Temporal analysis of hepatitis C virus cell entry with occludin directed blocking antibodies.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization

Characterization of OCLN FLAG insertional mutants.

<p>(A) Amino acid sequence of human OCLN EC1 and EC2 sequences with the location of each FLAG epitope insert marked. (B) 786-O cells were transduced with lentiviruses to express either GFP alone, wild type (WT) OCLN, or the indicated FLAG insertion OCLN mutants. These cell populations were then expanded and analyzed by FACS for expression of GFP, which is fused to each OCLN protein, (dark gray) and for OCLN expression after staining permeabilized cells with an OCLN antibody (light gray). (C) Lysates from these cells were probed by immunoblotting with either OCLN or β-actin specific antibodies. Approximate molecular weight (kDa) marker positions are indicated to the left of each blot. (D) To probe for cell surface accessibility of the FLAG epitope, transduced cells were fixed and nonpermeabilized cells were stained with the FLAG M2 monoclonal antibody. (E) These cells were then challenged with H77 genotype 1a HCVpp to determine their capacity to support HCV cell entry. ‘Relative infectivity’ refers to luciferase values measured two days post infection normalized to parallel VSVGpp infections, to control for variations in cell number, and set relative to luciferase values for infections of cells expressing wild type human OCLN. *<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001 (Mann-Whitney test).</p

Analysis of the timing of entry factor usage during HCV cell infection.

<p>Synchronized infections of OCLN EC2-F5 mutant transduced (A+B) HepG2+miR-122+CD81 cells that express an shRNA to silence endogenous OCLN expression or (C) 786-O cells. Cells were challenged with either (A+C) H77 1a HCVpp or (B) S52 3a HCVcc. To probe the relative timing of each step in the entry process, the antibodies or drugs specified on the x-axes were added at the indicated times relative to the shift of the culture temperature to 37°C. For each inhibitor or antibody, results are relative to infection when inhibitor was added 240 min post temperature shift, at which point inhibition was no longer observed for any of the inhibitors. (D–F) To directly compare the kinetics of action of inhibitors that acted post-binding, the results from the graphs on the left were further normalized for each inhibitor to the amount of infectivity that occurred when that inhibitor was most and least active, which are set to 100% and 0% inhibition, respectively. Variable slope (CD81 and CLDN1) and Boltzmann (OCLN and BafA1) sigmoidal curves were fit to the % inhibition points and the equations for these curves were used to calculate the time of half maximal inhibition (t1/2) shown on the right. **<i>P</i><0.01, *** <i>P</i><0.001 (Mann-Whitney test).</p

OCLN directed FLAG antibody inhibition is independent of endocytosis induction.

<p>(A) Subcellular localization, assessed by confocal microscopy, of wild type or EC2-F5 OCLN-GFP fusion proteins in 786-O cells following a two hour 37°C incubation in the presence of either 10 µg/ml isotype control, FLAG antibody, or FLAG Fab, as indicated. In each set, blue, green, and red represent Hoechst 33342 nuclear DNA staining, GFP fluorescence, and Alexa Fluor 594 wheat germ agglutinin (WGA) staining of the cell membrane, respectively. To the right of each set is a graph showing the quantification of GFP (green line) and WGA (red line) signal intensity across a representative cell cross section, indicated by the white line in the corresponding merged panel. A representative example of greater than three independent experiments is shown. (B) 786-O cells expressing either the EC2-F5 or F3 OCLN mutant were incubated with 10 µg/ml of FLAG M2 antibody for 15 min at 4°C. Antibody was then washed off, the temperature shifted to 37°C, and at the indicated time points cells were fixed and stained with secondary antibody for FACS analysis to determine surface accessible primary antibody. (C) 786-O cells expressing either wild type or the OCLN EC2-F5 mutant OCLN were challenged with HCVpp in the absence of antibody (white), or in the presence of FLAG M2 IgG (dark gray) or Fab (light gray). **<i>P</i><0.01, *** P<0.001 (Mann-Whitney test).</p

Mutant OCLN cell entry factor activities differ between HCV isolates.

<p>(A–G) HepG2+miR-122+CD81 cells that express an shRNA to silence endogenous OCLN expression or (H–K) 786-O cells were transduced to express either shRNA-resistant wild type OCLN (WT), GFP alone, or shRNA-resistant OCLN with the indicated FLAG insertional mutations and challenged with the indicated viruses in the absence of antibody (dark), in the presence of the M2 FLAG antibody (light), or, as positive inhibition controls, either an E2 monoclonal antibody, for HCVpp, or the HCV polymerase inhibitor 2′CMA, for HCVcc (white). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003244#s2" target="_blank">Results</a> are normalized to infection of cells expressing wild type OCLN for each virus in the absence of antibody. Gray boxes highlight mutants that exhibited different entry factor activities between isolates. *<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001 (Mann-Whitney test). Black asterisks represent statistically significant differences between the indicated value and cells expressing GFP alone. Light gray asterisks represent statistically significant differences between infections of the same cells without and with antibody.</p

The OCLN EC2-F5 mutant can complement HCV entry and tight junction defects in OCLN silenced HepG2 cells.

<p>(A) Immunoblots for either OCLN or β-actin of lysates from HepG2+miR-122+CD81 cell populations transduced to express either no target (NT) or OCLN specific shRNAs. Approximate molecular weight (kDa) marker positions are indicated to the left of each blot. (B) These cells were either mock transduced (Mock), or transduced to express either shRNA-resistant wild type (WT) or the EC2-F5 OCLN mutant along with either NT or OCLN specific shRNAs, and then challenged with either HCVpp (dark) or HCVcc (light). ‘Relative infectivity’ refers to values that are normalized to parallel VSVGpp infections, to control for variations in cell number, and set relative to infections of cells expressing wild type human OCLN. (C) To analyze tight junction formation, cells transduced as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003244#ppat-1003244-g004" target="_blank">Figure 4B</a> were grown for three days on collagen coated coverslips, and then stained for the bile canalicular marker MRP2 (red) or an isotype control and with Hoechst 33342 nuclear DNA stain (blue). A representative example of three independent experiments is shown. **<i>P</i><0.01, *** P<0.001 (Mann-Whitney test).</p

OCLN directed FLAG antibody inhibits HCVpp infection of 786-O cells.

<p>(A) Illustration of OCLN membrane topology. The region flanked by two conserved cysteine residues (C216 and C237) that was previously shown to be essential for HCV entry is marked by a dotted line and a star highlights the location of species-specific determinants of OCLN entry factor activity. F2–F5 indicate the location of the EC2 FLAG insertions that did not disrupt HCV cell entry factor activity. (B) 786-O cells expressing wild type OCLN, GFP alone, or the indicated FLAG insertion OCLN mutants were challenged with H77 genotype 1a HCVpp in the presence of 10 µg/ml of an irrelevant isotype control (dark gray) or the FLAG M2 monoclonal antibody (light gray). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003244#s2" target="_blank">Results</a> were normalized to parallel VSVGpp infections and set relative to infection of cells expressing wild type OCLN. (C) To demonstrate the specificity and dose dependence of FLAG antibody inhibition, 786-O cells expressing either the OCLN EC2-5 mutant or wild type OCLN were challenged with HCVpp in the absence of antibody (white), or in the presence of the indicated concentration of either isotype control (dark gray) or FLAG antibody (light gray). *** P<0.001 (Mann-Whitney test).</p