HCV infection leads to progressive liver disease, which has been associated with extrahepatic syndromes, including CNS abnormalities.3
There is a growing body of literature on mild neurocognitive impairment in chronic HCV infection that is independent of hepatic encephalopathy.27
However, there is a lack of studies to investigate whether cells of the CNS support HCV replication. In this study, we report that all of the essential HCV receptors are expressed on brain microvascular endothelial cells. Indeed, the microvascular endothelia were the only cell type in the brain that expressed all the factors required for HCV entry. To our knowledge, this is the first study to investigate the expression of HCV receptors in the human brain. Microvascular endothelial cells are a major component of the BBB28
and may provide a portal for HCV to infect the CNS.
Quantification of HCV RNA from matched samples of white and grey matter, cerebellum, medulla, liver, and plasma revealed that, in clinical samples with detectable brain HCV, the viral load was 1000- to 10,000-fold lower in brain compared with liver from the same subject. HCV RNA was detected in at least one brain region from 4 of 10 HCV-infected subjects, independent of HIV coinfection status. Although quantities of viral RNA from the brain and liver varied widely between clinical samples, in general a lower viral load was associated with a higher postmortem interval, suggesting RNA degradation in some samples over time. Variation between brain-, plasma-, and liver-derived HCV E1 and 5′ untranslated region sequences has previously been reported in this cohort, supporting the hypothesis that HCV replicates and evolves within the brain.29
However, care is needed when interpreting the physiologic relevance of detecting HCV RNA genomes. It is worth noting that 6 HCV-infected subjects had no detectable viral RNA in the brain, despite having comparable levels of viral RNA in the liver and plasma (Supplementary Materials and Methods
) to 4 subjects with no detectable HCV RNA in the brain.
There have been significant difficulties in visualizing HCV antigen-expressing hepatocytes in the liver that most likely reflect the low viral burden at a cellular level.30,31
Our quantification of HCV RNA in brain tissue compared with liver suggests that detecting HCV antigen in the brain will be technically challenging, and current imaging methodologies lack the sensitivity to reliably detect HCV-infected cells in the CNS. Indeed, our attempts to show NS3 or NS5A HCV antigen in brain or liver samples from subjects in this study have failed to provide robust signals (data not shown). Previous studies have reported the presence of HCV RNA in microglia and astrocytes isolated using laser capture microdissection.32,33
However, our experiments show that astrocytes and microglia lack expression of several receptors required for HCV entry.10
Our studies show that 2 independently derived brain microvascular endothelial cell lines, hCMEC/D3 and HBMEC, support HCVpp entry. Infection was inhibited by antibodies specific for CD81, SR-BI, claudin-1, or E2 glycoprotein, showing a common receptor-dependent entry pathway to that reported for hepatocytes and hepatoma-derived cell lines.34,35
These observations, along with our recent report that neuroepithelioma cell lines derived from peripheral tumors support efficient HCVpp infection,10
show that viral entry is not restricted to hepatocytes. Importantly, messenger RNA and protein profiling databases show that CD81, SR-BI, claudin-1, and occludin are expressed in epithelial and endothelial cells from various tissues,36,37
raising the possibility that other cell types may support HCV infection. Our data support the presence of functional entry receptors in BBB endothelial cells but not endothelial cells derived from umbilical vein and liver sinusoids.
Given the permissivity of brain endothelial cells for HCVpp entry, we investigated their ability to support HCVcc replication. HCV-infected hCMEC/D3 cells release low levels of virus that can infect hepatoma cells and showed evidence for a spreading infection that is CD81 dependent. Recent reports highlighting the role of ApoE in HCV assembly8,24
and the targeting of ApoE-containing nanoparticles across the BBB38,39
prompted us to investigate a role for ApoE in brain endothelial cell infection. Interestingly, antibodies targeting ApoE effectively neutralized HCV infection of hCMEC/D3 cells, despite modest neutralization of Huh-7 cells, suggesting a greater role for ApoE in virus infection of brain endothelial cells.
The BBB limits the passage of substances from blood to the CNS by the presence of tight junctions between endothelial cells and by receptor-mediated efflux transport systems that restrict the entry of hydrophilic molecules to the brain.28
Multidrug resistance proteins, including P-glycoprotein, restrict the transport of many drugs across the BBB, including antivirals that may contribute to the development of “sanctuary sites,” allowing pathogens (eg, HIV-1) to replicate in the brain of drug-treated patients.40
In the present study, HCV replication was inhibited by antiviral agents targeting NS3 protease and NS5B polymerase enzymes in vitro.
Disruption of BBB integrity can lead to an infiltration of pathogens, cytokines, and immune cells to the brain parenchyma as reported for HIV-1 and West Nile viruses.41,42
hCMEC/D3 infection led to increased HCV RNA and antigen expression over time, with a cytopathic effect that associated with TUNEL staining and increased permeability to the paracellular permeability marker FD-70. These data support a model in which HCV infection may compromise BBB integrity, with implications for brain homeostasis in HCV infection.
In conclusion, we show that brain microvascular endothelium expresses all the major viral receptors required for HCV infection. Two brain endothelial cell lines support HCV entry and replication, in which infection is inhibited by HCV receptor-specific antibodies, interferon, and specific antiviral agents. The observation that HCV-infected hCMEC/D3 cells release low levels of infectious virus and show evidence of apoptosis supports a model in which the BBB may provide an extrahepatic target for infection, and HCV may directly induce neuropathology in vivo.