HCV virions, lipid-enveloped nucleocapsids bearing the viral glycoproteins E1 and E2, appear to enter a host cell in a highly coordinated process involving components of the virus particle and numerous cellular factors7
. From the long list of putative HCV entry factors, strong evidence supports specific roles for the tetraspanin CD814
, and the tight junction protein CLDN16
. This list appears incomplete, however, as numerous human cell lines and all non-primate cell lines are resistant to HCV entry even when all three human factors are overexpressed3,6
(and data not shown).
To identify additional factors able to render non-human cells susceptible to HCV entry, we performed a cyclic retrovirus-based repackaging screen of a complementary DNA library derived from the highly HCV-permissive human hepatocarcinoma Huh-7.5 cell line8
. We screened for genes that rendered a non-permissive mouse embryonic fibroblast cell line, NIH3T3, infectable with HCV pseudoparticles (HCVpp), defective lentiviral particles that display the HCV glycoproteins and measure only viral entry9, 10, 11
(Supplementary Fig. S1a
and Supplementary Methods
). To maximize the likelihood identifying a novel component that would permit HCV entry into non-human cells, we utilized an NIH3T3 subclone overexpressing human CD81, SR-BI, and CLDN1. This screen identified human occludin (OCLN) as a potential novel HCV entry factor. OCLN is a four transmembrane domain protein present in the tight junction complex of polarized epithelial cells, where it likely functions to regulate paracellular permeability and cell adhesion12,13
. Expression of human CD81, SR-BI, CLDN1, and OCLN in NIH3T3 cells enhanced HCVpp infectivity by approximately 120-fold ( and Fig. S1c
OCLN expression confers susceptibility to HCVpp
To confirm that OCLN is required for HCV entry, we examined the relationship between its expression and HCV susceptibility in a variety of human cell lines. Naturally HCV-permissive human hepatocyte cell lines (Huh-7.5 and Hep3B) or those previously shown to lack other entry factors (hepatocellular carcinoma HepG2, and embryonic kidney HEK293T) were found to express readily detectable levels of endogenous OCLN (Fig. S1d
). In these cells further overexpression of OCLN did not enhance susceptibility to HCVpp (). Silencing of OCLN, however, inhibited HCVpp infection of Hep3B cells (), and both HCVpp and HCVcc infection of Huh-7.5 cells (, respectively), indicating that OCLN is essential for HCV infection of naturally permissive cells. This inhibition was HCV-specific, as infection with pseudoparticles bearing the vesicular stomatitis virus glycoprotein (VSVGpp) was not affected (values in are corrected for VSV-Gpp infectivity). It should be noted that OCLN knockdown resulted in incomplete inhibition of HCVpp infection for both cell lines. This is likely due to incomplete silencing or stable reservoirs of this protein that remained to contribute some entry functions. In contrast, the naturally HCV-resistant renal carcinoma cell line, 786-O, expresses high levels of the major HCV entry factors CD81, SR-BI, and CLDN1, but approximately 17-fold less OCLN than Huh-7.5 cells (Fig. S1d
). OCLN overexpression in 786-O cells specifically enhanced HCVpp infection by over 88-fold (). Furthermore, this OCLN-dependence of HCVpp infection was observed across a panel of diverse HCV genotypes (). Another HCVpp-resistant cell line, the HeLa cell-derived cervical carcinoma line TZM, was found to lack both endogenous CLDN1 and OCLN (>100 and 25-fold less mRNA than Huh-7.5 cells, respectively) (Fig. S1d
). Overexpression of these factors together increased HCVpp infectivity of TZM cells by 450-fold (). These results indicate that OCLN is an essential HCV entry factor.
OCLN silencing inhibits HCV entry
To further examine HCV entry requirements in non-human cells, we transduced murine NIH3T3 cells with all combinations of one, two, three, or all four human CD81, SR-BI, CLDN1, and OCLN. Expression levels were determined by expressing an mCherry/CLDN1 fluorescent protein fusion (Fig. S3a
) or by FACS staining for CD81, SR-BI (Fig. S3a
) and OCLN (Fig. S3b
). No combination of human factors enhanced VSVGpp infectivity (data not shown and results in are normalized to VSVGpp values. See Fig. S2
for representative raw data). As shown in , infectivity was not significantly altered by expression of any of the factors alone. Expression of all four entry factors, however, had a large impact on HCVpp permissiveness, with a 45-fold increase over naïve NIH3T3 cells. Furthermore, expression of all four factors, expressed as fluorescent protein fusions for CD81, CLDN1 and OCLN and un-tagged SR-BI, conferred HCV-susceptibility to mouse embryonic fibroblast cell lines, STO5 and L929, as well as the mouse hepatocyte cell lines, AML12, Hepa1.6, and H2.35, with enhancements of 4 to 85-fold over naïve cells (). Permissiveness to VSVGpp was not affected by expression of the human proteins. Attempts to infect these murine cells with HCVcc were unsuccessful, likely as a result of inefficient viral RNA replication in these cells, as previously documented2,14
. It is interesting that different murine cell lines exhibited varying degrees of HCVpp entry permissiveness after transduction with all four factors. HCV entry factor levels or cell determinants affecting lentivirus reporter expression do not appear to explain the observed differences (since the data were normalized to VSVGpp). Rather, these cell type specific differences may reflect real biological variability in other cellular machinery involved in HCV entry.
