Isolate-dependent use of claudins for cell entry by hepatitis C virus

Abstract

Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB-1), occludin (OCLN), and claudin-1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh-7.5) or CLDN6 (HuH6). Huh-7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6-dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1-specific neutralizing antibodies (Abs) fully abrogated infection of Huh-7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti-CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1-specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB-1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. Conclusion: These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1-specific therapeutic strategies. (HEPATOLOGY 2014;59:24-34) H epatitis C virus (HCV) is a highly variable, plus-strand RNA virus of the family Flaviviridae and a leading cause of liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. 1 The pronounced variability of HCV facilitates viral immune evasion and is attributable to enormous replication rate and error-prone RNA replication. Seven genotypes (GTs) and multiple subtypes are known, with genetic diversity being in the order of more than 30% between individual viral GTs. 2 Although the basic genome structure is conserved among HCV GTs, there are remarkable genotypedependent differences with regard to treatment response and pathophysiology of the infection. For instance, GTs 1 and 4 exhibit inferior response rates, when compared with GTs 2 and 3, in interferon-based therapy regimens, and GT3 virus infection shows a particularly strong association with liver steatosis. 3,4 Abbreviations: Abs, antibodies; CHC, chronic hepatitis C; CLDN, claudin; FACS, fluorescence-activated cell sorting; GTs, genotypes; HCV, hepatitis C virus; HCVcc, cell culture-derived HCV; HCVpp, HCV pseudoparticles; IgG, immunoglobulin G; miR-122, microRNA 122; MLV, murine leukemia virus; mRNA, messenger ...

Functional Analysis of Claudin-6 and Claudin-9 as Entry Factors for Hepatitis C Virus Infection of Human Hepatocytes by Using Monoclonal Antibodies

“…5C). Another study recently reported high levels of the main HCV entry factors but highly variable CLDN6 mRNA levels in liver biopsy specimens from HCV patients (17). Immunostaining of liver sections also demonstrated that, in contrast to CLDN6-positive seminoma, CLDN6 was not detected in human liver sections (Fig.…”

“…3 to 5). Interestingly, silencing of CLDN6 in Huh7.5 cells also did not affect HCV infection (17). Furthermore, these MAbs were unable to further decrease HCVpp entry into CLDN1-silenced Huh7.5.1 cells or when added simultaneously with the CLDN1-specific MAb to naive Huh7.5.1 cells, suggesting that CLDN6 and CLDN9 are not able to complement the absence of CLDN1 expression or blockage by the anti-CLDN1 MAb (Fig.…”

“…In contrast to previous reports (10, 11), we noted that HCV-JFH1 only infected 293T cells expressing CLDN1, suggesting that this strain cannot utilize CLDN6 or CLDN9. Interestingly, a recent study also reported that Huh6 cells, expressing CLDN6 but devoid of CLDN1, are resistant to HCVpp expressing genotype 2a glycoproteins in contrast to HCVpp of genotype 1 (17). Next, we assessed the ability of CLDN-specific MAbs to inhibit HCVpp entry into these cells by using a CD81-specific antibody as a positive control (18).…”

Apolipoprotein E Codetermines Tissue Tropism of Hepatitis C Virus and Is Crucial for Viral Cell-to-Cell Transmission by Contributing to a Postenvelopment Step of Assembly

“…Moreover, the functional relevance of each of these factors for HCV infection of human liver cells is supported by ample experimental evidence from different laboratories (8). Interestingly, CLDN6 and CLDN9 were shown to functionally substitute for a lack of CLDN1 in human nonliver cells (9)(10)(11). Therefore, these factors together with either CLDN1, -6, or -9 apparently make up the minimal requirements to render cells permissive for HCV entry.…”

Novel human anti-claudin 1 mAbs inhibit hepatitis C virus infection and may synergize with anti-SRB1 mAb

“…Two other members of the CLDN protein family (CLDN-6 and CLD-9) could be used alternatively for cell entry by those viruses with broad CLDN tropism (Haid et al, 2014) and thus could be used as additional targets for therapy. However, while some strains efficiently use CLDN-6 as well as CLDN-1, some other strains, such as JFH-1 and J6, solely use CLDN-1 to access cells and this can explain the efficacy of the above-mentioned anti-CLDN-1 antibodies.…”