The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit
Fig 1
PIV5-W3 protein synthesis and transcription are repressed with time p.i. in an IFN-independent manner.
Panel a) Monolayers of A549 cells were either mock infected or infected with PIV5 W3 at 10 pfu/cell in the presence or absence of Ruxolitinib (2μg/ml). At the times indicated the cells were metabolically labelled for 1h with [35S]-L-methionine. Polypeptides present in total cell extracts were separated by electrophoresis through a 4–12% SDS-PAG, and the labelled polypeptides visualized using a phosphorimager. The position of the NP and M polypeptides are indicated by asterisks (see S2 Fig). Note: PIV5 infection does not specifically switch off host cell protein synthesis and thus the relative levels of host cell protein synthesis in each track can be used as loading controls (S2 Fig). Panel b) The relative amounts of accumulated NP in the same samples shown in panel a) were visualized by immunoblot analysis. Also included as a control for IFN-induced MxA induction was a total cell lysate of mock-infected cells treated with IFN for 24h (control); immunoblotting for actin was used as loading controls. Panel c) Monolayers of A549 cells grown in 25cm flasks were infected with PIV5 W3 at 10 pfu/cell, RNA was extracted at 6, 12, 18, 24, 48 and 96h p.i. and subjected to total RNA sequencing following rRNA and mitochondrial RNA reduction. Directional sequence analysis was performed and the percentage of viral mRNA and genome reads were compared to the cellular reads at each time point. Error bars are based on three independent experiments. Note that although it is not possible to distinguish reads generated from viral mRNAs from those generated from antigenomes by directional sequencing, in the data presented here, because the proportion of antigenome reads cannot exceed that of the L gene extended over the whole genome, we can infer that the contribution of antigenome reads was <2% of the total mRNA/antigenome reads.