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

A DSP-based fusion assay. A DSP-based-fusion assay was performed as described previously 56, 57 . A fusion protein of Renilla luciferase and GFP was split into two fragments designated dual split proteins, DSP 1-7 and DSP 8-11 56 . Although each fragment lacks the activities as Renilla luciferase and GFP, they reassemble and become functional when they are expressed within an identical cell simultaneously. 293CD4/DSP 1-7 and 293FT/DSP [8] [9] [10] [11] cells constitutively expressing DSP 1-7 and DSP [8] [9] [10] [11] , respectively, were mixed and cultured together, and transfected with pcDNA3.1-CE2E1, pcDNA3.1-E2E1 or an empty pcDNA3.1 vector. At 32 h posttransfection, the cells were incubated with a low pH (pH5.1) culture media for 15 min to induce a cell-to-cell fusion by RV envelope proteins followed by the incubation with a standard culture media for 8 hours at 37 °C. Then the Renilla luciferase activity derived from the reassembled DSPs by a cell fusion were measured using the Renilla Luciferase Assay System (Promega, Madison, WI) and GloMax 20/20 Luminometer (Promega). The expression levels of Renilla luciferase were normalized by the total expression levels of E1 proteins detected by an immunoblotting as described above.