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. 2016 Feb 26;90(6):3257–3261. doi: 10.1128/JVI.03198-15

FIG 3.

FIG 3

Involvement of S1P/SKI-1 in LUJV assembly and production. (A) Schematic representation of the LUJV, LASV, and JUNV GPC cleavage site. The consensus sequence for S1P/SKI-1 cleavage is also shown. (B) 293T cells were transfected with plasmid pC-LUJV-GPC-FLAG or mutant forms thereof with RKLM changed to AAAA or RRRR. GPCs were detected with anti-FLAG antibody. Actin (used as a loading control) was also detected. (C) 293T cells were transfected with pC-LUJV-GPC-FLAG and treated with increasing concentrations of PF-429242 (0, 10, or 30 μM). At 48 h posttransfection, cell lysates were collected and the protein was detected by WB. Actin (used as a loading control) was also detected. (D) WT and mutant forms of LUJV GPC were analyzed for cell surface expression. Total cell lysate (top) and a cell surface biotinylated sample (bottom), pulled down by avidin beads, are shown. LASV ZΔ1 was used as a negative control. (E and F) GPC cleavage is important for its incorporation into VLPs. 293T cells were transfected with pC-LUJV-Z-HA, together with pC-LUJV-GPC-FLAG or its mutant forms, and at 48 h posttransfection, cell lysate and VLP fractions were analyzed by WB (E). 293T cells were transfected with pC-LUJV-Z-HA, together with pC-LUJV-GPC-FLAG, and at 6 h posttransfection, the culture medium was replaced with fresh medium containing increasing amounts of PF-429242 (0, 1, 10, or 30 μM). At 48 h posttransfection, cell lysate and VLP fractions were collected and the prepared samples were analyzed by WB (F). (G) Vero cells, which were seeded at 100% confluence, were infected with infectious LUJV at an MOI of 0.1, and the culture medium was replaced with fresh medium containing 30 μM PF-429242 at 1 h postinfection. After 48 h of incubation, the culture media were collected and infectious titers of LUJV were determined by the procedure described in the text. RNA was also extracted from the culture media for quantitative RT-PCR targeting the NP gene (S segment) to quantify LUJV genome copy numbers. Cell viabilities following the same treatment except virus infection are also shown at the bottom. TCID50, 50% tissue culture infective doses; DMSO, Dulbecco's modified Eagle's medium.