Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase
Fig 5
Analysis of replication-competent (RC) and replication-deficient (RD) MGs with mutated genome 3’-ends.
(A) Genome 3’-end of the wt MG and three mutant derivatives that either lack the 3’-terminal G residue (Δ1), the 3’-terminal two residues (Δ2) or carry an extra 3’-G residue (+G). (B-C) Corresponding reporter gene assays of lysates from cells transfected with MG variants illustrated in panel A, either as part of the (B) RC MG or (C) RD MG backbone. Mean activity values (± SEM) of the native 3’-leader MG (dark gray bars) and mutant MGs (light blue bars) were derived from 3 independent experiments with 3 technical replicates each; data for the wt MG were set to 100%. As negative control, the plasmid encoding the L gene was omitted during transfection (–L; black bars). (D-F) Corresponding two-step qRT-PCR of RC MG samples using the same cells as in panel B (for qRT-PCR setup, see S2C Fig); color code as in panel B and C. Mean 2-ΔΔCT values (± SEM) of viral mRNA (D), cRNA (E) and vRNA (F) derived from 3 independent experiments with 3 technical replicates each. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired Welch’s t test).