Fig. 6.

Tsg101 depletion decreases infectivity release. a Mov 127S cells were transduced with lentivectors encoding Sh-CT (negative control, lane 1) or two different ShRNAs against Tsg101 (Sh-Tsg101-1, lane 2 and Sh-Tsg101-2, lane 3) and were puromycin-selected. Western blotting analysis of cell lysates (20 μg of proteins) shows that efficient inhibition of Tsg101 expression (top panel) does not affect PrP levels (middle panel). GAPDH (lower panel) was used as a loading control. b 100 K pellets were harvested from Mov 127S cells transduced with Sh-CT, Sh-Tsg101-1 or Sh-Tsg101-(1 + 2). Transduced cells and the corresponding 100 K pellets were analyzed before (−) and after (+) PK digestion for Tsg101 (upper panel), Alix (middle panel) and PrP (lower panel). As shown in (a), depletion of Tsg101 did not affect PrP expression (compare lane 1 with lanes 2–3) nor PrP^res accumulation (compare lane 4 with lanes 5–6) in transduced cells. In sharp contrast, PrP and PrP^res were strongly reduced in 100 K pellets from Tsg101-depleted cells (compare lane 7 with lanes 8–9 and lane 10 with lanes 11–12, respectively). c Left panel percentage of Alix release from Sh-CT versus Sh-Tsg101 cells. Right panel percentage of PrP^res release from Sh-CT versus Sh-Tsg101 cells. The data are from three independent experiments. Values are given as mean ± SD. d Analysis of cell-associated infectivity by the scrapie cell assay. Serial 1/3 dilutions of cell homogenates (corresponding to 30, 10, 3.3 and 1.1 μg of proteins) from SH-CT (lanes 1–4) and Sh-Tsg101-1 (lanes 5–8) transduced Mov 127S cells were inoculated to target ovRK13 cells. PrP^res in the challenged cultures was analyzed 4 weeks later by immunoblotting. Note that PrP^res accumulation was similar in ovRK13 challenged with SH-CT or Sh-Tsg101 cell homogenates (compare lanes 1–4 with lanes 5–8) indicating that depletion of Tsg101 did not affect prion multiplication in the cells. M corresponds to molecular weight markers. e Analysis of 100 K pellet-associated infectivity by SCA. 100 K pellets from Sh-CT or Sh-Tsg101-1 transduced Mov 127S cells were inoculated to target ovRK13 cells and PrP^res in the challenged target cells was analyzed 4 weeks later by immunoblotting. Note the strong decrease of PrP^res signal indicating that the 100 K pellets from Tsg101-depleted cells are much less infectious (lane 3). No PrP^res was detected when recipient ovRK13 cells that did not express the PrP^C protein (-dox) were inoculated (lane 1). The graphic representation (right panel) shows the percentage of released infectivity in five independent experiments. Values are given as mean ± SD