A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells

Abstract

The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.

FIG. 1.

Hsp90 inhibition suppresses FHV protein A synthesis. (A) Drosophila S2 cells stably transfected with pS2FA (lanes 1 to 4), pS2LacZ (lanes 5 to 7), or pS2FB (lanes 8 and 9) were induced with Cu²⁺ and labeled with 100 μCi per ml [³⁵S]Met-Cys in the presence of 1 μM geldanamycin (GA), 10 μM lactacystin (Lact), or 100 μg per ml cycloheximide (CX) for 90 min. Radiolabeled proteins were immunoprecipitated with either HA-specific antibodies for FHV protein A (PtnA) and β-galactosidase or FHV protein B2 (PtnB)-specific antibodies, separated by SDS-PAGE, and analyzed by fluorography. (B) Quantitative data for the effect of geldanamycin and proteasome inhibitors (PI) on FHV protein A, protein B2, and β-galactosidase synthesis compared to no-inhibitor controls. Proteasome inhibitor results are composite data from experiments using either lactacystin or MG132. (C) Total lysates (lanes 1 to 4) or carbonate-resistant membrane fractions (lanes 5 and 6) from cells metabolically labeled with [³⁵S]Met-Cys in the presence of the inhibitors listed above the lanes. Lanes 1 to 4 correspond to similarly numbered lanes in A. Samples for lanes 5 and 6 were prepared by differential centrifugation as previously described (25) and represent cellular proteins tightly associated with intracellular membranes (5). The asterisk indicates a cellular protein whose degradation is inhibited by lactacystin. Results are representative of three independent experiments, and for B, results represent the means ± standard errors of the means relative to vehicle-only controls. MW, molecular weight (in thousands).

FIG. 2.

Hsp90 inhibition reduces FHV protein A mRNA association with polysomes. (A) Drosophila S2 cells stably transfected with pS2FA were induced with Cu²⁺ in the presence of dimethyl sulfoxide (DMSO) or geldanamycin (GA), cell lysates were separated by linear sucrose gradient density centrifugation, equal-volume fractions were recovered from the top (low density) to the bottom (high density) of the gradient, total RNA was recovered by phenol-chloroform extraction and ethanol precipitation, and FHV protein A (PtnA) mRNA content was analyzed by Northern blotting with a ³²P-labeled riboprobe (25). (B) Quantitative data for the effect of geldanamycin on protein A mRNA polysome association. The total protein A mRNA recovered was the sum of the densitometry analysis of lanes 1 to 8 (see above [A]). (C) Ethidium bromide-stained rRNA in sucrose gradient fractions from cells treated with dimethyl sulfoxide, geldanamycin, or the general translation initiation inhibitor hippuristanol (HIP). The quantitative values represent the percentages of total rRNA present in fractions 3 and 4 or fractions 7 and 8. Results are representative of two independent experiments, and for B and quantitative values in C, results represent the means from those experiments.

FIG. 3.

mRNA 5′ UTR does not affect geldanamycin-mediated suppression of FHV protein A synthesis. (A) Schematics of pS2FA and derivatives with modified 5′ UTRs. All plasmids contain an MT promoter and a simian virus 40 polyadenylation signal (A_n). The 5′ UTR of pS2FA contains 204 nucleotides (nt) with an EMCV IRES, whereas pS2FA-Gal L contains 168 nt with the yeast GAL1 leader (Gal L). The 5′ UTR of pS2FA-5′vUTR contains 39 nt with the authentic FHV 5′ UTR sequence (F). ORF, open reading frame. (B) Drosophila S2 cells stably transfected with pS2FA-Gal L or pS2FA-5′vUTR were induced with Cu²⁺ and labeled with 100 μCi per ml [³⁵S]Met-Cys for 90 min in the presence of vehicle only or decreasing geldanamycin (GA) concentrations (conc.). Radiolabeled protein A was immunoprecipitated with HA-specific antibodies, separated by SDS-PAGE, and analyzed by fluorography. Results are representative of three or four independent experiments, and the quantitative values are the means from those experiments relative to vehicle-only controls.

FIG. 4.

