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. Protein synthesis. A549 or A549 NPro cells were infected with rMP12, rMP12:S-Swap, rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP at a MOI of 3. Cells were labelled with 30 µCi [35S] methionine/cysteine for 2 h at the time points indicated, and cell extracts were fractionated by SDS-PAGE. The positions of the viral N and NSs proteins are shown. Total lane intensities were measured by densitometry and compared to the mock-infected sample for each virus time course as indicated. B. RNA synthesis. A549 cells were infected with rMP12 (panels a to d) or rMP12:S-Swap (panels e to h) at a MOI of 3. One hour prior to the time points indicated the uridine analogue 5-ethynyl uridine (EU) was added to the medium and then cells were fixed in 4% formaldehyde. Cells were processed using Click-iT RNA AF488 Imaging Kit (newly synthesised RNA stains green), and then reacted with anti-NSs antibodies and secondary goat anti-rabbit Alexa Fluor 633 antibody (red). As controls, mock-infected cells were left untreated (i) or treated with actinomycin D (Act D) at 5 µg/ml for 1 h prior to 5-EU treatment (j).