CLDN1 Knockout Cell Lines

Investigate the function of Claudin-1 (CLDN1) in hepatitis C virus (HCV) entry and its role as a critical co-receptor for viral infection, using CRISPR/Cas9-generated CLDN1 knockout (KO) cell lines.

1. HCV Entry and Host Receptors

HCV requires multiple host receptors for efficient entry into hepatocytes, including CD81, scavenger receptor class B type 1 (SR-B1), and tight junction proteins like Claudin-1 (CLDN1) and Occludin (OCLN). CLDN1 is a transmembrane protein that forms tight junctions, and its role in HCV entry has been suggested but not fully defined .

2. CLDN1 in Viral Tropism

CLDN1 expression is highly restricted to hepatic and certain epithelial cells, which correlates with HCV’s tropism for the liver. Previous studies using siRNA knockdown have shown reduced HCV entry upon CLDN1 depletion, but genetic KO models are needed for definitive mechanistic insights .

3. Gaps in Knowledge

Is CLDN1 essential for HCV entry, and does it act in conjunction with other receptors like CD81?

Can CLDN1 KO cell lines serve as a model to study viral-host interactions and therapeutic targets?

1. Generation of CLDN1 KO Cell Lines

Method: CRISPR/Cas9 genome editing was used to disrupt the CLDN1 gene in Huh7 human hepatoma cells. Two independent sgRNAs targeted exon 2 of CLDN1, and KO was confirmed by immunoblotting and DNA sequencing, showing complete loss of CLDN1 protein expression .

Validation: CLDN1 KO cells (CL-KO) were viable and maintained normal morphology, with no significant changes in lipid droplet distribution or cell viability compared to wild-type cells .

2. Functional Assays in CLDN1 KO Cells

HCV Entry Impairment: CL-KO cells showed a >100-fold reduction in HCVcc (genotype 2a JFH1) entry, as measured by intracellular HCV RNA levels and infectious titers in culture supernatants .

Receptor Redundancy: Co-knockout of CLDN1 and CD81 further abolished HCV entry, while single KO of CLDN1 or CD81 showed partial inhibition, indicating cooperative roles in viral entry .

Pseudotype Particle (HCVpp) Assays: CL-KO cells were resistant to HCVpp bearing HCV envelope proteins, confirming CLDN1’s role in the early entry step .

3. Mechanistic Studies

Co-receptor Interaction: CLDN1 co-immunoprecipitated with CD81 and SR-B1, forming a receptor complex critical for HCV attachment and internalization .

Tight Junction Integrity: CLDN1 KO disrupted tight junction structure, as shown by reduced transepithelial electrical resistance (TEER), but this did not directly correlate with HCV entry impairment, suggesting a specific role in viral receptor binding .

1. Mechanistic Insights

CLDN1 functions as an essential co-receptor for HCV entry through two key mechanisms:

Receptor Complex Formation: CLDN1 interacts with CD81 and SR-B1 to form a functional entry complex, enabling HCV particle attachment and fusion .

Host Tropism Determination: CLDN1’s hepatic-specific expression restricts HCV to liver cells, as non-hepatic cells expressing CLDN1 gain susceptibility to HCV infection .

2. Translational Significance

Drug Development: CLDN1 KO cells validate CLDN1 as a therapeutic target. Small-molecule inhibitors of CLDN1-HCV interaction (e.g., monoclonal antibodies) could prevent viral entry, especially in combination with other antiviral agents .

Viral Evolution Studies: CL-KO cells reveal that HCV strains with CLDN1-binding mutations may emerge under selective pressure, guiding the