We demonstrated that HCV entry is dependent on the target cell density, and this is most likely explained by increased CLDN1 and SR-BI expression in areas of cell-cell contact. CLDN1 and SR-BI demonstrated significantly elevated cell contact localization and expression in confluent cells (Fig. ), whereas mRNA levels were unaffected by the confluence status, suggesting posttranscriptional regulation of SR-BI and CLDN1 expression. In contrast, CD81 and OCLN demonstrated a uniform distribution at the plasma membrane of single cells, with no detectable enrichment at high density (Fig. ). Western blot analysis of total cell lysates confirmed that CD81 protein levels were unaffected by the confluence status. However, Huh-7.5 cells express relatively high levels of CD81, and we presume that modest changes in CD81 expression may be undetectable. These findings suggest that the Huh-7.5 cell density modulates CLDN1 and SR-BI protein expression and localization with possible consequences for HCV entry.
Cell surface expression of membrane proteins involves the sorting of proteins from the trans
-Golgi network to the plasma membrane domain and anchoring and/or retention at the cell surface, followed by endocytosis, transcytosis, or recycling (reviewed in reference 5
). Protein trafficking to the cell surface is largely mediated by peripheral membrane (PDZ domain) proteins, which interact with the C-terminal PDZ ligand binding domains of membrane proteins, including CLDN1 and SR-BI (23
). The mechanisms regulating membrane trafficking are incompletely understood; however, it has been demonstrated that adaptor proteins, such as PDZK1 and ZO-1 and -2, localize to sites of cell-cell contact and interact directly with CLDN1 and OCLN (14
There have been reports of CLDN1 cell contact localization in human, murine, and canine tumor and nontumor cell lines (16
). Indeed, human breast cancer cells display cell density-specific modulation of CLDN1 expression independently of mRNA levels. Hepatic SR-BI expression is posttranslationally regulated by the adaptor protein PDZK1 (27
); furthermore, by stabilizing SR-BI plasma membrane expression, PDZK1 determines SR-BI localization and function within the liver (42
SR-BI and CD81 surface expression levels have been reported to limit HCV infection (18
). Consistent with these findings, HCVpp demonstrated a significant increase in luciferase activity at cell confluence, when cell-cell contact was established between cells in the culture (Fig. ). The same effect was observed for HCVcc infection, with JFH-1 and J6/JFH infection increasing significantly when cellular contact was established (Fig. ). To assess whether cellular contact promoted the internalization of cell-bound HCVcc, we examined the ability of an anti-E2 antibody to inhibit infection of subconfluent and confluent cells when added at various times during the entry process. Previous reports have demonstrated that HCVpp and HCVcc escape the inhibitory activity of antibodies targeting HCV E2, CD81, and SR-BI approximately 17 to 20 min after entry initiation, consistent with the internalization rates reported in this study (3
). Interestingly, Meertens and colleagues reported that half-maximal HCVpp fusion requires 73 min, as determined by sensitivity to bafilomycin A (34
), suggesting a significant delay in virus internalization and subsequent fusion with early endosome membranes. We note that the inhibitory activity of C1 was reduced earlier at cell confluence than at subconfluence, with half-maximal inhibition being attained at 15 min and 30 min, respectively (Fig. ). Moreover, we observed that HCVcc internalization in subconfluent and confluent cells differed significantly during the first 30 min of the entry process and converged thereafter, suggesting that cellular contact modulates an early step in HCV internalization.
To further dissect the role(s) of CLDN1 and SR-BI in cell contact-mediated modulation of HCV entry, we studied HCVcc internalization in subconfluent Huh-7.5 cells transduced to overexpress CLDN1 or SR-BI. Cells transduced to overexpress CLDN1 were no more susceptible to JFH-1 HCVcc than parental cells (Fig. ); furthermore, CLDN1 overexpression had no detectable effect on virus internalization (Fig. ), despite a significant increase in cell contact expression levels. Taken together, these data suggest that CLDN1 expression levels do not limit the frequency or rate of HCVcc internalization into Huh-7.5 cells. CLDN1 is proposed to act at a late postbinding stage of viral entry (12
). Several reports have demonstrated that mutation or deletion of a domain(s) required for CLDN1 internalization has minimal effect on HCV entry (9
), suggesting that CLDN1 internalization per se is not essential for HCV particle entry. Our recent data suggest that CLDN1 may interact with CD81 and modulate HCV E1E2 interaction(s) and lateral particle trafficking to membrane domains amenable to internalization (H. Harris and M. Farquhar, unpublished data).
SR-BI overexpression enhanced HCV internalization (Fig. ), consistent with a model in which elevated SR-BI expression levels enhance HCV internalization in confluent cells. Silencing SR-BI expression reduced the infectivity of HCVcc (49
) and HCVpp bearing E1E2 glycoproteins from different genotypes (30
), while the neutralizing activity of patient sera increased as a result of low SR-BI levels (46
). Concomitantly, we observed that SR-BI overexpression promoted HCVpp entry and “attenuated” the neutralizing activity of MAbs and polyclonal antibodies (reference 18
and data not shown). These observations suggest that SR-BI expression levels not only modulate Huh-7.5 permissivity per se (Fig. ) (18
), but are rate limiting for HCVcc particle internalization (Fig. ). Compared to other enveloped viruses, HCVcc internalization is slow, which may reflect the time required to form higher-order protein complexes between CD81, SR-BI, CLDN1, and OCLN (10
). High SR-BI surface expression may facilitate the assembly of these complexes, promoting the internalization of cell-bound particles. Of note, the anti-E2 MAb (C1) used to neutralize surface-bound virus particles (Fig. , , and ) is reported to act by inhibiting E2-CD81 interactions (31
) and yet retains its neutralizing capacity after particle attachment (Fig. ). This is consistent with other observations that CD81 acts at a stage following the primary interaction of HCV particles and target cells (12
Persistence is a hallmark of HCV infection and is attributable to the ability of the virus to evade host cellular and humoral immune responses. The mechanism of action of HCV-specific nABs is incompletely understood; however, there have been several reports that antibodies inhibit HCV attachment to the cell surface by targeting the CD81 binding site and hypervariable region of glycoprotein E2 (1
). During viral entry, both regions need to be accessible to their cellular binding partners (6
) and may become transiently exposed to nAbs; consequently, the rate at which a virus particle internalizes may define its sensitivity to nAbs. Hepatocytes, which are the main reservoir of HCV replication, are tightly packed; furthermore, we have previously demonstrated high levels of SR-BI in healthy human liver (41
). It is interesting to speculate that the internalization rates and nAb escape we have observed in confluent Huh-7.5 cells, with high SR-BI and CLDN1 expression, may better represent hepatocytes in the liver. If this is indeed the case, the rate of internalization of virus particles in vivo may be rapid, offering further explanation for the ability of HCV to prevail in the face of a humoral immune response.