When one receptor closes, another opens: Claudins and the hepatitis C virus E1 glycoprotein
Authors: Che C. Colpitts, Mirjam B. Zeisel, Thomas F. Baumert
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 1059. Hepatitis C virus (HCV) entry is a complex, highly orchestrated process mediated by the viral envelope glycoproteins, E1 and E2. Indeed, both HCV envelope glycoproteins interact with heparan sulfate proteoglycans, and HCV E2 is well documented to bind to two cellular factors, CD81 and scavenger receptor class B type I (SR‐BI). In contrast, the roles and binding partners of E1 are largely unknown. Similarly, although many host cell factors are involved in HCV entry, their specific contributions to HCV entry remain enigmatic. The tight junction (TJ) proteins, claudin (CLDN)−11 and occludin (OCLN), are among these factors. CLDN1 is thought to mediate HCV entry by involvement in coreceptor complex formation with the tetraspanin, CD81.2,3 CLDN1, as an integral membrane protein, has two extracellular loops (ELs). A hydrophobic motif in EL1 that is highly conserved among claudin family members has been shown to mediate HCV entry.1 However, although HCV envelope glycoproteins have been shown to coimmunoprecipitate with CLDN14 and contribute to HCV pseudoparticle binding to CLDN1‐expressing cells,5 the potential mechanism of interaction between HCV particles and CLDN1 remains elusive. It is known that viral determinants dictate CLDN receptor usage. Whereas all HCV genotypes use CLDN1 as an entry factor, certain genotypes are capable of using CLDN6 and CLDN9.6‐9 However, genotype 2, 5, and 7 strains appear to depend entirely on CLDN1 for entry.7 Infection of Huh7‐derived cell lines by strains with broad CLDN tropism could be only partially inhibited by CLDN1‐specific antibodies,7 suggesting that escape from CLDN1‐specific antibodies by CLDN6 or CLDN9 receptor usage is possible for these genotypes. However, it is still unknown how escape occurs. Thus, an enhanced understanding of the CLDN1‐mediated entry process will be helpful to advance CLDN1‐directed therapies. In this issue of Hepatology, Hopcraft and Evans took advantage of CRISPR‐Cas9 genome engineering to generate CLDN1‐knockout (KO) Huh7.5 cells and investigate how CLDN1 mediates HCV entry.10 The CLDN1‐KO cells, which provided a strong selection pressure for mutant viruses with altered receptor usage, were used to select for an HCV genotype 2 variant capable of infecting CLDN1‐deficient cells. Importantly, CLDN1‐KO cells fully supported HCV replication and infectious virus release, confirming that the role of CLDN1 as a host factor is limited to viral entry. These cells were transfected with HCV genotype 2a (Jc1) to initially overcome the entry block. Interestingly, continuous passage of HCV Jc1 in these cells resulted in the selection of a virus with a single‐amino‐acid substitution in the E1 glycoprotein (H316N) capable of infecting CLDN1‐deficient cells. These data provide genetic evidence for a functional interaction between HCV E1 glycoprotein and CLDN. To investigate the entry mechanisms of the H316N mutant virus, the investigators used antibody studies and further CRISPR/Cas9 genome editing. A CLDN6‐specific antibody blocked entry of the H316N mutant, implying that it used CLDN6 as an entry factor instead of CLDN1. Furthermore, a combination of CLDN1 and CLDN6 antibodies inhibited infection by the H316N mutant more potently than the CLDN1‐specific antibody alone, further corroborating this finding. The H316N mutant virus, but not wild‐type (WT) Jc1, could also infect HuH6 human hepatoblastoma cells, which lack CLDN1 but express CLDN6.7 In contrast, CLDN1 and CLDN6 double‐KO Huh7.5.1 cells did not support entry by either the Jc1 WT or H316N virus. However, exogenous expression of CLDN9 restored the cells' susceptibility to the Jc1 H316N virus, but not to the WT virus. Interestingly, the H316N mutation, when introduced into a CLDN1‐dependent genotype 5 HCV strain, also rendered it capable of utilizing CLDN6 for entry, indicating that this mechanism of viral adaptation to use alternate entry factors may not be restricted to HCV genotype 2. However, whether this mutation may arise in HCV genotype 5 through cell culture adaptation remains to be determined. Overall, the H316N substitution confers the ability of previously CLDN1‐dependent viruses to utilize CLDN1, CLDN6, or CLDN9 for entry (Fig. 1). Furthermore, the H316N mutant virus was only slightly less fit than the WT virus, which may have clinical implications for antiviral therapy targeting CLDN1.Figure 1: Model of CLDN1 altered receptor use by Hopcraft and Evans.10 A single‐amino‐acid substitution in E1 renders previously CLDN1‐dependent HCV genotype 2 capable of using CLDN6 for cell entry.The simplest interpretation of these data is a direct physical interaction between E1 and CLDN, in which E1 H316 interacts with CLDN1 whereas E1 N316 is capable of interaction with CLDN1, CLDN6, and CLDN9. This corroborates the finding that CLDN1 can coimmunoprecipitate with E1 and E2.4 Along the same lines, subtle amino acid differences between CLDN1 and CLDN6/9 dictate receptor usage. Nine residues within the EL1 domain of CLDN1 are important for HCV entry: W30, I32, D38, EGLW51, C54, and C64.11 Of these, CLDN1 and CLDN6/9 differ in only two amino acids: I32 in CLDN1 is replaced by V32 in CLDN6/9, whereas D38 in CLDN1 is replaced by N38 in CLDN6/9. Recent work by Douam et al. supports the direct interaction model, given that E1 dictated the ability of HCV pseudoparticles to bind to CLDN‐expressing cells.5 Alternatively, E1 mutations could influence the association of E1 with E2, or indirectly influence interactions of E2 with host factors. Collectively, these data add another piece to the puzzle of how E1 may interact with CLDN1 during HCV entry. Of note, Hopcraft and Evans show that a CLDN1‐dependent strain (Jc1) is able to evolve to use CLDN6 or CLDN9 as a receptor. This is interesting because it demonstrates that the virus is able to change its host factor dependency under selection pressure. Viruses have been shown to evade antiviral immune responses or antiviral therapies targeting viral proteins, such as the polymerase or protease. In this study, Hopcraft and Evans extend this concept to the use of viral host dependency factors. CLDN1 has been uncovered as a therapeutic target, and several studies have shown that CLDN1‐specific peptides and antibodies have a marked antiviral activity.12 Moreover, it was recently demonstrated that a monoclonal antibody directed against CLDN1 cured HCV infection in a humanized mouse model without detectable resistance.12 Given that CLDN1 escape was not easily detected in human liver‐chimeric mice using the same strain as described in Hopcraft and Evans, it is likely that the low expression levels of CLDN6 and CLDN9 in human hepatocytes could preclude their efficient use as entry factors in vivo. Indeed, CLDN6 messenger RNA expression in liver of infected patients is highly variable,7 whereas protein expression was not easily detected in liver sections.9 Further studies in novel HCV animal models and ultimately in HCV‐infected patients are needed to determine the impact of CLDN1 escape for potential antiviral therapies targeting host cell entry factors. Collectively, the work of Hopcraft and Evans contributes significantly to the understanding of the molecular mechanism of viral entry and highlights the remarkable ability of HCV to adapt during the entry process.