Figure 1.

Lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) does not inhibit incorporation of respiratory syncytial virus (RSV) fusion protein (F) into vesicular stomatitis virus (VSV)-GP particles. 293T cells were transfected with expression plasmids for the wild type RSV F (F_wt), a codon optimized variant (F_syn) or a chimeric protein encoding the extracellular part of the codon optimized F and the transmembrane domain and the cytoplasmatic tail of VSV-G (F_syn:G) alone or in combination with an expression plasmid for LCMV GP. As controls, cells were left untransfected or the LCMV GP plasmid was transfected alone. (A) Cell lysates were prepared 24 h after transfection and analyzed via Western blotting for expression of RSV F (upper blot). As loading control an antibody against β-actin was used (lower blot); (B) cells were infected with the single-cycle infectious VSV*ΔG virus and supernatants were collected 24 h after infection. Virus was concentrated via low speed centrifugation through a sucrose cushion. Purified virus particles were analyzed via Western blotting for incorporation of RSV F (upper blot). As loading control, the same membrane was probed with an antibody against the VSV nucleoprotein (lower blot).We next analyzed the efficiency of incorporation of RSV F protein variants into VSV-GP particles by infection of transfected cells with replication defective VSV*ΔG virus, in which the gene encoding the envelope glycoprotein G is deleted and that expresses GFP. We purified newly generated virus particles via low speed centrifugation through a sucrose cushion and checked for incorporation of RSV F by Western blot analysis. As in F_wt transfected cells (Figure 1A), F protein was also not detectable in VSV*ΔG particles produced on these cells (Figure 1B). In contrast, high amounts of RSV F were detected in VSV*ΔG particles produced on F_syn or F_syn:G transfected cells (Figure 1B). However, fusion of F to the VSV-G transmembrane domain and cytoplasmatic tail did not increase incorporation into VSV-GP particles. The amount of VSV was normalized to the amount of N-protein on the Western blot analysis. We also found that the co-expression of LCMV GP did not limit incorporation of RSV F into viral particles, neither for F_syn nor for F_syn:G (Figure 1B). To study whether incorporation of RSV F into the viral particle alters the tropism of VSV-GP, we used VSV*ΔG particles either containing only RSV F_syn or F_syn together with LCMV GP to infect the African green monkey kidney cell lines Vero, and quantified GFP⁺ cells by FACS. VSV*ΔG particles produced on LCMV GP expressing cells or produced on naïve cells (mock) were used as positive and negative controls. Vero cells were efficiently infected with particles produced on GP expressing cells. However, VSV*ΔG particles with RSV F only were not infectious (Supplementary Figure S1).