Hit compounds target virus replication and/or virus egress, but not virus entry.

(A) Kinetics of N, Gn or S-Gn expression in RVFV-infected HeLa cells (MOI = 10) during a single replication cycle. Cells were fixed at various time points post-infection (PI) (0 h) as indicated in the x-axis. (B) Kinetics of N, Gn or S-Gn expression in RVFV-infected HeLa cells with hit compound treatment. (C) Time-of-addition assay in which HeLa cells were either mock treated or treated with the hit compounds at 2 h before prior to (−2), at the same time (0), or 2 h after virus infection. Cells were fixed at 13 h PI and immunostained to detect S-Gn expression. As a control for viral cell entry, cells were treated with 20 µM NH4Cl, a known inhibitor of endocytosis. (D) Time-of-addition assay in which HeLa cells were either mock treated or treated with the hit compounds at various times PI as indicated on the x-axis. Cells were fixed at 13 h PI and immunostained to detect S-Gn expression. (E) Relative changes in the RNA levels at cellular level or in the supernatants of compound treated cells at 12 h PI. Cells were mock treated or treated with the 4 hit compounds from 2 h prior to the start of infection through the entire duration of infection. In a duplicate experiment the percentage of cells expressing Gn and S-Gn was determined by HCI of the immunostained cells. The relative fold change was determined by normalizing data of compound treated cells with values of corresponding mock treated cells.