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

Sucrose gradient fractionation analysis of pseudotype viruses. The pseudotype virus particles from VSV FLuc -RV/CE2E1 and VSV FLuc -G in 10 ml stock solutions were precipitated using PEG-it Virus Precipitation Solution (System Biosciences, Mountain view, CA), and the precipitated virus particles were then resuspended in 0.5 ml of DMEM. RVLP, which have been reported previously 33 were generated and also precipitated and resuspended in 0.5 ml of DMEM, as were VSV FLuc -RV/CE2E1 and VSV FLuc -G. The resuspended VSV FLuc -RV/CE2E1, VSV FLuc -G and RVLP were layered onto 12 ml of a 20 to 40% (wt/vol) sucrose density gradient and fractionated by ultracentrifugation in an SW41 rotor (Beckman Coulter, Tokyo, Japan) at 145,000 × g for 2 h. Each 1 ml of the fractionated samples in the gradient was collected from the top to the bottom, and a small part of each fraction was diluted with an equal volume of DPBS. Next, the diluted samples were inoculated into the Vero cell culture media in the 96-well plates. For VSV FLuc -RV/CE2E1 and VSV FLuc -G, the RLU from the cells was detected after 24 h of incubation using the Bright-Glo Assay System (Promega) and POWER SCAN HT (BioTek, Winooski, VT), after which RLU was used as an indicator of infection with the pseudotype viruses. For RVLP, the RLU from the cells was detected after a 72 h incubation period using the Renilla Luciferase Assay System and GloMax 20/20 Luminometer, after which RLU was again used as an indicator of infection with RVLP.