Up‐regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication

Hepatitis C virus (HCV) is an important cause of chronic liver disease, with the severe consequences of hepatocellular carcinoma (HCC) and cirrhosis occurring in some patients(1, 2). When considering determinants of HCV persistence and propagation of infection, little consideration has been given to differences between cells within the liver. Recent studies have demonstrated HCV Core protein localized to discrete foci within HCC sections from patients, and laser captured microdissection samples indicated that HCV genomes exist at unexpectedly low average copy numbers per cell(3, 4). These observations suggest that HCV infection is not widespread throughout the liver, but rather selective or restrained in its target cells. In vitro studies of HCV rely heavily on the Huh7.5 cell line. This cell line was generated after Huh7 cells selected for harboring an HCV subgenomic replicon were cured of replicating viral RNA with interferon-α(5). The resulting cells were highly permissive for HCV replication when re-transfected with replicon constructs. As they support replication of the JFH1 viral isolate and produce infectious virus in tissue culture, Huh7.5 cells have propelled studies of the HCV life-cycle forward(6). Similar cell lines with increased HCV permissivity, like LH86 cells, have been directly isolated from patient samples, although HCV RNA levels are 1–2 log lower compared to Huh7.5 cells(7). The reasons for Huh7.5 cells being exceptionally permissive for HCV replication were attributed to a defect in RIG-I, a pattern recognition receptor that activates type I interferon expression during viral infection(8). However, recent studies found no RIG-I defects in novel cell lines also generated from Huh7 cells with increased permissiveness to HCV(9, 10). Thus RIG-I alone may not explain this phenomenon. The Hedgehog (Hh) pathway plays an important role during embryogenesis, normal tissue growth, regeneration after injury and carcinogenesis(11–15). Most hepatocytes in healthy adult livers do not express detectable Hh ligands Sonic hedgehog (Shh) or Indian hedgehog (Ihh), whereas some Hh ligand expression can be demonstrated in ductular-type cells(16). After injury, Shh expression increases, causing Gli family transcription factors to accumulate in Hh-responsive cells as part of the regenerative and fibrotic responses(14, 15). Hh pathway activation has also been observed in some HCC cell lines, although significant heterogeneity exists within actual tumors(16). A vigorous debate exists as to whether liver epithelial cells, such as cholangiocytes and hepatocytes, undergo epithelial-to-mesenchymal transitions (EMT) in injured livers, but some evidence supports this concept and suggests Hh-mediated regulation(17–21). In viral hepatitis patients, recent data suggests EMT may occur in response to infection(22). HCV infection of hepatoma cell lines in vitro alters cell polarity to expose gap junction complex proteins key to viral entry (23). To our knowledge, such studies have not addressed the effects of altered cell polarity on HCV replication, or mechanisms by which viral infection might promote EMT. We hypothesized that Huh7.5 cells are highly permissive for HCV because they possess a “transitional” phenotype skewed towards mesenchymal characteristics due to increased Hh pathway activity. We subsequently asked whether Hh pathway activation may create an environment conducive to viral replication and whether Hh pathway inhibition would inhibit HCV replication.