5, 6 EM of HCVsp-RG cells revealed the presence of several morphological alterations (Fig. 4B). A membranous web composed of small vesicles was identified in many cells (a). Multiple small vesicles connected to the membrane were observed (b). Multivesicular bodies (MVBs) accumulated internal vesicles (c). Submembranous thickening of cytoskeleton at the apical pole was visualized (d). CH5424802 price Remarkably, karmellae-like,

multilayer structures typical of membrane rearrangements associated with RNA replication by varied (+)RNA viruses15 were found specifically in HCVsp-RG cells (e). Some rare HCVsp-RG cells exhibited typical apoptosis-associated morphological alterations like formation of apoptotic bleeds (f). Immuno-EM

was performed to localize E1E2 Ag recognized by D32.10 in HCVsp-RG cells at the same culture time as morphological studies. Figure 5A shows that immunogold labeling for E1E2 was observed associated with 40-100 nm vesicles budding at the plasma membrane, resembling exosomes. To support potential association of E1E2 with exosomes, double-label immunogold EM experiments were performed using anti-E1E2/D32.10 (20 nm) and anti-HSC70 (5 nm). As shown in Fig. 5B, colabeling of HSC70 with E1E2 on the internal vesicles accumulated under the plasma membrane was observed. No immunolabeling with D32.10 was detectable in the noninfected HepaRG control cells (data not shown). The differentiation-inducible properties and the typical features of fully functional mature hepatocytes exhibited by HepaRG cells5, 6 make them attractive candidates for infection with naturally circulating HCV selleckchem particles isolated from PRKD3 chronically infected patients.7 Interestingly, the infection was primed in progenitors, whereas relatively robust sustainable replication and propagation of the infection only occurred in fully differentiated HepaRG cells with HCV RNA amplification up to 6 log10 for at least 1 to 2 months. Remarkably, the presence of 1% NHS during the infection process of HepaRG cells with HCVsp resulted in a more rapid

internalization and steady HCV RNA production in culture supernatants from 3 up to 9 weeks. This supports a possible synchronization of infection through serum factors such as high-density lipoproteins (HDLs), which have been shown to facilitate the entry of HCVpp and HCVcc into target cells.13 The endocytosis of viral particles could thus be accelerated by suppression of a time lag in which cell-bound virions are not internalized. These conditions appeared therefore optimal for mimicking natural infection. Because of the weak, very transient, delayed, and often artifactual detection of negative-strand viral RNA in infected cells, the HCV RNA amplification in the culture medium as enveloped complete virions10 and the detection of HCV structural proteins in the cells were used as infectivity assays.