Fig. 4.

Generation of DI-rich preparations of PIV5-VΔC. (A) A549/pr(IFN-β).GFP cells were infected at 5 PFU/cell with PIV5-VΔC vM0 or vM2 stocks, or mock-infected, in the presence of cycloheximide (50 μg/ml). Two hours later, monolayers were harvested and analysed for input virus NP and actin by SDS-PAGE and immunoblotting. (B) A549/pr(IFN-β).GFP cells were infected at an MOI of 10 PFU/cell with PIV5 wt vM0, PIV5-VΔC vM2 or 50:50 mixtures of both of these viruses, or mock-infected. At 20 h p.i. the monolayers were radioactively labelled with [³⁵S]methionine and the labelled polypeptides in total cell extracts were visualised by SDS-PAGE analysis and autoradiography. The positions of the viral NP and M proteins are highlighted with an *. (C) A549/pr(IFN-β).GFP cells were infected at an MOI of 10 PFU/cell with PIV5 wt vM0, PIV5-VΔC vM2 or 50:50 mixtures of both of these viruses. At 20 h p.i. cells were fixed and the distribution of NP was visualised by immunostaining and fluorescence microscopy. (D) Total RNA was prepared from either mock-infected 293 cells or 293 cells infected at an equivalent PFU/cell with either PIV5-VΔC vM0 or PIV5-VΔC vM2, and then subjected to RT-PCR as described in Materials and methods. Reverse transcription was performed with primer A or B, and then PCR was carried out with primer pairs B + C (vRNA primers) or A + C (copyback primers). Primers B and C are in opposing orientations and permit amplification of any PIV5 RNA generated by authentic replication. Primers A and C are both from the same strand and can only generate a PCR product if the template has switched strands. Products were analysed on a 1.2% agarose gel and the presence of vRNA and copyback molecules is indicated. The faint DNA fragment seen in the mock-infected sample with the copyback primers has a similar but distinct mobility to that seen with the copyback fragment and is non-specific.