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. 2011 Aug;85(15):7613-21.
doi: 10.1128/JVI.00212-11. Epub 2011 Jun 1.

Impact of Intra- And Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

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Free PMC article

Impact of Intra- And Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

Sandra Ciesek et al. J Virol. .
Free PMC article

Abstract

Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN(low) subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN(low) cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.

Figures

Fig. 1.
Fig. 1.
HCVpp can utilize human OCLN variants for efficient cell entry. (A) Schematic representation of OCLN, with stars indicating the positions of amino acid changes caused by known nonsynonymous SNPs in the occludin coding region and circles indicating the positions of two alanine residues that are replaced by glycine in the mouse homologue and responsible for the inferior (co)receptor function of murine OCLN in HCVpp assays. (B) Flow cytometric analysis of GFP-expression in 786-O cells transduced to express GFP-tagged wild-type OCLN, human OCLN variants, and CLDN1 at the time when susceptibility to HCVpp entry was assessed. (C, D) Above-mentioned 786-O cells expressing OCLN variants were challenged with envelope-deficient pseudoparticles (NoEnv), HCVpp strain H77, and VSV-Gpp. Cells were lysed, and luciferase activity was determined 3 days after infection. NoEnv (C) and HCV (D) infectivity relative to VSV-G infectivity (mean ± standard deviation) in a representative experiment, with 3 replicates per condition, is shown, with the value determined in mock-transduced cells set to 1. Statistically significant (P < 0.05) differences from mock-transduced cells are indicated by asterisks. ECS, extracellular space; ICS, intracellular space; wt, wild type.
Fig. 2.
Fig. 2.
Characterization of clone#1, an OCLNlow yet HCV replication- and assembly-competent Huh-7.5 subclone. (A) Immunoblot for OCLN and beta-actin in parental Huh-7.5 cells, Huh-7.5 cells expressing an irrelevant shRNA (sh-irrelevant) or occludin-specific shRNA (sh-OCLN), and clone#1, a subclone from the Huh-7.5/occludin-specific shRNA population chosen for subsequent experimentation. (B) Immunoblot for CLDN1 and beta-actin in Huh-7.5 cells expressing an irrelevant shRNA and clone#1. (C) Parental Huh-7.5 and clone#1 cells were inoculated with supernatant containing Jc1-Luc reporter virus. Luciferase activity was assayed 48 h postinfection. Mean values and the standard deviations from a representative experiment performed in triplicate are shown. (D) Parental Huh-7.5 and clone#1 cells were inoculated with HCVpp and VSV-Gpp. HCVpp infectivity relative to VSV-Gpp infectivity, with the value detected in Huh-7.5 cells set to 100% (means and standard deviations of 3 replicates), is shown. (E) Parental Huh-7.5 (open squares) and clone#1 (closed squares) cells were electroporated with Jc1-Luc RNA transcripts, and luciferase activity over the following 72 h was monitored as indicated. Mean values from a representative experiment done in duplicate are shown. (F) To assess efficiency of viral particle production, supernatant from Huh-7.5 and clone#1 cells electroporated with Jc1-Luc RNA transcripts was collected after 72 h, filtered, and used to inoculate naïve Huh-7.5 cells. Luciferase activity was assessed 48 h after inoculation. “Mock” indicates the background luciferase reading detected in uninfected cells. Mean values and the standard deviations from a representative experiment done in quadruplicate are shown. Significant differences from parental Huh-7.5 are indicated by asterisks. RLU, relative light units.
Fig. 3.
Fig. 3.
OCLN overexpression does not alter HCV permissivity of Huh-7.5 cells. (A) Immunoblot for OCLN and beta-actin in parental Huh-7.5 cells and Huh-7.5 cells transduced to overexpress OCLN-GFP (OCLNhigh). (B) Parental Huh-7.5 and OCLNhigh cells were inoculated with supernatant containing Jc1-Luc reporter virus. Luciferase activity was assayed at 48 h postinfection. Mean values and the standard deviations from a representative experiment performed in triplicate are shown. (C) Parental Huh-7.5 (open squares) and OCLNhigh (closed triangles) cells were electroporated with Jc1-Luc RNA transcripts, and luciferase activity over the following 72 h was monitored as indicated. Mean values from a representative experiment done in duplicate are shown. (D) Supernatant from Huh-7.5 and OCLNhigh cells electroporated with Jc1-Luc RNA transcripts was collected after 72 h, filtered, and used to inoculate naïve Huh-7.5 cells. The mock value represents background luciferase readings in uninfected cells. Luciferase activity was assessed 48 h after inoculation. Mean values and the standard deviations from a representative experiment done in duplicate are shown.
Fig. 4.
Fig. 4.
HCVcc can utilize human OCLN variants for efficient cell entry. (A) Flow cytometric analysis of GFP expression in clone#1 cells transduced to express GFP-tagged wild-type OCLN, human OCLN variants, and CLDN1 at the time when susceptibility to HCVcc was assessed. (B) Above-mentioned clone#1 cells expressing OCLN variants were challenged with genotype 2a Jc1-Luc HCVcc, and luciferase activity was measured 48 h after inoculation. Mean values and the standard deviations from three independent experiments each done in duplicate are shown. Significant differences from mock-transduced cells are indicated by asterisks. (C) Transgene-expressing cells were challenged with genotype 1a/2a (structural region of genotype 1a) H77/JFH1 chimeric HCVcc in a standard limiting dilution assay (12 dilution steps, 8 wells per dilution). Cells were fixed and stained for NS5A expression 48 h after inoculation, and the TCID50/ml was determined. Results from a representative experiment are shown.
Fig. 5.
Fig. 5.
HCVcc can utilize murine OCLN for cell entry with reduced efficiency. (A) Flow cytometric analysis of Venus expression in clone#1 cells transduced to express Venus-tagged human or murine OCLN variants as well as chimeric OCLN with residues 223 and 224 exchanged between the human and murine homologues at the time when susceptibility to HCVcc was assessed. (B, C) Above-mentioned clone#1 cells expressing the respective OCLN variants were challenged with genotype 2a Jc1-Luc HCVcc (B) and a Jc1-Luc variant adapted to utilize murine CD81, with efficiency equal to that of human CD81 (C) (4). Luciferase activity was measured 48 h after inoculation. Mean values and the standard deviations from at least three independent experiments each done in duplicate are shown. Significant differences from mock- and human OCLN (hOCLN)-transduced cells are indicated by asterisks and pound signs, respectively. mOCLN, murine OCLN.
Fig. 6.
Fig. 6.
Cell-to-cell spread of HCV is undetectable in the absence of OCLN. (A) As detailed in the text, a fully H77/JFH1-infected population of nonfluorescent Huh-7.5 donor cells was put in coculture with uninfected target cells (parental Huh-7.5 [left] or clone#1 [right]) harboring a tagRFP-NLS-IPS reporter (22). Briefly, in uninfected cells tagRFP fluorescence is localized in the cytoplasm in a punctuate mitochondrial pattern; upon HCV infection, the tagRFP-NLS is cleaved from its IPS anchor in the mitochondrial membrane, and fluorescence relocates to the nucleus driven by the NLS fused to the tagRFP. Nuclei were costained with DAPI (4′,6-diamidino-2-phenylindol). (B) Percentage of infected cells, as indicated by nuclear localization of tagRFP per well after 96 h of coculture using as target cells Huh-7.5, clone#1, or clone#1 expressing different OCLN variants. Means and standard deviations from a representative experiment done in duplicate are shown, and significant differences from parental Huh-7.5 are indicated by asterisks.

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