Figure 6.

R682G and S813Y mutations facilitate rVSV-S replication. (A) HEK293T-hACE2 cells were infected with the SΔ19 Rep virus with R682G or R682G-S813Y mutation. Virus titer in the culture supernatant was measured (moi=0.1, 3 dpi, n=4). (B) Virus-infected cell lysate (moi=1, 1 dpi) was subjected to immunoblotting with anti-S2 antibodies. (C) The viral particles in the culture supernatant were digested with TPCK-trypsin (2 μg/ml at 37°C for 30 min) and analyzed by immunoblotting. (D) SΔ19 beta variant (B.1.351) gene with and without R682G mutation was inserted into the VSVΔG-GFP DNA vector and co-transfected with all the required helper plasmids in HEK293T-hACE2 cells to rescue recombinant VSVΔG-SΔ19 (B.1.351)/G virus. Virus replication was monitored by GFP expression. The rescued viruses were collected to infect BHK21-hACE2 cells with limiting dilution, and further amplified in HEK293T-hACE2 cells. (E) The Spike protein expression pattern of the replication-competent SΔ19 (B.1.351-R682G) virus was examined by immunoblotting alone with SΔ18 and SΔ18 (B.1.351) pseudoviruses for comparison. Scale bar: 100 μm. *P < 0.05.