Figure 3
From: Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging
VP24 169–176A does not support trVLP production. (a) A schematic of the EBOV transcription/replication-competent virus-like particle (trVLP) assay. In producer cells (p0), initial transcription of the tetracistronic minigenome (4xMG) by the T7 polymerase produces a viral RNA (vRNA) template, which is then replicated, via complementary RNA (cRNA) intermediates, by the EBOV ribonucleoprotein complex, consisting of L, NP, VP35, and VP30. From the minigenome, the ribonucleoprotein complex also transcribes Renilla luciferase, VP40, GP1,2, and VP24 mRNA, which is then translated by host cellular machinery. NP encapsidates the vRNA, along with VP35, VP30, VP24, and L, and the nucleocapsids interact with the matrix protein VP40 and the glycoprotein GP1,2, to form trVLPs. trVLPs are capable of infecting target cells (p1) pre-transfected with the EBOV ribonucleoprotein complex and mounting a second round of replication. Renilla luciferase signal in producer cells is proportional to EBOV minigenome replication and transcription, and in target cells it is additionally proportional to the amount of trVLPs produced and capable of infecting target cells. Notably, VP24 is required for the production of infectious trVLPs. (b,c) HEK 293T producer cells were co-transfected with p4cis-vRNA-RLuc (p4cis), encoding wild-type (WT) VP24, VP24-3x-stop, or VP24 169–176A, as well as pCAGGS-luc2, encoding Firefly luciferase as a transfection control, pCAGGS-T7, pCAGGS-L, pCAGGS-NP, pCAGGS-VP35, and pCAGGS-VP30. trVLPs in the supernatant were harvested and used to infect HEK 293T target cells pre-transfected with pCAGGS-Tim1, pCAGGS-L, pCAGGS-NP, pCAGGS-VP35, and pCAGGS-VP30. Data are presented as relative light units (RLU) on a log scale with the Renilla luciferase signal in target cells (p1) minus the Renilla luciferase signal in producer cells (p0) normalized to WT VP24 (b) or with the Renilla luciferase signals in producer (p0) and target cells (p1) indicated separately (c). The Renilla luciferase signal in producer cells was normalized against the average Firefly luciferase signal. The means and standard error of the mean for 3 independent experiments are shown (n.s., not significant; ***p ≤ 0.001; ****p ≤ 0.0001).
