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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

Figure 6

Inhibition of host cell protein and RNA synthesis.

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).

Figure 6