Splicing diversity of the human OCLN gene and its biological significance for hepatitis C virus entry

Abstract

Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.

FIG. 1.

Genomic organization of the human 15q13.2 region. (A) The map shows the full-length OCLN gene with exons 1 to 9 and an inverted partly duplicated OCLN pseudogene (LOC647859) that includes exons 5 to 9. The HCV-binding MARVEL domain encoded by exons 3 and 4 of OCLN is underlined. (B) Structure of OCLN protein isoforms. Protein domains, exons, and translation start and stop sites are indicated. AA, amino acid.

FIG. 2.

Protein expression of OCLN in human liver. Western blot showing expression pattern of OCLN protein isoforms in normal human liver samples. The observed OCLN protein isoforms are matched with their expected protein sizes (52.7 to 59.1, 31.6, and 23.3 kDa).

FIG. 3.

Analysis of subcellular localization of recombinant OCLN protein isoforms. (A) Confocal imaging of OCLN-HaloTag constructs transiently transfected into HeLa cells. Untransfected cells in the same field serve as negative controls. Antibody against α-tubulin was used to stain the cytoskeleton (green), while DAPI was used to stain the cell nuclei (blue). Images are obtained at 63× magnification with immersion oil. (B) Schematic representation of OCLN protein isoforms. EC1 and EC2 mark extracellular parts of the OCLN protein.

FIG. 4.

Splice forms of human OCLN have different abilities to mediate HCV entry. Human 786-O cells were mock transduced or transduced with human pTRIP-TagRFP-OCLN splice variants as indicated and then challenged in parallel with HCV and control VSV-G pseudoparticles (HCVpp and VSVGpp, respectively). HCVpp infectivity is reported as the titer of HCVpp divided by the titer of VSVGpp, after subtraction of the signals from infection with nonenveloped pseudoparticles (Env-pp). HCVpp infectivity is normalized to the signal in Huh-7.5 cells. Means and standard deviations from at least triplicate experiments are shown. wt, wild type.

FIG. 5.

Expression of alternative splice forms of human OCLN does not significantly affect HCV entry. Human 786-O cells were mock transduced or (co)transduced with human Venus/YFP-WT-OCLN or TagRFP-OCLN splice variants as indicated and then challenged in parallel with HCV and control VSV-G pseudoparticles (HCVpp and VSVGpp, respectively). HCVpp infectivity is reported as the titer of HCVpp divided by the titer of VSVGpp, after subtraction of the signals from infection with nonenveloped pseudoparticles (Env-pp). HCVpp infectivity for the 786-O cell populations expressing the wild type or the splice variants at the indicated mean fluorescent intensities (MFIs) is normalized to the signal in Huh-7.5 cells. Means and standard deviations from at least triplicate experiments are shown. N/A, not applicable.