Overexpression of Tsg101 accelerates entry kinetics of rLCMV-LASVGP.

<p>(A) Over-expression of a MYC-tagged recombinant Tsg101 by transient transfection of HEK293 cells. Cells were transfected with recombinant Tsg101 (Tsg101) or empty vector (control). After 36 hours total cell lysates were probed by Western-blot with antibodies recognizing Tsg101 or the MYC epitope. The MYC-tagged form of Tsg101 runs at a slightly higher apparent molecular mass, due to the presence of the tag (*). (B) Overexpression of Tsg101 accelerates the entry kinetics of rLCMV-LASVGP. Cells transfected with recombinant Tsg101 (Tsg101) or empty vector (Control) as in (A) were cooled on ice for 30 min and rLCMV-LFVGP added at an MOI of 3. After incubation for 1 h on ice, unbound virus was washed off, and cells shifted to 37°C. At different time points, medium containing 20 mM ammonium chloride was added. After a total of 16 h, cells were fixed and LCMVNP detected by intracellular staining with mAb 113 to LCMV NP, combined with Alexa 488-labeled secondary antibody. NP positive cells were quantified by flow cytometry as in Fig. 5D. Note the delay in the fusion kinetics relative to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g001" target="_blank">Fig. 1A</a> due to incubations of cells with ice cold medium after the washes. Data presented are means (n = 3+SD). (C) Entry kinetics in cells depleted of Tsg101. HEK293 cells were transfected with siRNA to Tsg101 or control siRNA as in Fig. 5A, resulting in >90% reduction of Tsg101 protein expression after 72 hours (data not shown). Cells treated with siRNA for 72 hours were cooled on ice for 30 minutes and rLCMV-LFVGP added at an MOI of 3. After removal of unbound virus entry kinetics was assessed as in (B). Data presented are means (n = 3+SD).