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

The efficiency with which RV is able to infect different cell lines differs dramatically; however, it is not known which steps restrict RV infection in cells that do not support infection by this virus. Pseudotype viruses are useful tools for analyzing the susceptibility of specific viruses separately from their intracellular replication processes. A pseudotype virus with RV envelope proteins has been reported previously 31 . This is a lentiviral pseudotype virus based on the simian immunodeficiency virus (SIV). However, the SIV vector failed to incorporate the intact forms of RV envelope proteins 31 . Therefore, the SIV pseudotype virus bearing modified RV envelope proteins with the cytoplasmic tail of the VSV G protein was used in the study 31 . In the present study, a new 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 pseudotype virus bearing the intact forms of the RV envelope proteins was generated using a VSV pseudotype system. Although the E1 protein alone causes membrane fusion and supports virus entry, the E2 and C proteins play roles in RV entry. After E1 protein binding to a receptor, RV enters cells by endocytosis 44 . Exposure to the low pH environment in early endosomes induces conformational changes in the E1 and E2 proteins and membrane fusion 34, 36 concomitantly with a solubility change in the C protein 36 . The C protein also has a supportive role in membrane fusion 31 . The mechanism of fusion enhancement by the C protein is still unclear, but the C protein likely supports the maturation or stabilizes either E2 and E1 or their interactions during intracellular transport to the cell surface 31 . As expected, co-expression of the C protein promoted the production of infectious VSV pseudotype virus with RV envelope proteins (VSV-RV/CE2E1). Infection by the novel VSV-RV/CE2E1 pseudotype virus was indeed mediated by RV envelope proteins, and the virus underwent a similar entry process to that of the authentic RV as previously reported 35, 44 .