Farquhar MJ, Hu K, Harris HJ, Davis C, Brimacombe CL, Fletcher SJ, Baumert TF, Rappoport JZ, Balfe P, McKeating JA.
Journal of Virology, 86:4305-4316, 2012.
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CD81 and claudin-1 are essential cellular factors for hepatitis C virus (HCV) entry however their precise role has yet to be determined. In this study we show that high affinity anti-CD81 monoclonal antibodies inhibit infectivity even after the virus has entered the cell, suggesting a role for intracellular CD81 in HCV infection. Confocal imaging reveals a limited pool of intracellular CD81 that, via endocytosis from the plasma membrane, increases following the addition of antibody.
We show that, in a manner analogous to HCV entry, CD81 endocytosis occurs in a clathrin and dynamin dependent manner. Association of CD81 with claudin-1 is important for entry of HCV (Harris, 2010) and this study reveals that co-endocytosis of CD81 and claudin-1 is promoted both by receptor specific antibodies and by viral particles. In addition we show that internalized CD81 and claudin-1 localize to Rab5 expressing early endosomes, the site of HCV fusion within the host cell. Our data supports a model where HCV stimulates trafficking of the CD81-claudin-1 receptor complex to promote particle internalization.
Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I and tight junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. In this study, we demonstrate that anti-CD81 antibodies inhibit HCV infection at late times post virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domain, however, CD81 lacks such motifs leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin and dynamin dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 co-endocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model where HCV stimulates receptor trafficking to promote particle internalization.
Figure 1. Anti-CD81 mAbs neutralize HCV post internalization.
(A) Schematic illustration of experimental design. (B) Time course of J6/JFH escape from proteinase K (PK), and from the anti-CD81 mAbs JS81 and 2s131. The time for J6/JFH to escape 50% of the neutralizing effects of PK and anti-CD81 mAbs (t1/2) was calculated relative to the maximum inhibition observed at 120 minutes.
PK stripping of virus from the cell surface shows the rate of internalization (t1/2 = 18 minutes in this experiment). By 40 minutes essentially all of the virus is internalized, yet addition of anti-CD81 mAb 2s131 at this time still is > 50% effective in neutralizing virus.
Figure 2. CD81 internalization.
(A) Total surface biotinylated protein (lane 1), biotin-labelled protein internalized at 4°C (lane 2) or at 37°C for 60 minutes in the presence of irrelevant mouse immunoglobulin (lane 3) or anti-CD81 2s66 (lane 4) were captured by streptavidin pulldown. Precipitates (P, upper panel) and whole cell lysates (WCL, lower panel) were subjected to SDS-PAGE and reactivity with anti-CD81 assessed by Western blotting. The data shown are representative of two separate experiments, from which the intracellular CD81 relative to total surface biotinylated CD81, as determined by densitometric analysis is determined. Results were compared using a Students T test (**p < 0.01). (B) Huh-7.5 cells expressing AcGFP.CD81 were incubated with control mouse immunoglobulin, equimolar amounts of 2s66 or Fab for 1 h at 37°C. After fixation, ten or twenty cells were imaged and intracellular CD81 fluorescence quantified as a percentage of total fluorescence ± SEM. Data were compared using a non-parametric ANOVA (Dunn’s test, Kruskal Wallis ANOVA, ***p < 0.001). We found no significant difference when 10 or 20 cells were analyzed.
Figure 3: CD81 internalization is clathrin and dynamin dependent.
Huh-7.5 cells expressing GFP-control, GFP-Eps15-EH29, or Dynamin2-K44A-GFP (transdomininant mutants of their respective proteins) were incubated with Alexa-594 labelled Transferrin (Tfn-594) or anti-CD81 2s131 (2 µg/ml) for 60 minutes at 37°C. Intracellular Tfn and CD81-Ab fluorescence in ten selected GFP-Eps15-EH29 and Dynamin2-K44A-GFP expressing cells was measured and presented relative to the intracellular fluorescence in control GFP expressing cells. Tfn and CD81 internalization relative to control was compared using a non-parametric ANOVA (Dunn’s test, Kruskal Wallis ANOVA, * p < 0.05, ** p < 0.01, ***p < 0.001).
Eps15 is a regulatory protein essential for the formation of clathrin coated pits, whereas Dynamin2 is necessary for the scission of vesicles from the plasma membrane. By repressing these two processes (which are known to be involved in Transferrin trafficking) with these transdominant mutants we can demonstrate that they have a role in CD81 trafficking.
Figure 4: HCV promotes CD81 and claudin-1 endocytosis.
Huh-7.5 cells expressing AcGFP.CD81 were mock infected (control) or infected with HCVcc J6/JFH, either untreated, heat inactivated, or pre-treated with an anti-HCV Ig or a control Ig. Cells were fixed and CD81 or claudin-1 detection performed. Intracellular protein/antibody fluorescence was quantified as a proportion of the total fluorescence ± SEM in a sample population of 10 cells, where each circle on the graphs represents an individual cell. Data were compared using a non-parametric ANOVA (Dunn’s test, Kruskal Wallis ANOVA, * p < 0.5, ** p < 0.01, *** p < 0.001). These data are representative of two other experiments which showed comparable levels of statistical significance.
Those cells in which infection is occurring show clear evidence of increased levels of intracellular CD81 and claudin-1.
Huh-7.5 cells were incubated with ATTO 647N labelled anti-CD81 2s66 (blue) and ATTO 565 labelled anti-claudin-1 (red), for 30 min at 37°C, washed and real-time imaging on a 1 μm Z section through the center of the cell performed over a period of 5 min (~4 frames/second). CD81 and claudin-1 can be seen trafficking at the plasma membrane prior to their endocytosis from the cell surface.
CD81-claudin-1 fusion with early endosomes.
Huh-7.5 cells expressing GFP-Rab5 (green) were incubated with 647N-anti-CD81 2s66 (blue) and 565-anti-claudin-1 (red) for 30min at 37°C, washed and real-time imaging of a 1 μm Z section through the center of the cell performed over a period of 5 min. Co-localization of all three fluorescence signals results in a white color, suggesting fusion with early endosomes.