Figure 4.

UCA1 Expression Specifically Targets Oncolytic Vaccinia Virus Cell-to-Cell Spread and Increases the Oncolytic Effect of OVV-LG

(A and B) KFlow, UCA1-KFlow, and Empty-KFlow cells were infected with OVV-LG (MOI = 10) for 30 min. After ample washes, the number of plaque-forming units (PFUs) of OVV was determined by titration using RK-13 cells (A), and viral luciferase activity was determined (B) (n = 3). (C–E) Gene expression in KFlow, UCA1-KFlow, and Empty-KFlow cells after either mock infection or infection with OVV-LG (MOI = 5). RNA was extracted at 1, 2, 4, 6, 8, 10, and 12 h postinfection (hpi). qRT-PCR was performed with primers specific for M1L (C), G8R (D), A5L (E), and GAPDH (C–E, as an internal control) (n = 3). (F) One-step growth curve. KFlow, UCA1-KFlow, and Empty-KFlow cells were infected with OVV-LG (MOI = 5). Cells (CAV) and supernatant (extracellular enveloped virus [EEV]) were collected, and the number of OVV PFUs was determined by titration using RK-13 cells (n = 3). (G) KFlow, UCA1-KFlow, and Empty-KFlow cells were treated with nocodazole (25 nM), colchicine (2.5 nM), and cytochalasin D (KFlow and Empty-KFlow, 25 nM; UCA1-KFlow, 50 nM) and infected with OV-LG (MOI = 0.001). Representative plaques formed under semisolid medium were photographed and calculated with the analysis application measurement module (Keyence) (n = 10). Data with error bars represent mean ± SEM.