Characterisation of endogenous Claudin‐1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock‐in cells

Abstract

Background Information. Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival.Results. Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1 +/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs.Conclusions. The Huh7.5.1 TagRFP-CLDN1 +/+ edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging.Significance. Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.

Occludin stalls HCV particle dynamics apart from hepatocyte tight junctions, promoting virion internalization

“…Next, we tested whether the EGFP-OCLN protein retained its functional features. Using a transwell system to assess for permeability coefficient as previously done (22,32), we found that the relative permeability of the wild-type or EGFP-OCLN edit cells did not show significant differences (Figure 1E), indicating that the addition of the EGFP tag does not affect the OCLN's impermeability function. As a control EDTA treatment was impairing the permeability coefficient of both cell lines.…”

“…Our extensive characterization of these cells showed that they retained similar features as their parental non-edited lineage, including permissiveness to HCVcc infection. Of note, tagging the CLDN1 entry factor using the same CRISPR-based approach led the cells to be fully resistant to the infection (22).…”

“…Of note, tagging the CLDN1 entry factor using the same CRISPR‐based approach led cells to be fully resistant to the infection. [ 22 ] Together, Huh7.5.1 EGFP‐OCLN edit cells represent a functional tool to interrogate for OCLN distribution over time as well as a reporter cell line for OCLN expression‐level variation.…”

“…To decipher the spatiotemporal dynamics and role of OCLN during HCV entry, we aimed to obtain a HCV permissive cell line expressing endogenous levels of fluorescent OCLN proteins, as EGFP-OCLN overexpression perturbs its distribution (28). To this end, the genome of Huh7.5.1 cells was edited using a CRISPR/Cas9 knock-in approach as previously described (22,29,30). Briefly, cells were transfected with a plasmid coding for the spCas9 protein, a DNA PCR product coding for a guide RNA (gRNA) targeting the ATG region of the ocln gene, and an EGFP "donor" plasmid providing the DNA template for homologous recombination (see Figure 1A and Material & Methods for details).…”

“…[28] To this end, the genome of Huh7.5.1 cells was edited using a CRISPR/Cas9 knock-in approach as described. [22,29,30] Briefly, cells were transfected with a plasmid coding for the spCas9 protein, a DNA PCR product coding for a gRNA targeting the ATG region of the ocln gene, and an EGFP "donor" plasmid providing the DNA template for homologous recombination (see Figure 1A and Materials and Methods for details). Upon sorting and clonal cell selection, a monoclonal Huh7.5.1 EGFP-OCLN edit cell line showed the EGFP sequence inserted on all alleles in the start codon region of the ocln gene (Figure 1B).…”