Figure 1

Construction and characterisation of oncolytic HSV viruses expressing shRNA. (a) Schematic representation of the oncolytic HSV genome and RNAi expression cassettes used in this study. Generation of OncoVEX viruses is described by Liu et al., 2003 and generation of replication-defective HSV-1 vectors is described by Anesti et al., 2008. (b) BHK cells were dually infected with a non-replicating HSV CMVeGFP virus and either Onc U6shGFP or Onc U6shLacZ, at an MOI of 1.0, and incubated for 24 hrs. The cells were digitally photographed using an UV-inverted microscope (Nikon Eclipse TE200) and Lucia Imaging (MV-1500 version 4.6). (c,d,e) BHK-LacZ cells ²⁹ were infected at various MOIs (1, 0.1, and 0.01) with oncolytic HSV viruses expressing shRNA (Onc U6shLacZ) or pre-miRNA (Onc miR-LacZ) against lacZ, or the negative control viruses Onc U6shGFP and Onc miR-neg. Infected cells were harvested at 24, 48 and 72 hrs and β-galactosidase protein levels were assessed by high sensitivity β-galactosidase assay (Stratagene). B-galactosidase levels (%LacZ) were expressed as a percentage of the expression measured in the cells infected with the negative controls (Onc U6shGFP for Onc U6shLacZ, and Onc miR-neg for Onc miR-Lac. (f) B-galactosidase activity (units/ml) in BHK-LacZ cells infected with Onc U6shGFP and Onc miR-neg control viruses at an MOI 1.0, and incubated for 24 hrs, 48 hrs and 73 hrs, or at an MOI 1.0, 0.1 and 0.01, and incubated at 72 hrs. BHK-LacZ cells constitutively express lacZ and thus, β-galactosidase levels are a good indication of cell survival. All P values were obtained using unpaired student t test. (g) BHK-LacZ cells were infected with OncoVEX, Onc U6shLacZ or Onc miR-LacZ at an MOI of 1.0, 0.1 and 0.01. MTS cell viability assay was performed at 24, 48 and 72 hours post-infection. Cell death increases with time and MOI of virus. Expression of shLacZ or miR-LacZ does not significantly affect cytotoxicity caused by the oncolytic virus. (h) BHK-LacZ cells were infected with OncoVEX, Onc U6shLacZ or Onc miR-LacZ. Growth curves were prepared by counting plaque forming units at 24, 48 and 72 hours post-infection. Expression of shLacZ or miR-LacZ does not significantly influence the growth of the oncolytic virus.