Expression of human OCLN and human CD81 determines HCV species tropism
To investigate which of the four entry factors are responsible for HCV species-specific tropism, we performed additional experiments in Chinese hamster ovary (CHO) cells, which express low endogenous levels of SR-BI, CLDN1 and OCLN (Fig. S4b
). In CHO cells overexpression of all four human factors specifically enhanced HCVpp infection by 60 to 336-fold (Figs. and S4a
). While omission of CD81, CLDN1, or OCLN abolished HCVpp entry, a low level of endogenous SR-BI in CHO cells provided some HCVpp entry function when the other three factors were transduced (Fig. S4a
). However, since overexpression of SR-BI further enhanced entry by 3-fold in the context of the other human proteins (Fig. S4a
), we still considered CHO cells to be an excellent environment in which to study the species-specific functions of each factor. To do so, we transduced CHO cells to express every combination of human and mouse CD81, SR-BI CLDN1, and OCLN. In all combinations, mouse SR-BI functioned equivalently to that of human origin (). This shows for the first time that mouse and human SR-BI are equally capable of mediating HCV uptake. In CHO cells mouse CLDN1 appears slightly more functional for HCVpp entry than the human protein (). This is in agreement with a slight preference observed when either of these CLDN1 proteins, which are 90% identical, were expressed in human 293T cells6
. These observations were confirmed by transducing mouse NIH3T3 cells with human CD81 and OCLN and all permutations of mouse and human SRBI and CLDN1 (). While SRBI displayed little to no species specificity, NIH3T3 cells expressing mouse CLDN1, in addition to the other three entry factors, were slightly more permissive to HCVpp than the same cells expressing human CLDN1. While the determinants for this CLDN1 preference have not yet been examined, it remains clear that CLDN1 does not contribute to the restriction to HCVpp entry in mouse cells. It is noteworthy that none of the mouse cells examined above express sufficiently high levels of either murine SR-BI or CLDN1 (Fig. S4d and S4e
), thus explaining their dependence on transduction of these proteins for HCVpp permissivity.
In contrast, CD81 and OCLN exhibited human specific HCV entry factor functions in CHO and NIH3T3 cells. CHO cells expressing either of these human proteins in the context of the other three mouse proteins were slightly more permissive to HCVpp than cells expressing only the mouse proteins (). Furthermore, expression of both human CD81 and OCLN, together with murine SR-BI and CLDN1, rendered CHO cells as infectable with HCVpp as cells expressing all four human factors (). These results agree with our previous finding that mouse and hamster CD81, when expressed in the CD81-deficient human hepatocellular carcinoma cell line HepG2, support only low level HCV entry (greater than 10-fold reduced from human CD81)15
. Importantly, these data indicate that CD81 and OCLN represent the minimal human-specific entry factors, at least in the context of mouse and hamster cells.
For CD81, the species-specific difference in HCV-entry activity between rat and human proteins maps exclusively to its large extracellular loop15
. To determine the regions of OCLN that are responsible for the functional difference observed between mouse and human proteins, which are 91% identical, we expressed chimaeric OCLN molecules in 786-O cells and assayed HCVpp permissivity. In these experiments, a mouse chimaera bearing the second extracellular loop (EC2) of human OCLN was as active as the full-length human protein and at least 4-fold more active than the mouse homologue (, respectively). Conversely, a human OCLN mutant with the EC2 of the mouse protein functioned similarly to the full-length mouse factor in HCVpp infectivity (). These data suggest that the human-specific determinants of OCLN's HCV entry factor functions are entirely contained within EC2.
This study represents a major step forward in understanding both HCV host cell entry and HCV species tropism. All human, murine, or hamster cell lines that we tested became permissive for HCV entry when engineered to express the molecules CD81, SR-BI, CLDN1, and OCLN. HCV entry into primary mouse hepatocytes might be complicated by expression of additional dominant negative restriction factors absent in cell lines, which is experimentally difficult to address at this point. Our data suggest that OCLN completes the list of cell-type specific HCV entry factors; any other factors required for HCV entry must be ubiquitously expressed, or at least conserved between a wide range of human and mouse cells. The fact the OCLN is a major component of the tight junction complex further highlights the significance of this structure and cell polarity to HCV entry. We previously showed that CLDN1 acts late in the entry process, just prior to virion internalization6
. The intimate association of CLDN1 and OCLN at the tight junction suggests that both these factors may function in a similar time frame. The use of multiple uptake factors with distinct cell surface distributions strengthens the hypothesis that HCV follows a coordinated entry pathway similar to that of coxsackievirus B. This virus initially interacts with a primary receptor (decay-accelerating factor) on the luminal cell surface, followed by lateral migration of the virus–receptor complex to the tight junction, where interaction with the coxsackie and adenovirus receptor is immediately followed by uptake into the host cell16
. Strikingly, coxsackievirus B entry also requires OCLN17
, further suggesting similar entry mechanisms of this virus and HCV. Recent work by Brazzoli et al. supports this step-wise model of HCV entry, demonstrating that initial engagement of CD81 on the cell surface, by either fluorescently labeled CD81 antibodies or soluble forms of the HCV glycoproteins, is followed by GTPase-dependant actin rearrangements that allow lateral movement of the CD81-bound complex into areas of cell-cell contact overlapping with both CLDN1 and OCLN localization18
The study of HCV pathogenesis and the development of urgently needed effective antivirals and therapeutic and/or preventative vaccines targeting this virus has been severely hampered by the lack of convenient inbred small animal models capable of supporting HCV infection and replication. Numerous blocks will certainly need to be overcome before complete viral replication in a mouse can be achieved. HCV RNA replication in mouse cells is inefficient1,2
, and the ability of such cells to support virion assembly is unknown. Our results clearly demonstrate, however, why mouse cells are unable to support HCV entry. This major block to HCV replication in murine cells can now be overcome simply by the expression of human CD81 and OCLN in the context of mouse CLDN1 and SR-BI, providing a clear foundation upon which a mouse model for HCV infection can begin to be constructed.