Implicaton of tetraspanin‐enriched microdomains (TEMs) in hepatitis C virus
Authors: Paloma Sanz‐Cameno, Samuel Martín‐Vílchez, Yolanda Rodriguez‐Muñoz, Ricardo Moreno‐Otero
Journal: Hepatology (2008)

Recent experimental approaches have highlighted several key factors involved in hepatitis C virus (HCV) entry into host cells. Nonetheless, there is still a lack of understanding in how all of them could interplay to confer HCV invasiveness. In a clever attempt to elucidate it, Reynolds et al. have undertaken an in-depth analysis of the liver expression and location of principal receptors described for HCV: the scavenger receptor class B type I (SR-BI), the tetraspanin CD81, and the tight junction protein claudin-1.1 Additionally, the authors also assessed the receptors' possible modulation by virus infection in both patients with chronic hepatitis C, as well as in infection models of primary liver cells or hepatic cell lines. Such a proposal has interestingly revealed the hepatocyte basolateral colocalization of all the abovementioned receptors in concordance with the conceptual viral accession by sinusoidal blood through its previous liver concentration by dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin (DC-SIGN) and liver/lymph node–specific intercellular adhesion molecule 3–grabbing nonintegrin (L-SIGN) at the surface of sinusoidal cells. Moreover, colocalization implied the presence of claudin-1 beyond tight junctions, and this expression was observed to be induced in hepatocytes by virus infection but not in sinusoidal cells. Reynolds et al. attributed the increase of colocalization to claudin-1 induction through a messenger RNA (mRNA)-independent mechanism that could involve posttranslational regulation. Finally, the authors concluded that claudin-1 overexpression in chronic hepatitis C (CHC)-infected tissues could facilitate intrahepatic viral spread. Despite these interesting results, many questions still remain regarding how all described receptors interact to allow virus entry. In addition to the colocalization of HCV receptors, the latest studies have reported their reciprocal association and the relevance of the claudin-1 pattern of distribution on HCV infectivity.2, 3 As described for other HCV receptors, claudin-1 also shows a widespread tissue expression, neither of which explains HCV tropism. Claudin-1 belongs to the integral membrane protein family involved in tight junction formation through their interaction with other cytoplasmatic proteins that contain a PDZ motif (named for postsynaptic density protein, Drosophila disc large tumor suppressor, and zonula occludens-1 protein), which renders a physical barrier that prevents paracellular permeability of solutes and avoids lateral membrane distribution of molecules in polarized cells.4 Recently, other claudin family members have been implicated as cofactors for HCV entry5, 6; therefore, it would be worthy to investigate the functional consequence of HCV binding in hepatocyte polarization. However, studies have shown the expression of intracellular forms of claudin-1 and membrane plasmatic ones outside tight junctions, which could mediate HCV entry regulation.2, 3 But what regulates claudin-1 expression levels and cell distribution? As described above, claudin-1 has been observed to be associated with CD81 at both membrane plasmatic and intracellular vesicles. Furthermore, other tetraspanin molecules, such as CD9 and CD151, have also been shown to be associated with nonjunctional claudin-1,7 whose functional significance is still unknown. In light of the widely accepted implication that tetraspanins orchestrate macromolecular membrane complexes with functional significance,8 as well as being involved in membrane trafficking,9 could the expression and location of claudin-1 be regulated by CD81 or any CD81 partner? How does HCV binding to these receptors influence their distribution and functionality? As shown for claudin-1, there is previous evidence related to tetraspanin regulation of synthesis and stability of their molecular partners in a tetraspanin web, also known as tetraspanin-enriched microdomains (TEMs). Consequently, the claudin-1 relocation and expression increase observed by Reynolds et al. in patients with CHC could reflect the adjustment of TEM-mediated regulation by the influence of HCV infection. In this way, we thought that it would be very useful to assess the molecular constitution, distribution, and functionality of TEMs, in livers of both healthy individuals and patients with CHC, as well as to investigate the consequences of HCV arrangement, reproduction, and release on tetraspanin web organization and functionality. By doing so, we could appreciate a notable redistribution of the CD81 hepatocyte membrane expression in patients with CHC, from the basolateral zone, which is described to be mainly located in human epithelial cells,10 to the apical pole, that could imply some CD81 polarization lost (Fig. 1A). This finding was associated with a basolateral CD151 expression increase, as described by Reynolds et al. for claudin-1. Interestingly, such CD151 distribution in CHC liver samples mainly coincided with the site of HCV receptor colocalization reported by Reynolds et al. and with our own results related to the HCV-E2 protein binding pattern to the liver (Fig. 1A), which was more restricted than that observed for CD81. Furthermore, we also appreciated the presence of CD151 in the membrane domains that bind HCV-E2 envelope protein (Fig. 1B), which is in clear concordance to the recently reported tetraspanin–claudin-1 associations and with the known tetraspanin-tetraspanin associations. As shown for other TEM members, such as EWI-2 or CD19,11 the regulation of claudin-1 expression, stabilization, and location by tetraspanins could operate at the posttranslational level and be affected by HCV infection. Moreover, the CD151 depletion caused a notable destabilization of newly synthesized claudin-1, as was recently demonstrated.7 Once more, CD151 tissue expression cannot be used to explain HCV hepatotropism, but it accounts again for the implication of TEMs in HCV infection, especially when claudin-1 has been characterized as a TEM member. In this respect, the explanation for HCV tropism has arisen from the absence of hepatocyte expression of the CD81 partner Ewi-2wint, another TEM constituent, which has been shown to abolish HCV pseudo-particles (HCVpp) infection (Rocha-Perugini et al., unpublished data). Tetraspanin implication in HCV infection. (A) Immunohistochemical staining of tetraspanins CD81 and CD151 showed marked differences in their pattern of expression between control and HCV liver tissue samples. There was an evident redistribution of CD81 expression to whole hepatocyte membrane in CHC patients jointed to an increase of CD151, which was mainly related to sinusoidal poles of hepatocytes and vascular endothelium (Original magnification, x200). HCV-E2 binding to control or CHC liver specimens mimicked the CD151 staining pattern (Original magnification, ×250). Figure exemplifies representative staining experiment from different analyzed patients. (B) Tetraspanin immunoprecipitation experiments, prior recombinant HCV-E2 binding to Chang Liver lysates (2 μg/mL), showed the participation of CD151 in HCV-E2 binding (in addition to the previously described CD81) in either, tetraspanin complexes permissive lysis conditions (Brij96) or in lysis buffer conditions that disrupt tetraspanin complexes (Triton X-100). Representative experiments are illustrated. All these data together tempted us to speculate that the characteristic hepatocyte TEMs could represent a scaffold whereby the described HCV receptors, and probably other unknown factors, assemble space-temporally to form the active entry complex. Furthermore, by mediating tight-junction regulation, TEMs could also be involved in cell polarization control, epithelial-mesenchymal transition, and cancer progression, all processes that could be sensitively altered by HCV. Therefore, a detailed study of the constitution and functionality of hepatic TEMs could help in the better understanding of the HCV vital cycle and in its pathophysiological repercussions. Reagents provided by doctors F. Sanchez-Madrid and J. Dubuisson. Paloma Sanz-Cameno*, Samuel Martín-Vílchez*, Yolanda Rodriguez-Muñoz*, Ricardo Moreno-Otero*, * Digestive Diseases Service and CIBEREHD-Instituto de Salud Carlos III, Hospital Universitario de La Princesa, Universidad Autónoma de Madrid, Madrid, Spain.