Characterization of the DENV-faR reporter virus.

(A) Schematic of the DENV reporter virus genome. The first 103 nucleotides of the capsid coding sequence were duplicated, because they contain the circularization sequence 1 (CS1; blue bar) that is essential for DENV RNA replication. Thus, the far red reporter is fused N-terminally with ~34 amino acid residues of the capsid protein that contains a nuclear localization sequence and C-terminally with the sequence encoding for the 2A cleavage factor (PCS) of the Thosea asigna virus, thus generating the authentic N-terminus of the capsid protein. (B) A549 cells were infected with the DENV-faR reporter virus or the parental wildtype (wt) lacking a reporter gene at a MOI of 1 TCID50/cell. At time points specified in the bottom, culture supernatants were harvested and titers of infectious virus were determined by limiting dilution assay. (C) Single-step growth curve of DENV-faR in A549 cells. Cells were infected at a MOI of 10 TCID50/cell and harvested at multiple time points. Viral RNA was quantified by RT-qPCR and infectivity released from cells was monitored by limiting dilution assay. Graphs display a representative result out of 3 independent experiments. Values correspond to the mean of triplicate measurements and error bars. (D) Kinetics of activation of IFIT1 transcription and IFN-λ secretion. A549 cells were infected with DENV-faR at a MOI of 10 TCID50/cell and harvested at time points specified in the bottom of the graph. IFIT1 transcription was determined by RT-qPCR, whereas IFN-λ secretion was quantified by ELISA. IFIT1 mRNA values were normalized to GAPDH mRNA levels.