For comparison, HCV from patient sera or chimeric mice was 30%-80% ApoB associated. A third difference between virus produced in tissue culture and virus produced in animal models is its specific infectivity. Virus produced in tissue culture (FL-J6/JFH variant; HCV in cell culture [HCVcc]) has a specific infectivity of approximately 1 TCID50/1,230 genomes, whereas the specific infectivity of virus produced in either mice or chimpanzees was 1 TCID50 per 10-150 viral genomes. We determined specific infectivity of the
two viral variants, expressed buy Tanespimycin as TCID50/RNA copies at various time points (Fig. 7E). The specific infectivity of the JFH-HS increased gradually in the first 21 days, then reached a plateau, whereas the specific infectivity of JFH-FBS did not change. The average specific infectivity of virus produced by cells that were fully differentiated in HS was 1 TCID50 per 236 viral genomes, compared to an average specific infectivity of 1/2513 for JFH-FBS (Fig. 7F). The overall specific infectivity of JFH-HS is now similar to the highest
specific infectivity that was reported previously for HCVcc, corresponding to the small low-density peak in that study. In this study, we investigated the effects of HS on tissue culture of Huh7.5 cells. We compared the standard tissue culture protocol, using media supplemented with 10% FBS, to the use of media supplemented with 2% HS. Cells cultured in HS media undergo rapid growth arrest and show increased expression of hepatocyte
differentiation markers (α1AT and ALB). In HS-supplemented media, the expression of cell-contact EGFR inhibitors list medchemexpress proteins claudin-1, occludin, and e-cadherin was also increased. These factors are indicative of differentiated epithelial cells. Because previous reports have shown that claudin-1 and occludin are entry factors and confer infection of nonpermissive cell types,[20, 21] the increase in claudin-1 and occludin likely plays a role in the increase in viral titers in HS media. The level of expression of other HCV-entry receptors (CD81, SR-B1, and NPC1L1) did not change when Huh7.5 cells were cultured in media with HS. Expression of key lipid metabolism regulators (LXR-α, PPAR-α, and PPAR-γ) was increased, and consistent with this, the lipid droplet content of these cells was highly increased. We showed that VLDL secretion was restored, a complex process that requires the integration of various biogenesis, modification, and transportation steps. All these factors have been implicated in the life cycle of HCV, and, in particular, HCV has been shown to hijack the VLDL secretion machinery for egress. Consistent with this, we have shown that under these new tissue culture conditions, production of JFH-1 increased more than 1,000-fold. The virus produced under these conditions more closely emulates HCV that is found in serum of patients and animal models, was associated with ApoB, had a lower density, and was highly infectious.