Nicola F Fletcher, Jian Ping Yang, Michelle J Farquhar, Ke Hu, Christopher Davis, Qiuchen He, Kimberly Dowd, Stuart C Ray, Sophie E Krieger, Johan Neyts, Thomas F Baumert, Peter Balfe, Jane A McKeating and Flossie Wong-Staal.
Gastroenterology, 139: 1365-74. 2010.
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Viruses initiate infection by attaching to molecules or receptors at the cell surface, whose expression can define cellular or tissue permissivity. Recent evidence shows a number of host cell molecules to be important for Hepatitis C virus (HCV) entry: tetraspanin CD81, scavenger receptor class B member I (SR-BI) and the tight junction proteins Claudin-1 (CLDN1) and occludin. RNA profiling and protein expression studies demonstrate that the HCV entry factors are expressed in a variety of tissues, but with the highest expression in the liver. Reports of cognitive defects and nuclear magnetic resonance abnormalities in HCV infected people led us to screen a series of neural and brain-derived cell lines. We found two independently derived neuroepithelioma cell lines which can support HCV pseudoparticle entry (fig 1) and express all 4 HCV receptors (fig 2). This infection was neutralized by antibodies specific to CD81, SR-BI and CLDN-1 (fig 3), demonstrating a common entry pathway for infection for both neuroepithelioma and hepatoma cell lines. Furthermore, both neuroepithelioma cell lines will support HCVcc infection, though at a 130-330 fold lower level than the Huh-7.5 hepatoma cell line. In summary, these data demonstrate that HCV infection in vitro is not restricted to cells of the liver and raises the possibility of HCV infecting the cells of the CNS.
Background & Aims: Hepatitis C virus (HCV) establishes chronic infections in 3% of the world’s population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication.
Methods: We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism.
Results: Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells.
Conclusions: Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to liver-derived cells, so HCV might infect cells of the CNS in vivo.
Figure 1. HCVpp infection of neuroepithelioma cells.
HCVpp-H77 (white, left) and VSV Gpp (grey, right) infection of a panel of neurally derived cell lines, with the Huh-7.5 permissive hepatoma cell line and the non-permissive 293T cell line as controls. Data is presented as the mean specific infectivity, where the virus specific RLU is expressed relative to a no env RLU ± SD. The dotted line represents the mean specific infectivity + 3 SD for 293T cells and provides a threshold for defining permissivity to HCVpp entry. This data is representative of two independent experiments. Three of the neuroepithelioma cell lines are clearly infectable (blue).
Figure 2. Viral receptor localization in hepatoma and neuroepithelioma cells. Four of the cell lines in Figure 1 were stained for CD81, CLDN1 and Occludin expression and imaged by laser scanning confocal microscopy. Receptor localization was either at the plasma membrane or intracellular. All 3 receptors are detectable in Huh-7.5, SK-N-MC, MC-IXC and SK-PN-DW but not in U87. The scale bar represents 10µm.
Figure 3: Receptor dependency of HCVpp infection of neuroepithelioma cells.
Huh-7.5 or MC-IXC cells were incubated with 5µg of a neutralizing anti-receptor antibody specific for CD81, SR-BI or CLDN1 Treated cells were incubated with HCVpp-H77 for 72h and luciferase activity measured to assess infectivity. The antibodies show similar activity with both cell types (though anti CLDN1 is more effective in blocking MC-IXC infection) suggesting similar receptor dependencies in liver and brain.