Fig. 1: Production and characterization of immature prM-containing TBEV.
From: Irreversible furin cleavage site exposure renders immature tick-borne flaviviruses fully infectious
a Schematic overview. LoVo cells (Δfurin) were infected with TBEV (MOI = 2). At 12 h post-infection (hpi), unbound virus was removed, the cells were washed with PBS, and fresh medium was added. At 48 hpi, prM-TBEV virus was harvested. b Western blot analysis of TBEV and prM-TBEV. Proteolytic processing of m-TBEV by furin yielded a completely cleaved prM protein. Without furin, the prM-TBEV sample contained a majority of immature prM-containing particles, as confirmed by the band corresponding to uncleaved prM protein. c Viral titres of m-TBEV and prM-TBEV determined by plaque assay, compared with viral RNA levels from quantitative PCR with reverse transcription (RT-qPCR). d Susceptibility of mammalian (PS, VERO, BHK-21) and tick (IRE/CTVM19) cell lines to prM-TBEV and m-TBEV. e, f Role of furin during host cell entry. LoVo cells were infected with TBEV and prM-TBEV (MOI = 0.1). The viral load in supernatant was determined by plaque assay, and infectivity of the prM variant was visualized using mCherry_prM-TBEV and mCherry_TBEV. g, h To confirm that furin was necessary during entry steps of prM-TBEV infection, PS cells were pretreated with the furin inhibitor decanoyl-RVKR-CMK (100, 50, 25, and 0 μM). Cells were then infected with mCherry variants of the virus, as described above. At 48 hpi, infection was visualized, and the percentage of inhibition was determined by plaque reduction assay. Each image is representative of two separate experiments (n = 3). Dashed lines correspond to the detection limit of the plaque assay. Scale bars = 200 μm. Data from experiments presented in c–e are from biological duplicates or triplicates (n = 3) presented as mean values ± SD. The statistical significance was calculated using Mann-Whitney test for comparing two groups; ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Schematic elements in panel a and selected annotation graphics were created in BioRender. Ruzek, D. (https://BioRender.com/4n4b27g).
