Figure 5

VP24 is critical for EBOV genome encapsidation. (a) A schematic of the EBOV RNA immunoprecipitation assay. Transcription of the tetracistronic minigenome (p4cis), encoding VP24-3x-stop, by the T7 polymerase produces viral RNA (vRNA) template that can be encapsidated by NP with a C-terminal FLAG/HA tag (NP-FH), VP35, and VP24, supplied in trans. (b–g) HEK 293 cells were co-transfected with p4cis-vRNA-RLuc (p4cis), encoding VP24-3x-stop, as well as pCAGGS-T7, pCAGGS-NP-FH, pCAGGS-VP35, and wild-type (WT) pCAGGS-VP24 (b–d) or a VP24 mutant: pCAGGS-VP24 V170A, pCAGGS-VP24 N171A, pCAGGS-VP24 V170/N171A, or pCAGGS-VP24 169–176A (e–g). Lysates were immunoprecipitated with mouse anti-FLAG or an isotype control (Iso) antibody, and RNA was purified and then quantified via RT-qPCR. The total concentration in pg/ul of EBOV minigenome RNA precipitated was normalized against the total concentration of EBOV minigenome RNA in whole cell lysates for each transfection (b,e). Immunoprecipitation (IP) and whole cell lysate (WCL) fractions were subjected to Western Blot (WB) with mouse anti-FLAG, mouse anti-VP35, rabbit anti-VP24, or rabbit anti-β-tubulin antibodies (c,f). Western blots were cropped for presentation; full-length blots are available in Supplementary Fig. 9. The total concentration in pg/ul of cellular RNA in IP fractions was quantified using a Low Abundance RNA Quantification kit (d,g). The means and standard error of the mean for 8 (b) and 6 (e) independent experiments are shown, with the exception of the minigenome/T7-only control, for which 7 (b) independent experiments are shown (**p ≤ 0.01; ****p ≤ 0.0001).