Figure 3. Differential uptake of eHAV versus nonenveloped HAV by pDCs.
(A) pDCs were exposed to equal quantities of gradient-purified eHAV or nonenveloped HAV, and cell-associated viral RNA and supernatant IFN-α levels were determined at intervals. Circles and squares represent cells from 2 individual donors, respectively. (B) pDCs were incubated with eHAV in the presence or absence of annexin V (2 or 10 μg/ml), and cell-associated viral RNA was determined at intervals by RT-qPCR. (C) IFN-α produced by pDCs exposed to concentrated supernatant fluids from mock- or HM175/p16-infected cell cultures in the presence or absence of annexin V (2 μg/ml). (D) IFN-α produced by pDCs exposed to gradient-purified eHAV following inactivation by UV light or cultured with eHAV in the presence of 100 μM HAV 3Cpro inhibitor (3C-inh) (EC90 = 62 μM, CC50 = 3 mM). Efficiency of UV inactivation and 3C inhibitor treatment. Huh-7.5 cells were infected with UV-inactivated eHAV or nontreated eHAV in the presence of 3C-inh, and cell-associated HAV RNA was measured by RT-qPCR and compared with that in cells infected with untreated HAV at 48 hours. (E) pDCs were transfected with HM175/18f genomic RNA in the presence and absence of IRS-661 or subgenomic HAV-luc RNA or a replication incompetent variant HAV-Luc-Δ3D. Supernatant IFN-α levels were measured by ELISA. Results represent the mean ± SEM (n = 2 or 3 cultures) obtained with pDCs from single donors.