Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

Sakata, Masafumi; Tani, Hideki; Anraku, Masaki; Kataoka, Michiyo; Nagata, Noriyo; Seki, Fumio; Tahara, Maino; Otsuki, Noriyuki; Okamoto, Kiyoko; Takeda, Makoto; Mori, Yoshio

To generate VSV GFP -RV/CE2E1, 293T cells, cultured in 6-well collagen-coated plates (8.0 × 10 5 cells/well), were transfected with the pcDNA3.1-CE2E1 expression plasmid using branched polyethylenimine (Sigma-Aldrich). At 32 h posttransfection, the cells were infected with VSV GFP -G at a multiplicity of infection (MOI) of 3.0 13, 16, 22, 30, 63 . The VSV GFP -G genome lacks the G gene, which is replaced with the GFP gene 13 . On the viral envelope the VSV GFP -G particles contain the G protein, which is provided in trans using the pC-VSV-G expression plasmid 13 ). The cells were washed four times with DMEM and incubated with DMEM containing 10% FBS. After 24 h, the culture supernatants containing VSV GFP -RV/ CE2E1 were harvested and centrifuged at 10,000 × g for 5 min at 4 °C to remove the cell debris. VSV FLuc -RV/ CE2E1 was generated similarly to VSV GFP -RV/CE2E1 using VSV FLuc -G, which encodes the FLuc gene, instead of the GFP gene 14, 28 , at a MOI of 0.3. VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1 were also generated similarly to VSV GFP -RV/CE2E1 and VSV FLuc -RV/CE2E1, respectively, using pcDNA3.1-E2E1 instead of pcDNA3.1-CE2E1. VSV GFP -G, a gift from Dr. M. A. Whitt (University of Tennessee, TN), and VSV FLuc -G were propagated as reported previously 13, 14 . VSV GFP -∆G and VSV FLuc -∆G, which lack envelope proteins, were generated using pcDNA3.1 + (Thermo Fisher Scientific) instead of pcDNA3.1-CE2E1 13, 14 . VSV FLuc -MLV/Env was generated similarly to VSV FLuc -RV/CE2E1 using pFBASALF 58 instead of pcDNA3.1-CE2E1. VSV GFP -MV/FH and VSV FLuc -MV/ FH were also generated similarly to VSV GFP -RV/CE2E1 and VSV FLuc -RV/CE2E1, respectively, using pCXN-Ed-F and pCA7PS-Ed-H 30, 59, 60 instead of pcDNA3.1-CE2E1. To generate VSV GFP -MV/FH and VSV FLuc -MV/FH, a fusion-blocking peptide (Z-D-Phe-Phe-Gly) (The Peptide Institute, Osaka, Japan) was used to inhibit syncytium formation during the production of these pseudotype viruses, as reported previously 30, 59 . VSVΔG/GFP-*G 63 Electron microscopy image of viral particles. The stock solution of VSV GFP -RV/CE2E1 was centrifuged at 1,500 × g for 10 min at 4 °C twice to remove cell debris. The viruses were collected by centrifugation at 120,000 × g for 1 h at 4 °C onto 20% (wt/vol) sucrose cushions 9 . The pellets were resuspended in phosphate-buffered saline (PBS). The ultracentrifugation step was repeated. After fixation with 2% paraformaldehyde in PBS, the viruses were placed on a carbon-coated grid for 45 s, rinsed with distilled water, stained with 2% phosphotungstic acid solution and observed with an electron microscope (TEM-1400, JEOL, Tokyo) operating at 80 kV as previously reported 64 . Flow cytometry for detection of the E1 protein. 293T Immunoblotting for detection of the E1 protein. 293T cells were transfected with pcDNA3.1-CE2E1 or pcDNA3.1-E2E1 expression plasmids. At 36 h posttransfection, the cells were analyzed by immunoblotting using anti-RV E1 or anti-GAPDH antibody as previously reported 33 . The signal intensity of the E1 protein was normalized by that of GAPDH.