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

RV indicated that many cell lines were nearly non-susceptible or non-permissible, or both, to RV infection. To assess the susceptibilities of individual cell lines to infection with RV, pseudotype VSVs bearing RV envelope proteins were generated. The VSV pseudotype virus genomes lack the G gene, which is replaced with a reporter protein gene, the green fluorescent protein (GFP) or firefly luciferase (FLuc) gene 8, 14, 18, 19 . The pseudotype viruses were thus expected to show similar behaviors to RV during the process of virus entry, and the subsequent processes of gene expression are dependent on the VSV replication machinery. In the initial experiment, only RV envelope E2 and E1 proteins were provided in trans to generate the GFP gene-and FLuc gene-encoding pseudotype viruses, VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1, respectively, like other VSV pseudotype viruses 13, [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . The infectivity titers for VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1 were 10-fold higher than those of the counterpart control viruses, VSV GFP -∆G and VSV FLuc -∆G, respectively, which lack envelope glycoproteins, in Vero cells ( Fig. 2A, B) . Although this suggests that RV envelope proteins contribute to the infectivity of the pseudotype viruses, they seem to have little practical application because of their low infective titers. Co-expression of the Capsid (C) protein resulted in production of the pseudotype viruses, VSV GFP -RV/CE2E1 and VSV FLuc -RV/CE2E1 and these pseudotype viruses showed higher infectivity titers than VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1, respectively ( Fig. 2A, B) . The infectivity titers for VSV GFP -RV/CE2E1 and VSV FLuc -RV/CE2E1 were 50-200-fold higher than those of VSV GFP -∆G and VSV FLuc -∆G, respectively. An experiment indicated that the RV C protein promotes fusion activity in RV envelope (E1 and E2) proteins by supporting the maturation or stabilizing either E2 and E1 or their interactions during intracellular transport to the cell surface 31 . We have confirmed the enhance effect by the C protein on fusion by RV envelope proteins. The surface expression level of the E1 protein with the C protein was similar to that without the C protein (Fig. 2C ). The total amounts of the E1 protein in cells were also similar between cells co-expressed with or without the C protein (Fig. 2D) . Nevertheless, the level of cell-to-cell fusion was increased by ~two-fold by co-expressing the C protein (Fig. 2E ). Although the detailed mechanism was unclear, the data demonstrated that the RV envelope protein expressed on the cell surface showed a better fusion activity than that expressed without the C protein. Thus, in the following experiment, the C protein was provided together with RV envelope E2 and E1 proteins. However, it should be noted that co-expression of the C protein with the E1 and E2 proteins may produce empty non-infectious RV-like-particles (RVLP). Indeed, an electron microscopic assay revealed both bullet-shaped (~61 ± 7 nm × 173 ± 18 nm; n = 5) and spherical particles (~73 ± 12 nm in diameter; n = 8), which were corresponding to VSV and RV virions, respectively 9, 32 . This suggests that pseudotype VSV and RVLP were contained in the VSV GFP -RV/CE2E1 stocks (Fig. 2F) . No particles showed a combined shape of the bullet and spherical forms. Although the VSV pseudotype virus genome was expected to be incorporated into the bullet shaped particles, it was confirmed by the following