IFNs promote the establishment of persistent Ad infection.
(A–C) HDF-TERT cells were infected with dl309 (A), Ad5-WT (B) or Ad5-mut1 (C) at 25 virus particles/cell. Infected cells were cultured in the presence and absence of IFNα or IFNγ. Growth media plus or minus IFNs were replenished every 5 days. Periodically, infectious virus yields were determined by plaque assay (A, B) and viral DNA replication was quantified by qPCR (C). (D and E) The viability of infected cells was determined by trypan blue staining at the indicated times, and plotted as a percentage of total cells (D) or the number of total viable cells (E). (F) Immunofluorescence assays were used to determine the percentage of Ad protein-positive cells. At 113 days post-infection, Ad5-infected HDF-TERT cells treated with IFNγ were immunostained for Ad early proteins (E1A, DBP, E4-ORF3) and late proteins (pVII, Hexon). Cells were scored positive or negative for the expression of viral proteins, and the percentages of 250 cells in 5–6 random views were plotted. E and L stand for early and late protein expression, respectively. (G and H) The change from persistent infection to lytic infection upon withdrawal of IFNγ was examined. At 107 days post-infection, Ad5-WT-infected HDF-TERT cells treated with IFNγ (shown in panel B) were seeded into new plates in duplicate. When the cells reached ~80% confluence, IFNγ was removed from one set and IFNγ was maintained in the second set. Cells were subcultured at day 5 and growth medium was replenished at day 10. Ad5 DNA replication was quantified by qPCR (G) and infectious virus yield was determined by plaque assay (H) at days 5, 10 and 15. The plots represent the average of two independent experiments.