Moreover, quantification of intracellular selleck chemicals HBV DNA at an MOI of 50 demonstrated that the magnitude of HBV replication in PMHs was 106 HBV DNA copies per well; it reached a peak around day 3 PT and
lasted more than 9 days (Fig. 1E). In comparison, the transduction of HepG2 cells was more efficient because intracellular HBV DNA quantification was 2 log higher than that in PMHs (Fig. 1E). Moreover, by using a recombinant baculovirus for green fluorescent protein, we found that the transduction efficiency at an MOI of 50 was clearly higher in HepG2 cells versus PMHs, with more than 70% and 50% of the cells transduced, respectively (data not shown). When we looked at extracellular HBV DNA after PMH transduction, we observed a peak of secretion on day 6 PT with 8 × 105 copies per well (Fig. 1F). Finally, the results also showed that the amount of the intracellular baculovirus genome was maximum on day 1 PT, and it decreased thereafter but was still detectable by Southern blot analysis on day 9 PT (Fig. 1C). The differences in the kinetics of accumulation and clearance of both baculoviral and HBV nucleic acids suggest that
the half-life of encapsidated HBV DNA is longer than that of the baculoviral genome, as previously described.12 It has previously been shown that HepG2 cells transduced with HBV baculoviruses produce infectious HBV particles.12, EPZ-6438 purchase 18 The supernatant from HBV baculovirus–transduced PMHs was first characterized by CsCl gradient analyses. Most HBV DNA was found in fractions with a density range of 1.24 and 1.20 g/cm3 (fractions 9-12; Fig. 2A). This indicates that most HBV DNA was released within Dane particles, which have been shown to peak at 1.21 g/cm3 in isopycnic density analysis.18 Electron microscopy analyses confirmed the presence of Dane particles as well as spheres and filamentous particles (Fig. 2B). Altogether, these data showed that HBV-transduced
PMHs secreted newly produced HBV particles in 3 typical forms.19 Concentrated supernatant from HBV baculovirus–transduced PMHs was then used to inoculate freshly prepared PMHs or HepaRG cells,20 but none of these cells showed convincing signals of HBV infection within 15 days after inoculation (data not shown). The last step was the evaluation of the potentiality of our system Metformin datasheet for antiviral therapy testing. First, the antiviral activity of lamivudine was tested. Nucleos(t)ide analogues such as lamivudine are able to specifically inhibit HBV viral polymerase activity and thus prevent the secretion of mature HBV particles, whereas other steps of the viral life cycle, such as entry or antigen secretion, are not targeted by such a treatment.21 As expected, HBV DNA secretion (Fig. 3A), but not HBsAg (Fig. 3B), was inhibited by lamivudine treatment in a dose-dependent manner in PMHs transduced with Bac-HBV-1.