by immunofluorescence (Figure 5A) and western blotting (Figure 5B), 40 h post-infection. Interestingly, we observed a reduction in HCVcc infection level in cells expressing EWI-2FLAGfur, whereas it remained unmodified in control cells (Figure 5C). It is worth noting that in HAEWI-2FLAG cells, the HCVcc infection level was similar to that of pcDNA3.1 cells, indicating that ectopic expression of HAEWI-2FLAG does not affect HCV infection. In addition, flow cytometry analyses ruled out any potential bias due to different levels of CD81 cell surface expression on clones (Figure 5D). Altogether, these results indicate that EWI-2wint produced in Huh-7/EWI-2FLAGfur cells inhibits HCV infection.
EWI-2wint blocks HCV entry into target cells
The life cycle of HCV can be divided into three major steps : entry of the virus into its target cells by receptor-mediated endocytosis, cytoplasmic and membrane-associated replication of the RNA genome, and assembly and release of the progeny virions.
To analyze the effect of EWI-2wint expression on HCV genome replication, we compared the efficiency of RNA replication and virus production between transfected cell lines expressing or lacking EWI-2wint. We transfected equal amounts of RNA corresponding to the full-length JFH1 genome [6] into the different cell lines (Figures 6A and 6B). Immunofluorescence analyses of transfected cells showed similar positivity for HCV anti-core antibody at 40 h post-transfection. Flow cytometry analyses using an anti-NS3 mAb showed that, although slightly stronger in Huh-7 cells, viral expression was similar in cellular clones that lacked or expressed EWI-2wint (Figure 6B), indicating that EWI-2wint does not affect HCV genome replication. Next, to assess the effect of EWI-2wint on the assembly and/or egress of particles, the culture medium of each transfected cell line was harvested and used to infect naive Huh-7 cells. Through core-specific immunofluorescence staining, we found that Huh-7 cells were similarly infected by virus produced from all transfected cellular clones (data not shown), suggesting that EWI-2wint does not affect HCV assembly and release.
Discussion
Here, we identified a novel CD81 associated protein, EWI-2wint, which is able to inhibit HCV entry into target cells by blocking the interactions between HCV glycoproteins and CD81. This finding indicates that the early steps of HCV entry into its target cells involve tight control of CD81 accessibility to the viral particle.
EWI-2wint may inhibit HCV entry by reducing E1E2-CD81 interactions for a number of possible reasons. EWI-2wint may reduce CD81 accessibility to envelope glycoproteins by steric hindrance. Alternatively, the association of EWI-2wint with CD81 may induce conformational modifications in CD81, blocking the binding of HCV heterodimers. Although the exact role of CD81 in the course of virus infection is not well defined, it is a key molecule required for productive infection by HCVpp and HCVcc of Huh-7 cells [3], [4], [5], [6], [7], [10], [32]. Moreover, most recently CD81 was shown to be required for the infection of primary hepatocytes by serum-derived HCV [33]. Previous studies have suggested that CD81 may not be required for the first attachment step, but rather function as a post-attachment entry co-factor [34], [35], [36]. Very recently, it has been shown that CLDN-1, 6 and 9 membrane proteins localized at tight junctions, are additional entry factors for HCV [11], [12], [37], indicating that HCV entry might involve a complex multistep process. CD81 might potentially be required for a post-binding step such as escorting the particle into the endocytic pathway or priming it for the pH triggered fusion mechanism. The association of EWI-2wint with CD81 could block such entry stages. Since our preliminary results suggest that CD81 and EWI-2wint likely colocalize in an endosomal compartment (unpublished data), EWI-2wint might exert its inhibitory effect on CD81 functionality in the endosomes. In addition, EWI-2wint may interfere with actin polymerization potentially required for HCV entry. Indeed, HCV replication requires microtubule and actin polymerization [38] and CD81 engagement leads to actin rearrangement [39], [40]. Furthermore, it has also recently been shown that CD81 and EWI-2 interact with Ezrin, an actin-linking ERM protein [23]. Lastly, we cannot exclude the possibility that EWI-2wint blocks other signaling pathways necessary for viral entry.
EWI-2wint is a cleavage product of the EWI-2 protein. The proteolytic cleavage of EWI-2 Ig1 domain occurs downstream of the RGR amino acids. A site directed mutagenesis analysis showed that RXR (where X is any amino acid) correspond to the consensus site recognized by the protease involved in this cleavage (unpublished data). Further analyses using protease inhibitors and small interference RNAs should enable us to identify this protease. Pulse-chase experiments in CHO cells showed that the cleavage occurs after N-glycan maturation (unpublished data), suggesting that it likely involves a Golgi resident protease. We showed that a RGRR furin cleavage motif made by insertion of an Arg residue into the EWI-2 sequence allows efficient production of EWI-2wint and additional cleavage product(s) in Huh-7 cells. As shown here, EWI-2 can be cleaved at RGR site to produce EWI-2wint, and it is likely that a furin-like protease may be responsible for this cleavage. Such a protease may be expressed differently among cell types, such that liver cells lacking the enzyme are consequently more sensitive to HCV infection. On the other hand, the accessibility of EWI-2 to protease(s) may be cell-type specific and might be modulated by other components of the tetraspanin webs that vary in different cell types [14], [16].
Although several molecules have already been implicated in the entry process of HCV, it is likely that additional specific factors regulate its entry into hepatic cells [41]. Silencing of endogenous EWI-2 in Huh-7 cells did not significantly affect HCVcc infection (data not shown) indicating that while being a major partner of CD81, EWI-2 is likely not involved in the HCV entry process. Here, we showed that EWI-2wint, present in some cell lines and absent from hepatic cells, is able to inhibit HCV entry. The production of a mAb directed against EWI-2wint will be essential to further analyse the expression profile of EWI-2wint in various tissues and cell lines. Nevertheless, our results suggest that, in addition to the presence of specific entry factors in the hepatocytes such as CD81, SR-BI, CLDN-1 and additional unidentified molecule(s), the absence of a specific inhibitor can also contribute to the hepatotropism of HCV. Interestingly, 293T cells, that are permissive for HCV entry when they express CLDN-1 [11], do not express EWI-2wint (Table 1). Further experiments with HCV non permissive cells expressing all known positive entry factors will be necessary to determine if the silencing of endogenous EWI-2wint is sufficient to induce HCV permissivity.
In conclusion, our observation constitutes a basis for the rational design of new antiviral molecules. Moreover, it represents a new mechanism that contributes to the regulation of the entry of a pathogen into a host cell.
|
|
| |
| |
|
|
|
