Figure 5. Overexpression of Tsg101 accelerates entry kinetics of rLCMV-LASVGP.

(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 Fig. 1A 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).