15 g/mL, reflecting the reproducibility MG 132 of our infection system. No particles could be detected when HCV infection was performed in the presence of D32.10 (all the values were under the cutoffs, not shown). These results indicate that the D32.10 mAb efficiently inhibits HCVsp infection of HepaRG hepatocytes. To assess whether the differentiated HepaRG cells could indeed become persistently infected with HCVsp, cells were frozen at D56** p.p. after primary infection. The HCV-infected HepaRG cells were then thawed, plated at low density (4 × 104/cm2), and subjected either to 1 (P1) or 3 (P3) subcultures before forcing induction of the differentiation process (Fig. 3A). The supernatants
were then collected each week and analyzed as above. Figure 3B shows that extracellular HCV RNA could be detected only during the differentiation (“D”) stage between 14 and 28 (P1) or 35 to 56 (P3) days. Interestingly, earlier (D14 instead of D35) and higher (4.5 log10 instead of 3.3 log10 copies/mL) titer virus levels were observed after one rather than three subcultures. This suggests that successive phases of Osimertinib research buy proliferation (“P”) before induction of the differentiation process (no splitting at confluency) resulted in an advantage of noninfected over HCV-infected
HepaRG cells. When we analyzed the HCV particles on sucrose gradient released in the culture media collected at D28 (P1) and D49 (P3) as a pool corresponding to 4.7 log10 copies of HCV RNA/mL, the total amount of HCV RNA cosedimented with core antigen and E1E2 in association with apoE and apoB at densities between 1.18 and 1.20 g/mL (peak II, Fig. 3C). In these experimental conditions of fractionation,7, 10 no reactivity was detected at low density (1.06 g/mL). However, a major peak of defective particles containing only E1E2 envelope associated with lipoproteins (apoE and apoB) sedimented filipin at intermediate densities of 1.14-1.15 g/mL (peak I, Fig. 3C).14 Taken together, these data demonstrate that the HepaRG cells remained
persistently infected by HCVsp (HCVsp-RG cells) and could produce larger amounts of empty apoE/apoB-associated E1E2 than apoE/apoB-associated complete HCV particles only when still differentiated. To identify ultrastructural modifications induced by HCVsp infection, EM analysis was performed. The HCV-infected HepaRG cells frozen at day 56** after plating (infection 1: Fig. 1A,b) were thawed, seeded at low density, cultivated 1 week until confluence, reseeded (P1, Fig. 3A), and then maintained without splitting after confluency up to day 28. At this time, EM examination of noninfected HepaRG cells revealed characteristics typical of normal human hepatocytes (Fig. 4A). Apical and basolateral poles as well as tight junctions between two adjacent hepatocyte-like cells (H) were clearly identified (Fig. 4A). Bile canalicular structures with microvilli protruding into the lumen and no alterations in the endoplasmic reticulum (ER) were observed.