The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging
A. Effect on PKR and p62. A549 cells or A549 cells treated with 5 µg/ml actinomycin D were infected with rMP12 or rMP12:S-Swap at a MOI of 3, or mock-infected. Cell extracts were prepared at the time points indicated, proteins fractionated by SDS-PAGE, and blots probed with anti-N, anti-NSs, anti-PKR, anti-p62, and anti-tubulin antibodies as indicated. B. Induction of interferon. A549 cells were infected with rMP12, rMP12ΔNSs:eGFP, rMP12:S-Swap or rMP12:S-SwapΔNSs:eGFP at MOI of 5, and supernatants harvested at 18 h p.i. After UV treatment, 2-fold dilutions were applied to A549-NPro cells for 24 h, before infection with EMCV. Monolayers were stained with Giesma after a further 96 h. C. The amount of IFN produced is expressed as relative IFN units (RIU), defined as RIU = 2N where N = the number of two-fold dilutions of the supernatants that protected the reporter cells from EMCV challenge.