RESULTS Hsp90 inhibition and FHV protein A synthesis in Drosophila S2 cells. We have previously shown that the inhibition of protein Hsp90 FHV suppresses accumulation in Drosophila S2 cells transfected fa PS2FA stable, leads an MT promoter CVOL terminal HA-tagged protein expression plasmid. We in Substantially observed TW-37 that the treatment with geldanamycin, a selective inhibitor of Hsp90, not remove protein A mRNA accumulation in transfected S2 cells fa PS2FA is stable, measured 12 h after induction of Cu2. As to determine whether the gel Schte protein structure is partially due to reduced synthesis, we examined the effects of geldanamycin in 35S metabolic labeling experiments. We induced Drosophila S2 cells transfected fa PS2FA is stable with Cu2 2 h, the cells were incubated with Met for 90 min in the presence of inhibitors and Cys immunpr Zipitiert Volll Nts-protein A from cell lysates with an antique Rpern HAspecific. Treatment with suppressed geldanamycin protein A synthesis by 80% compared to controls, but had negligible Ssigbar effects on the synthesis of the entire cellular Ren protein with membrane or cellular’s other proteins associated.
In contrast, the translation inhibitor cycloheximide completely Constantly suppresses the synthesis of the two proteins elongation A and total cellular Ren protein. The inhibitory activity of t of geldanamycin, because she had induced a minimal effect on the production of Cu2 galactosidase according to the accumulation of reporter protein activity and t leads S2 cells. We also examined whether lactacystin, an irreversible inhibitor of the proteasome nachgewiesenerma Abzuschw en Chen partially protein accumulation in the absence of Hsp90 inhibition w Is during the incubation for 12 mediates h, also the effect of geldanamycin reduced to a protein synthesis.
Lactacystin had no significant influence on the mediated repression by geldanamycin full protein L Nge A synthesis but increased the recovery of certain cellular Rer proteins Called 35S. Similar results were obtained with the reversible proteasome inhibitor MG132, suggesting that geldanamycin not to a rapid deterioration proteasome A. newly synthesized protein mediates precisely examine the relationship between Hsp90 inhibition and FHV protein A synthesis, we conducted a dose and-effect analysis. Geldanamycin suppressed protein A synthesis in a dose–Dependent, calculated with an IC50 of 92 nM, about two times h Ago than the calculated IC50 value for geldanamycin-mediated suppression of protein A accumulation in Drosophila S2 cells.
Different experimental conditions in the IC 50 values were determined, for example, to make the synthesis of more than 90 minutes against the accumulation of more than 12 hours a direct quantitative comparisons difficult. Nevertheless, these results are strongly suggest that geldanamycin suppressed FHV-cellular protein A general synthesis without interruption Ren Translation. To determine whether inhibition of Hsp90 also affects the synthesis of other viral proteins, we examined the effect of geldanamycin for FHV B2 protein synthesis, which will be translated from subgenomic RNA3 and functions as a suppressor of RNA interference in plants, nematodes and insects. We generated Drosophila S2 cells transfected fa PS2FB is inducible with a Cu2 FHV B2 protein expression of plasmid stability, and examined the B2 protein synthesis by metabolic labeling and Immunpr Zipitation 35S.