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

Human non-immune cells are generally susceptible to VSV-RV/CE2E1, whereas immune cells are much less susceptible than non-immune cells. We analyzed VSV-RV/CE2E1, VSV-G, and VSV-∆G for their infectivity levels in various human cell lines and Vero cells. To compare the different pseudotype viruses, the infectivity titers were standardized by the genome copy numbers in the virus stocks. The genome copy numbers in VSV GFP -RV/CE2E1, VSV GFP -G, and VSV GFP -∆G stocks used in the experiments [8] [9] [10] [11] , respectively, were mixed and cultured together. The cells were transfected with pcDNA3.1-E2E1, pcDNA3.1-CE2E1 or the empty vector and incubated for 32 hours. (D) The cell lysates were subjected to immunoblotting with anti-RV E1 and anti-GAPDH antibodies. The signal intensity of the E1 protein in the cells were 5.02 × 10 10 , 1.15 × 10 11 , and 4.29 × 10 10 , respectively, per milliliter. Also the genome copy numbers in VSV FLuc -RV/CE2E1, VSV FLuc -G, and VSV FLuc -∆G stocks used in the experiments were 9.35 × 10 9 , 2.81 × 10 10 , and 1.21 × 10 10 , respectively, per milliliter. VSV-G infectivity titers were generally much higher than those of VSV-RV/ CE2E1 (Fig. 4) . Not surprisingly, these data suggest that RV envelope proteins were less efficiently incorporated and/or less functional in VSV-based pseudotype virions than the authentic VSV G glycoprotein. Nonetheless, the VSV GFP -RV/CE2E1 infectivity titers were significantly higher than VSV GFP -∆G in many cell lines (Fig. 4) . VSV GFP -G infectivity titers in 293T, Huh7, NJG, JAR, and JEG3 cells were similar to that in Vero cells (Fig. 4A ), as were the infectivity titers of VSV GFP -RV/CE2E1 in 293T and NJG cells in Vero cells (Fig. 4A ). In contrast, the infectivity titers of VSV GFP -RV/CE2E1 in Huh7, JAR, and JEG3 cells were ~10-times greater than they were in Vero cells (Fig. 4A ). Vero cells are commonly used for propagating RV and it is well accepted that this cell line is susceptible to RV. These data therefore suggest that these non-immune cell lines (293T, Huh7, NJG, JAR, and JEG3) are similarly or more highly susceptible to RV than Vero cells. VSV GFP -G infectivity titers were reduced by 10 to 1,000-fold in HeLa, FLC-4, FaDu, Detroit562, HSQ89, and A549 cells, when compared with the titer in Vero cells (Fig. 4A, B) . Therefore, these cell lines may be less susceptible or less permissible to VSV infection. Even when considering these observations, the infectivity titers of VSV GFP -RV/CE2E1 in these cells were significantly higher than those of VSV GFP -∆G (Fig. 4B) , suggesting that these cell lines are also susceptible to RV. Similar experiments were performed using VSV FLuc -RV/CE2E1, of which the infection was highly sensitively quantified by the luciferase activity. In these experiments a VSV G protein-specific antibody, which neutralized VSV FLuc -G infection efficiently, was used to eliminate the possible effect by the residual VSV FLuc -G used for the production of VSV FLuc -RV/CE2E1. Data with VSV FLuc -RV/CE2E1 and the VSV-G neutralizing antibody confirmed the negligible or small effect by the residual VSV FLuc -G and the high susceptibility of non-immune cells to VSV FLuc -RV/ CE2E1 (Fig. 4C ).