Figure 2.

Properties of the VSV-RV/CE2E1 pseudotype virus. (A) Infectivity of VSV^GFP-RV/E2E1, VSV^GFP-RV/CE2E1, and VSV^GFP-∆G. Vero cells were infected with VSV^GFP-RV/E2E1, VSV^GFP-RV/CE2E1 or VSV^GFP-∆G, and at 24 h post-infection (p.i.) the cell infectious units (CIUs) were determined by counting the number of GFP-expressing cells. (B) Infectivity of VSV^FLuc-RV/E2E1, VSV^FLuc-RV/CE2E1, and VSV^FLuc-∆G. Vero cells were infected with VSV^FLuc-RV/E2E1, VSV^FLuc-RV/CE2E1, or VSV^FLuc-∆G, and at 24 h p.i. the luciferase activities in the cells were measured.(C) Effect of the C protein on the cell surface expression of the E1 protein. Expression of the E1 protein on the surface of 293 T cells transfected with pcDNA3.1-E2E1 or pcDNA3.1-CE2E1 were detected by flow cytometry using anti-RV E1 antibody. The left panel shows percentage of cells expressing the E1 protein on the cell surface, and the right panel shows expression level of the E1 protein in the positive cells indicated as a mean fluorescent intensity (MFI). (D, E) The total amount of the E1 protein in cells co-expressed with or without the C protein. 293CD4/DSP_1–7 and 293FT/DSP_8–11 cells constitutively expressing DSP_1–7 and DSP_8–11, respectively, were mixed and cultured together. The cells were transfected with pcDNA3.1-E2E1, pcDNA3.1-CE2E1 or the empty vector and incubated for 32 hours. (D) The cell lysates were subjected to immunoblotting with anti-RV E1 and anti–GAPDH antibodies. The signal intensity of the E1 protein in the cells was normalized by those of GAPDH. The full-length images are shown in Supplementary Fig. 2. (E) The cells were incubated with low pH (pH 5.1) media for 15 min and then were cultured with the standard culture media for 8 h. The Renilla luciferase activity derived from fusion cells were measured and normalized by expression levels of the E1 protein determined in (D). (F) Electron microscope image of particles in the VSV-RV/CE2E1 stock solution. Purified virions in the VSV^GFP-RV/CE2E1 stock solution were fixed with 2% paraformaldehyde, and then were negatively stained with 2% phosphotungstic acid solution. The arrows indicate spherical particles. Bar indicates 100 nm. (G, H, I) Viral particle densities. VSV^FLuc-G (G), VSV^FLuc-RV/CE2E1 (H), and RV virus like-particles (RVLPs) (I) were fractionated in a sucrose-gradient by ultracentrifugation. Twelve fractions were collected from top to bottom of the gradient. VSV^FLuc-G, VSV^FLuc-RV/CE2E1, and RVLP were detected by infecting Vero cells with individual fractions from the gradient and measuring the luciferase activities in the cells. (B, E G–I) RLU, relative light unit of luciferase activity. (A-E, G-I) Data represent the mean values ± standard deviations for triplicate samples. (A-E) The significant differences were determined by two-tailed t-tests.