Electron microscopy imaging of purified L-A particles and their interaction with GST–Ded1p. (A) L-A particles were purified from a wild-type strain by a conventional CsCl gradient method or (B) from a Ded1p–PA-containing strain by affinity purification, in which L-A particles were eluted in one step by 2 M MgCl₂. Virus particles were then imaged by transmission electron microscopy. Bar, 100 nm. (C) Direct interaction of Ded1p with L-A particles. CsCl-purified L-A particles were incubated with glutathione beads precoated with GST–Ded1p (lane 1) or GST (lane 2). Lane 3 represents 33% of the input L-A particles. After incubation and washes, beads were analyzed as described in Figure 2A.

L-A dsRNA co-precipitates with Ded1p–PA. (A) L-A dsRNA co-precipitates specifically with Ded1p–PA. Extracts made from yeast strains harboring Ded1p–PA (lane 1) or Dbp5p–PA (lane 2) were incubated with IgG–Sepharose. The bound RNAs were extracted with phenol/ chloroform, precipitated (Ppt.) by ethanol, separated by agarose gel electrophoresis and visualized by ethidium bromide (EtBr) staining (top panel, lanes 1 and 2). Total RNAs (Total) prior to immunoprecipitation were extracted and used as positive controls (lanes 3 and 4). The identity of the L-A dsRNA was verified by northern blotting using either ³²P-labeled positive-strand probe (middle panel) or negative-strand probe (bottom panel). (B) The L-A dsRNA co-elutes with Gag. The experiment was done as in Figure 1D, except that the RNA was extracted from MgCl₂-eluted fractions, separated by agarose gel electrophoresis and then visualized by EtBr staining.

Ded1p accelerates the rate of in vitro negative-strand RNA synthesis. (A) Ded1p promotes negative-strand RNA synthesis but not positive-strand RNA synthesis. Positive-strand RNA-containing (Log Phase) or dsRNA-containing (Stationary Phase) L-A particles were incubated in the presence of either GST (lanes 1 and 4) or GST–Ded1p (lanes 2 and 5). Lane 3, dsRNA virus alone. After incubation, RNAs were extracted and analyzed by denaturing agarose gel electrophoresis (lanes 1 and 2) and autoradiographed. (B) Time–course study of Ded1p-promoted negative (–) RNA synthesis. Aliquots of the reaction in (A) using the positive (+)-strand RNA-containing L-A particles (Log Phase) were withdrawn at various time points and analyzed in native agarose gel electrophoresis.

Affinity-purified L-A particles are active in synthesizing L-A RNA. (A) The co-precipitated L-A dsRNA is resistant to RNase treatment. Ded1p–PA-bound IgG beads were incubated with pancreatic RNase in the presence of a ³²P-labeled L-A transcript as described in Materials and Methods. After incubation, RNAs were recovered and run on an agarose gel, which was stained with ethidium bromide (top panel) and autoradiographed (bottom panel). (B) In vitro synthesis of the L-A transcript. L-A particles were bound to IgG beads precoated with Ded1p–PA (lane 1) or Dbp5p–PA (lane 2) for in vitro RNA polymerase activity assay. The ³²P-labeled RNA products were separated on a denaturing agarose gel and autoradiographed. RNA size markers (kb) are indicated to the left. (C) Production of both positive (+) and negative (–) RNA by affinity- purified virions. The virus-synthesized RNA from lane 1 of (B) was used as a probe to hybridize to vector DNA alone (lane 1), vector DNA containing L-A cDNA (lane 2), single-stranded DNA containing the (+)-strand L-A cDNA (lane 3) and single-stranded DNA containing the (–)-strand L-A cDNA (lane 4). (Top panel) Ethidium bromide (EtBr) staining; (bottom panel) autoradiogram of the Southern blotting result.

Interaction of the L-A Gag protein with Ded1p. (A) Ded1p interacts specifically with Gag. Extracts prepared from yeast strains containing Ded1p (lane 1), Ded1p–PA (lane 2), Dbp3p–PA (lane 3) or Dbp5p–PA (lane 4) were incubated with IgG–Sepharose. After washes, IgG-bound proteins were eluted with acetic acid and analyzed by SDS–PAGE. Molecular size markers are indicated (kDa). (B) Resistance of Ded1p–Gag interaction to RNase treatment. Ded1p–PA-bound IgG beads were treated with 10 µl of RNase cocktail (1 mg/ml RNase A and 20 000 U/ml RNase T1; Ambion.) and 10 µl of micrococcal nuclease (1 mg/ml; Worthington Biochemical), incubated at 30°C for 30 min (lane 1) and analyzed as in (A). Lanes 2 (30°C) and 3 (4°C) are Ded1p–PA-bound IgG beads without RNase treatment but incubated for 30 min at the indicated temperatures. (C) In vitro binding of Ded1p with Gag. In vitro synthesized ³⁵S-labeled Gag was incubated with glutathione beads precoated with either GST–Ded1p (lane 1) or GST alone (lane 2). Materials bound were analyzed by SDS–PAGE followed by autoradiography. Lanes 3—7, 4% (lane 3), 1% (lane 4), 0.5% (lane 5), 0.25% (lane 6) and 0.125% (lane 7) of the input ³⁵S-labeled Gag. (D) Ded1p interacts strongly with Gag. Extracts from Ded1p–PA-containing strain was incubated with IgG–Sepharose. The IgG-bound proteins were then sequentially eluted with increased concentrations (0.05–4.5 M) of MgCl₂ as shown.