Hsp90 suppression does not accelerate FHV protein A degradation. (A) Basal FHV protein A stability in transfected Drosophila S2 cells. Cells stably transfected with pS2FA were induced with Cu²⁺ and labeled with 125 μCi per ml [³⁵S]Met-Cys for 90 min, washed extensively, and cultured with cSDM in the absence of Cu²⁺ for 4 days with daily passages to maintain normal cell proliferation. We harvested equivalent numbers of cells at 24-h intervals and analyzed FHV protein A (PtnA) by immunoprecipitation (IP) and fluorography (top), total ³⁵S-labeled protein by fluorography (middle), and total cellular protein by immunoblotting (WB) for tubulin (bottom). (B) Cells stably transfected with pS2FA were induced with Cu²⁺ and labeled with 250 μCi per ml [³⁵S]Met-Cys for 90 min; cultured in cSDM in the presence of no inhibitor (lanes 2 to 5), geldanamycin (GA) (lanes 6 to 8), or geldanamycin plus lactacystin (Lact) (lanes 9 to 11); and harvested at 3, 6, and 12 h, and immunoprecipitated protein A (top) and total ³⁵S-labeled protein (bottom) were analyzed by fluorography. MW, molecular weight (in thousands) (C) Quantitative analysis of protein recovery at 12 h (Fig. 4B, lanes 5, 8, and 12) compared to the time-zero (t=0) control (see B, lane 2). For cellular protein quantitation, we used the radiolabeled band indicated by the arrow in B. *, the P value was <0.02 compared to the vehicle-only control. Results are representative of at least three independent experiments, and for C, results represent the means ± standard errors of the means relative to untreated controls at time zero. DMSO, dimethyl sulfoxide.

FIG. 5.

Newly synthesized FHV protein A rapidly associates with cellular membranes in Drosophila S2 cells. (A) Cells were incubated with PBS and 0.01% saponin on ice for 10 min and centrifuged at 10,000 × g for 5 min to recover the supernatant fraction (S) and the resultant pellet, which was extracted in an equal volume of RIPA buffer and centrifuged at 10,000 × g for 5 min to remove nuclei and insoluble cellular debris and to recover a final pellet (P) fraction. Equal-volume fractions were separated by SDS-PAGE and immunoblotted with antibodies against the cytosolic proteins Hsp83 and tubulin, the mitochondrial membrane protein VDAC, the mitochondrial matrix protein Hsp60, and FHV protein A (PtnA). (B) Cells stably transfected with pS2FA or pS2LacZ were induced with Cu²⁺, incubated with 125 μCi per ml [³⁵S]Met-Cys, and harvested at 15, 30, 45, 60, and 90 min. Cells were separated into cytosolic and membrane fractions as described above, and FHV protein A and β-galactosidase were immunoprecipitated with HA-specific antibodies and analyzed by fluorography. (C) Cells stably transfected with pS2FA were induced and labeled as described above for 90 min in the presence or absence of 1 μM geldanamycin (GA) and separated into cytosolic (C) and membrane (M) fractions. Full-length FHV protein A was immunoprecipitated and analyzed by fluorography. Results are representative of at least four independent experiments.

FIG. 6.

Hsp90 inhibition does not alter FHV protein A membrane association in Drosophila S2 cells. (A) Schematic of pulse-chase fractionation protocol. (B) Cells stably transfected with pS2FA or pS2LacZ were induced with Cu²⁺; pulse labeled with 500 μCi per ml [³⁵S]Met-Cys for 15 min; chased with cSDM and no inhibitor, 1 μM geldanamycin (GA), or 1 μM geldanamycin plus 10 μM lactacystin (Lact); harvested at 15-min intervals; separated into cytosolic (C) and membrane (M) fractions; and analyzed by immunoprecipitation as described in the legend of Fig. 5. (C) Quantitative temporal analysis of full-length FHV protein A recovery from the membrane fraction after pulse labeling and treatment with vehicle only (closed circles) or 1 μM geldanamycin (GA) (open circles) compared to the time-zero (t=0) control (Fig. 5B, lane 4). Results are representative of two independent experiments, and results for C represent the means from those experiments.

FIG. 7.

Hsp90 inhibition suppresses the synthesis of retargeted FHV protein A. (A) Schematics of pS2FA, which encodes wild-type mitochondrial targeted protein A, and pS2FA-P450, which encodes a chimeric protein A retargeted to the endoplasmic reticulum. The amino-terminal coding sequences are shown, and the transmembrane domains are underlined. The endoplasmic reticulum targeting signal from the yeast cytochrome P450 oxidoreductase is italicized, and the residues in boldface type are encoded by the unique BspEI sites used to generate the chimeric junctions (26). ORF, open reading frame. (B) Drosophila S2 cells stably transfected with pS2FA-P450 were induced with Cu²⁺ and labeled with 100 μCi per ml [³⁵S]Met-Cys for 90 min in the presence of decreasing geldanamycin (GA) concentrations (conc.). Radiolabeled endoplasmic reticulum-targeted protein A (ER-PtnA) was immunoprecipitated with HA-specific antibodies, separated by SDS-PAGE, and analyzed by fluorography. Results are representative of at least three independent experiments, and the quantitative value is the mean from those experiments relative to the vehicle-only control.