Human occludin is a hepatitis C virus entry factor required for infection of mouse cells

Abstract

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lentiviral particles bearing HCV glycoproteins (HCVpp) or HCV produced in cell culture (HCVcc) (A.P., M.E. and C.M.R., unpublished observations), suggesting that there is a block at the level of entry.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.

Figure 1. OCLN expression confers susceptibility to HCVpp

a, Human cells were transduced with human pTRIP-mCherry-CD81 (CD81), pTRIP-Cerulean-CLDN1 (CLDN1), pTRIP-Venus-OCLN (OCLN), both CLDN1 and OCLN, or mock transduced, then challenged in parallel with HCVpp and VSVGpp. HCVpp infectivity is reported as the titer of HCVpp divided by the titer of VSVGpp, after subtraction of the signals from infection with non-enveloped pseudoparticles (Env⁻pp). Pseudoparticles of different HCV genotypes were used to infect naive (black) or OCLN-expressing (white) 786-O cells (b). c, Mouse cells transduced with human pTRIP-mCherry-CD81 (CD81), pTRIP-SR-BI (SR-BI), pTRIP-Cerulean-CLDN1 (CLDN1) and pTRIP-Venus-OCLN (OCLN) (4×) or mock transduced; challenged and HCVpp infectivity calculated as above. ND, not determined. Means and standard deviation (SD) of at least triplicate experiments are shown.

Figure 2. OCLN silencing inhibits HCV entry

Huh-7.5 (left) and Hep3B (right) cells transfected with irrelevant (grey), CD81 (checkered), or OCLN (black) specific siRNA pools were stained with CD81 (a and b) and OCLN (c and d) antibodies. Expression was analyzed by flow cytometry; the ratio between specific and isotype control staining is shown. Concurrently, siRNA-treated cells were challenged with HCVpp (e and f) and HCVcc (g). HCVpp infectivity was calculated as described in methods; HCVcc infection was quantified by the ratio of viral protein (NS5A) to isotype control staining. All samples are normalized to irrelevant-siRNA treatment. dMFI, difference in mean fluorescence intensity. Means and SD of at least triplicate experiments are shown.

Figure 3. Expression of human OCLN and human CD81 determines HCV species tropism

a, Mouse NIH3T3 were transduced with combinations of human pTRIP-CD81 (CD81), pTRIP-SR-BI (SR-BI), pTRIP-mCherry-CLDN1 (CLDN1) and pTRIP-OCLN (OCLN). Hamster CHO (b) and NIH3T3 (c) cells were transduced with combinations of human (H) and mouse (M) pTRIP-mCherry-CD81 (CD81), pTRIP-SR-BI (SR-BI), pTRIP-Cerulean-CLDN1 (CLDN1) and pTRIP-Venus-OCLN (OCLN). d, Diagram of OCLN membrane topology. e, 786-O cells were transduced with mouse/human OCLN chimaeras; all chimaeras were N-terminally tagged with Venus yellow fluorescent protein. Transduced cells (a, b, c and e) were challenged with HCVpp and VSVGpp encoding GFP reporters and HCVpp infectivity calculated and normalized as described in methods. Means and SD of at least duplicate experiments are shown.