Fig. 3

Zika infection induces autophagy and inhibition of autophagy decreases lipid droplets. A MDCKs were infected with ZIKV at MOI 1 for 6, 12, 24 and 48 h. Immunofluorescence for LC3 was used as a marker for induction of autophagy. More than minimal punctation was never seen in mock-infected cells. Activation of autophagy, seen early during infection at 6 and 12 h as evidenced by bright punctation, drops by 24 h (less puncta) and is restored to initial levels by 48 h. B MDCKs were pretreated with autophagy inhibitor wortmannin at 50 mM, 1 h before infection at MOI 1 for 48 h. Lipid droplets were stained with ORO during immunofluorescence. Wortmannin partially blocks the induction of lipid droplets by zika (Compare Zika + wort to Zika). Viral replication is also decreased in the presence of wortmannin as detected by immunofluorescence. C This reduction in lipid droplets is statistically significant as analyzed by ImageJ (Compare wort + Zika to wort). Quantifications are analyzed in more than 200 cells for each condition. D MDCKs were pretreated with autophagy inhibitor wortmannin at 50 mM, 1 h before infection at MOI 1 for 48 h. Viral replication was measured by using an Anti-E protein primary antibody (green). Wortmannin reduced the production of Zika E protein (Compare Zika to Wort + Zika). E qPCR measuring ZIKV NS1 mRNA was performed to verify the effects of ATV on viral replication. ATV pretreatment reduced ZIKV replication approximately 50%. F Quantification of ZIKV E protein expression shows statistically significant decrease %) in infection following wortmannin treatment. Percentage of infection. calculated as 100 x (cells expressing ZIKV E protein (green/total cells)