Pharmacological and antibody treatment to either antagonize or promote Hh signaling in liver cells at different points in the pathway had the same direct effects on HCV replication. Included in the current study was GDC-0449, an Hh pathway antagonist already in preclinical development for other indications, which we have shown can inhibit HCV replication. The observation that HCV replication is closely associated with Hh pathway activity has not been previously appreciated. Certainly it has never been explored as an explanation for why Huh7.5 cells are exceptionally permissive for HCV replication. Hh pathway components were

previously identified in a functional genomic primary screen, but were not identified in the secondary screen.31 This may be due to the broad changes in the intracellular environment induced by Hh pathway activation that may not be detected in single gene small interfering RNA (siRNA) knockdowns. Carfilzomib manufacturer It may be that the intracellular changes that occur upon AZD4547 in vivo Hh pathway activation and its possible association with EMT are part of the same continuum when considering an environment conducive to HCV infection. The concept of a continuum is supported by the mixed phenotype we detected in LH86 cells compared with Huh7.5 cells and how that correlates with the original observation that LH86 cells were less permissive than Huh7.5 cells.7 One of the key remaining questions is at what level

does Hh pathway activation exerts its effects on HCV replication. Although we used multiple agonists and antagonists, these agents all act close to the “top” of the pathway. We suspect the changes may occur further downstream and are likely to involve other pathways known to be associated with Hh in promoting an environment conducive for HCV replication. Our results may be further cause to reevaluate the model of HCV infection in the liver. Liang et al.3 demonstrated in their analysis of explanted livers from HCV patients with HCC that Core protein was detected in a minority of cells despite this advanced-stage

of disease. Perhaps Huh7.5 cells are representative of a minor population of liver cells that retain an MCE active Hh pathway.18 HCV may preferentially infect and replicate in this minority population of cells so hospitable to efficient replication. Infection of these cells combined with a possible contribution of the resulting interferon release may induce further Hh pathway activation, including increased Shh ligand production. Moreover, increased Shh ligand may serve as a paracrine factor that allows neighboring cells to become more easily infected by promoting a “transitional” phenotype, exposing the gap junction complexes to facilitate viral entry and/or altering the intracellular environment. The association between HCV and the Hh pathway will not alone settle the vigorous debate regarding the occurrence of EMT within the liver.