Producing large amounts of infectious HCV virions in cultured cells has been difficult, especially for the most prevalent and clinically problematic genotype 1, which in part relates to its poor ability to replicate in vitro
and the subsequent appearance of cell culture-adaptive mutations interfering with its propagation and infectivity. Here, we produced HCV particles of genotype 1 containing a genome previously shown to be highly infectious in vivo 
. Their ability to infect human liver slices demonstrates the biological relevance of the particles produced in this in vitro
system. Two major features underlie independence from HCV replication, which avoided adaptive mutations typically associated with HCV propagation in cell culture: first, the unique and robust strategy for producing HCV genomes in the cytoplasm independent of HCV replication, and second, the WNV subgenomic replicon that created an appropriate cellular environment for HCV RNA translation as well as particle assembly and release.
HCV particle formation likely took place within membrane rearrangements derived from those induced by the WNV subgenomic replicon, as suggested by immuno-gold electron microscopy results. We also observed that the release of HCV particles by BHK cells was enhanced by lineage I WNV 
and serotype-2 dengue virus 
subgenomic replicons, but not by one of Semliki Forest virus 
, an alphavirus belonging to the Togaviridæ
family (Fig. S1B
). This indicates that, beyond similarities in genomic organizations and sequences 
, the increased production of infectious HCV could result from common functional properties conserved amongst members of the Flaviviridæ
family rather than strict sequence specificity of the proteins encoded by the flavivirus subgenomic replicons. In BHK-WNV cells, this possibility is further substantiated by the lack of correlation between HCV production and translation of the WNV subgenomic replicon, upon inhibition of the latter's activity. The replication of flaviviruses and HCV induce similar membrane rearrangements in the cytoplasm of infected cells 
, and our data confirmed that flaviviruses also infect hepatocytes 
. In Huh-7.5 cells, cholesterol metabolism has been implicated in HCV replication 
and lipid droplets in its assembly 
. Likewise, WNV replication involves cholesterol metabolism 
and, for dengue virus particle formation, the interaction of the viral core with lipid droplets 
. As the mechanisms involved in the production of HCV by hepatocytes are still debated, these similar features perhaps underlie part of the BHK-WNV cell permissiveness for HCV particle formation.
The correlation between reversal of membrane rearrangements and loss of HCV particles production (not shown) suggests that these rearrangements, and perhaps related cellular changes (e.g. cholesterol metabolism and lipid droplet formation), are playing a major role in the permissiveness of BHK-WNV cells. However, the down or up regulation of other cellular factors could be involved as well. Thus, several intracellular mechanisms involved in innate immunity interfere with flavivirus propagation [e.g. 57]
, and knockdown of interferon stimulating mechanisms or signaling pathways enhance WNV 
and HCV 
productions in cell culture; WNV 
and HCV 
proteins have been shown to directly target such pathways. Here we cannot exclude that such a mechanism took place prior to or upon expression of HCV genes. However, the introduction of a BHK cell-adapted WNV subgenomic replicon into naïve BHK-21 cells rendered them rapidly permissive for the production of WNV, whereas that of HCV appeared after many more passages (not shown). One possible interpretation is that co-evolution of WNV subgenomic replicon and BHK cells under antibiotic selection led to the regulation of additional cellular factors, probably involved in fine tuning WNV replication and/or translation, but absolutely required for the production of infectious HCV. This prompted us to identify such cellular factors in BHK-WNV cells and test their relevance with the JFH-1 strain/Huh-7.5 cells paradigm, the results of which will be presented elsewhere.
The entry assay with particles produced in BHK-WNV cells (HCVrp) requires only the delivery of the associated RNA molecule into the cytoplasm of the target cell where it can be translated at sufficient levels to trigger the dual bacteriophage RNA polymerase amplification system. Thus, the target cell needs to be permissive only for viral entry, and possibly a limited number of post-entry steps (e.g. RNA uncoating). Most importantly, the non-involvement of RNA replication for the signal readout allows assessment of the entry permissiveness of diverse cell types, independent of their ability to support HCV replication. This represents a significant advantage over the HCVcc system that also relies on viral spreading to amplify the read out signal, and the involvement of only HCV structural proteins and RNA clearly distinguishes this system from HCVpp, which is based on non-HCV protein and nucleic acid platform.
We had previously observed that both ASGP-R subunits were required for internalization of HCV materials into hepatocellular carcinoma as well as non-target cells 
. Here we show that these subunits are involved in delivering HCV reporter RNA into HCV-permissive hepatic cells. It is not yet known whether the role of ASGP-R in HCV uptake relates to incomplete maturation of E1 and/or E2 carbohydrate residues, as previously observed 
, or involves another mechanism 
. HCV has been reported to enter cultured cells via
clathrin-coated pits 
, and ASGP-R internalization follows the same path 
. Yet, ASGP-R can be targeted to various intracellular compartments including ER 
, which leaves open the possibility that this receptor plays a role at an early as well as a late step of the HCV entry process and RNA delivery.
As inter-genotypic differences and cell-adaptive mutations could affect viral production in hepatic cells, the BHK-WNV paradigm provides an alternative model to produce wild type virus for in vivo or ex vivo studies without the concern that adaptive mutations develop. It could also present major advantages for deciphering mechanisms of viral translation, assembly, release and entry, including involvement of non-structural genes in viral production independent of their role in replication.