FIG 6.

Accumulation of ZIKV sfRNAs is essential for productive infection in human cells. (A) ZIKVAr ΔSLI-II infection is quickly cleared in A549 cells. Immunofluorescence shows ZIKV antigen positivity and DAPI staining in A549 cells infected at an MOI of 10 with WT or mutant ΔSLI-II virus. Cytopathic effect was observed with the WT virus at 72 hpi. (B) ZIKV RNA genome accumulation as a function of time assessed by RT-qPCR for the WT and mutant as indicated. (C) Northern blot showing the appearance of sfRNAs as a function of time from 2 to 24 h after infection of A549 cells with WT virus. RNA markers are shown on the left, and sfRNAs are indicated on the right. (D) Induction of cytokines produced in A549 cells infected with ZIKVAr WT or ZIKVAr ΔSLI-II under the same conditions as for panel A. IFN-β, TNF-α, and IL-6 mRNAs were evaluated by RT-qPCR and are expressed as fold induction with respect to the noninfected control and relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Shown are the means with SD of two replicates. Statistics were performed with an unpaired t test, with P values represented as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ns, not significant. (E) Induction of ISG in ZIKVAr WT- or ZIKVAr ΔSLI-II-infected cells. MxA, OAS-1, ISG-15, and IRF-7 mRNAs were evaluated by RT-qPCR and expressed as fold induction with respect to the control and relative to GAPDH mRNA. Shown are the means with SD of two replicates; statistics were performed as described for panel D. (F) STAT2 is efficiently degraded after 24 h of infection with WT or mutant ΔSLI-II virus in A549 cells. Western blots show human STAT2, viral structural capsid protein, nonstructural protein NS3, and GAPDH control.