Fig 2.

TOSV NSs does not suppress cellular general transcription. (A) 293 cells were mock infected (a to c, k to m) or infected with TOSV at an MOI of 1 (n to p), RVFV MP-12 at an MOI of 3 (d to f), or rMP12-TOSNSs at an MOI of 3 (g to i). From 23 to 24 hpi, cells were treated with 1 mM 5-ethynyl uridine (5-EU) to label newly synthesized RNA. As a control for transcriptional suppression, cells were treated with 5 μg of ActD/ml concurrently with the 5-EU treatment (a to c). After fixing and permeabilization, labeled RNA was detected by click chemistry with an Alexa Fluor 594-coupled azide (red). To visualize expression of viral proteins, the cells were stained with either a polyclonal anti-RVFV (a to i) or anti-TOSV (k to p) antibody, followed by an Alexa Fluor 488-coupled secondary antibody (green). The cells were analyzed by fluorescence microscopy. (B) 293 cells were mock infected or infected with TOSV at an MOI of 1. From 14 to 16 hpi, the cells were treated with 0.5 mM 5-EU to label newly synthesized RNA. As a control for transcriptional suppression, cells were treated with 5 μg of ActD/ml concurrently with the 5-EU treatment. After fixing and permeabilization, labeled RNA was detected by click chemistry with an Alexa Fluor 647-coupled azide. To visualize expression of viral proteins, the cells were stained with a polyclonal anti-TOSV antibody, followed by an Alexa Fluor 488-coupled secondary antibody. The cells were analyzed by flow cytometry.