Helen J Harris, Christopher Davis, Jonathan G L Mullins, Ke Hu, Margaret Goodall, Michelle J Farquhar, Christopher J Mee, Kitty McCaffrey, Stephen Young, Heidi Drummer, Peter Balfe and Jane A McKeating.
Journal of Biological Chemistry, 285:21092-102. 2010.
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Current evidence suggests that CD81, SR-BI and tight junction proteins Occludin and CLDN1 (plus CLDN6 and 9) are critical factors defining HCV entry. We have previously shown that CLDN1 associates with CD81 in a variety of cell types. To ascertain the role of these complexes in HCV entry, we investigated the relationship between various members of the CLDN family and CD81 by employing FRET and stoichiometric imaging methodologies.
Only CLDNs 1, 6, 9 and 12 co-localized with CD81 in a defined organization at the plasma membrane, consistent with a 1:1 molar relationship or stoichiometry, FRET studies confirmed these interactions for CLDNs 1, 6 and 9, but not 12. In contrast, several members of the CLDN family associated with Occludin, independent of viral receptor activity.
Mutation of residues 32 and 48 in CLDN1 ablated its association both with CD81 and viral receptor activity. Importantly, mutation of the same residues in receptor inactive CLDN7 resulted in a protein which associated with CD81 and facilitated viral entry into 293T cells.
The main site of HCV replication is the human liver, where the primary site of infection is the highly polarized Hepatocyte. Since CLDNs associate with other proteins to form tight junctions in polarized cells we investigated the location of CLDN1-CD81 complexes in polarized hepatocyte cell line, HepG2. CLDN1-CD81 complexes were detected at the basolateral surface of polarized HepG2 cells but were absent from apically located tight junctions. This observation is consistent with a model where virus engagement of CLDN1-CD81 at the basolateral surface initiates the particle internalization process.
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multi-step process involving tetraspanin CD81, scavenger receptor class B member I (SR-BI) and the tight junction proteins Claudin-1 and Occludin. CD81 and SR-BI interact with HCV encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there is minimal data supporting Claudin-1 association with HCV particles, raising questions on its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by FRET and imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
Figure 1: Analysis of CLDN-CD81 interactions. 293T cells were made to express a red-tagged CD81 (DsRED-CD81, r.CD81) with a diverse panel of green tagged CLDNs (AcGFP-CLDN, g.CLDN). The association of r.CD81 with each g.CLDN was evaluated by regression analysis. Representative scatter plots illustrating the relationship for each r.CD81-g.CLDN pair are shown. The modest association seen for CLDN12 was not confirmed by FRET (which detects proteins < 10nm apart).
Only the known HCV receptors, CLDN1, CLDN6 and CLDN9 show any real association with CD81.
Figure 2: Effect of mutations in CLDN1 and CLDN7 EC1 on CD81 and Occludin association. 293T cells were transfected to express AcGFP (g) and DsRED (r) fluorescent tagged wild type and mutant forms of g.CLDN and r.CD81 (A) or r.Occludin (B) and the degree of association between fluorophore tagged proteins assessed by FIR and FRET analysis. (C) 293T cells were transfected with AcGFP and DsRed tagged versions of wild type and mutant CLDN constructs to assess the effect of EC1 mutations on CLDN-CLDN cis-interactions. Median FIR and FRET values from ten individual cells are presented.
Receptor inactivating mutations of CLDN1 lost association with CD81, whereas the complementary activating mutations in CLDN7 gained CD81 association.
Figure 3: Effect of cell polarization on CLDN1-CD81 and CLDN1-CLDN1 association. HepG2 cells transfected to express AcGFP (g) and DsRED (r) fluorescent tagged g.CLDN-r.CD81 and g.CLDN1-r.CLDN1 were allowed to polarize over a period of three days. Apical bile canalicular structures were identified by staining with anti-ZO-1 and visualized with an alexa-633 (red) conjugated secondary anti-rabbit Ig (A). Representative scatter plots of g.CLDN-r.CD81 and g.CLDN1-r.CLDN1 at the basolateral surface (B) and tight junction (C) are shown.
FRET occured between g.CLDN1-r.CLDN1 at both basolateral and tight junctions. Basolateral membrane pools of g.CLDN1-r.CD81 demonstrated a 25% FRET, in contrast r.CD81 was largely excluded from tight junctions, with no detectable CLDN1-CD81 complexes (<1% FRET).
In summary, CLDN1 homotypic association(s) were comparable at the basolateral and tight junction membranes of polarized HepG2 cells, however g.CLDN1-r.CD81 association was not detected in the tight junction domains of HepG2 cells. These data are consistent with our previous report (click here) demonstrating a role for basolateral pools of CLDN1 in HCV infection.