The complexities of hepatitis C virus entry☆
Article Outline
Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP, Rice CM.
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp) or HCV produced in cell culture (HCVcc) (A.P., M.E. and C.M.R., unpublished observations), suggesting that there is a block at the level of entry. Here we show, using an iterative complementary DNA library screening approach, that human occludin (OCLN) is an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for the HCV-susceptibility of human cells, because its overexpression in uninfectable cells specifically enhanced HCVpp uptake, whereas its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors CD81 (ref. 4), scavenger receptor class B type I (SR-BI, also known as SCARB1) and claudin-1 (CLDN1). Although the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, both OCLN and CD81 must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process, and provides an important advance towards efforts to develop small animal models for HCV.
[Abstract reproduced by permission of Nature 2009;457:882–886]
One of the greatest barriers to understanding hepatitis C virus (HCV) pathobiology is the lack of immune competent small animal models that support virus infection. Without such models, the evaluation of antiviral compounds and vaccine candidates is considerably delayed. The chimpanzee is the only non-human host that supports HCV infection and is not widely available for use in most laboratories. The recent paper by Ploss and colleagues showing the tight junction (TJ) associated molecule occludin as an entry factor which, together with the already known co-receptors, allows HCV to infect murine cells is an exciting discovery that removes one barrier to the development of transgenic animals supporting HCV replication [1].
In these studies, Ploss and colleagues applied the same screening approach that succeeded in identifying the TJ associated protein claudin-1 (CLDN1) as a co-factor for HCV entry [2]. In the earlier study, using the non-permissive human 293T cell as the screening target, Evans and colleagues demonstrated that introducing CLDN1 into human 293T cells rendered them permissive for HCV pseudoparticle (HCVpp) entry. In this new study, the target cell was a murine NIH3T3 cell line, engineered to express the three known HCV (co)-receptor molecules (scavenger receptor BI (SR-BI), CD81 and CLDN1). Screening of these cells after transduction with a human liver cell derived cDNA library identified a second TJ associated protein, human occludin, as an essential factor for HCVpp entry. Multiple combinations of human and murine (co)-receptor molecules were expressed in CHO or NIH3T3 cell backgrounds and screened for their ability to confer HCV entry, leading to the conclusion that human CD81 and occludin are required for HCVpp infection.
Several TJ proteins have been reported to act as primary receptors for a range of viruses, including: junctional adhesion molecule (JAM-A) for reovirus [3] and feline calicivirus [4], coxsackie and adenovirus receptor (CAR) for coxsackievirus and adenovirus [5]. Recent work with coxsackievirus group B (CVB) which utilises Decay Accelerating Factor (DAF) and CAR cell surface molecules to enter cells has highlighted the complex and dynamic properties of intercellular junctions [6]. CVB binds to its primary receptor DAF at the luminal surface of intestinal epithelial cells and this interaction triggers a signalling cascade that promotes actin-dependent relocalization of the virion–DAF complex to the TJ, where secondary interaction(s) with CAR induce particle internalization [6]. Occludin has been shown to have an important role in CVB internalization, where siRNA silencing led to a 9-fold reduction in virus infection, suggesting that occludin has an important role in the trafficking of CAR and other TJ associated proteins that are utilized by CVB to enter cells. Using this method Liu and colleagues have recently shown a similar effect of occludin silencing on HCV entry [7].
This two-step model for CVB entry informs our thinking on the potential multi-step process of HCV internalization that will involve at least four molecules SR-BI, CD81, CLDN1 and occludin, whose roles are currently unclear. To add further complexity to the HCV entry process, there is increasing evidence that HCV associates with host lipoproteins. It is not clear how lipoprotein components contribute to HCV infectivity, however, particle assembly and entry are interwoven with lipid metabolism, with two lipoprotein receptors, low density lipoprotein receptor and SR-BI, implicated in HCV attachment and entry (reviewed in [8]). Indeed, the engagement of HCV lipoviral particles with SR-BI may initially be mediated via its associated lipoproteins before the subsequent interaction of the viral glycoproteins with SR-BI and CD81. Recent studies using fluorescence resonance energy transfer (FRET) [9] and biomolecular fluorescence complementation (BiFC) methodologies [10], [11] have reported CLDN1-CD81 protein complexes at the plasma membrane of permissive hepatoma cells. Perturbation of CLDN1-CD81 complexes with Protein Kinase A (PKA) antagonists [12] and monoclonal antibodies specific for CD81 [9] suggest a role for these complexes in HCV entry. It is interesting to speculate whether CLDN1 association with occludin at TJs has an additional role in the viral entry process by directing the trafficking and internalization of virus–receptor complexes.
In summary, these studies remove one of the primary barriers to HCV propagation in mouse cells. However, these engineered receptor permissive cells failed to support the infection of cell culture-derived HCV particles, consistent with previous reports that murine cells support relatively low levels of HCV RNA replication and fail to assemble and release infectious HCV particles [13]. Taken together these data suggest that we are one step nearer to a small animal model for HCV replication; however the next steps, which will involve defining the human specific determinants that will be required to modulate viral replication and particle assembly in murine cells, will remain a significant challenge for the next few years.
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☆ The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
PII: S0168-8278(09)00455-3
doi:10.1016/j.jhep.2009.06.014
© 2009 Published by Elsevier Inc.
