Translation of the L-Species dsRNA Genome of the Killer-associated Virus-like Particles of

Virus-like particles containing the L (Pl)-species of double-stranded RNA (dsRNA) were isolated from Saccharomyces cerevisiae, and the translational activity of the virus-like particle-derived dsRNA was analyzed in the wheat germ cell-free system. Denaturation of the dsRNA immediately prior to in vitro translation resulted in the synthesis of one major and at least three minor polypeptides, whereas undenatured dsRNA, as expected, did not stimulate [YSlmethionine incorporation into polypeptides, but actually slightly inhibited endogenous activity. The major in vitro translation product of the denatured L-dsRNA was shown to be identical with the major L-dsRNA containing virus-like particle capsid polypeptide on the basis of three criteria: co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and tryptic peptide analysis. We have therefore established that the L-dsRNA

and the role each plays in encapsulation and in toxin production and immunity.
In the experiments reported here we have utilized a direct in vitro translational assay to determine the coding capacity of the L-dsRNA genome.
For the sake of clarity in discussion, we have called polypeptides derived from dsRNA-containing VLPs &V-P, (S. cerevisiae virus polypeptides) where ScV-Pl is the major L-dsRNA-containing VLP capsid polypeptide.Polypeptides derived from in vitro translation of denatured L-dsRNA are designated L-P,.L-P1 is the major polypeptide product and it co-migrates with ScV-Pl.The data presented here show that these two polypeptides share all major tryptic peptides, and that the L-dsRNA genome codes for at least its own major capsid polypeptide.The implications concerning the basis of the dependence of M-dsRNA on L-dsRNA are discussed.(33,(37)(38)(39).Seven grams of raw wheat germ were ground moderately hard with 7 g of 120.pm acid-washed glass beads in a cold mortar for 1 min and then extracted with 25 ml of lysis buffer consisting of 40 rnM KAc, 2.0 rnM magnesium acetate, 20 mM Hepes, 2 mM CaCl,, 1 rnM dithiothreitol, pH 7.2.The lysate was centrifuged at 23,000 x g for 10 min, and 0.01 volume of 1 M Hepes, pH 7.2, and 0.01 volume of 100 rnM magnesium acetate were added while mixing.After centrifugation at 23,000 x g for 20 min the supernatant was immediately applied to a column ( 2 Optima for these were 100 mM potassium acetate and 40 pg/ml of RNA for denatured dsRNA and 160 mM potassium acetate and 100 pg/ml of RNA for total polysome-derived RNA.The concentrations of all other components used were: 1.6 mM magnesium acetate, 0.16 mM spermidine-free base, 40 pg/ml of creatine phosphokinase, 3 mg/ml of creatine phosphate, 0.6 mg/ml of ATP, 0.1 mg/ml of GTP, 1.4 m&r dithiothreitol, 0.44 rnM concentration of each of 19 nonradioactive amino acids, 1 +M nonradioactive methionine, and [SsS]methionine to a final specific radioactivity of 4.5 x lo5 cpmlpmol. Reaction mixtures were incubated at 22" for 3 h.Samples for the determination of radioactivity in trichloroacetic acid-precipitable material and for SDS-polyacrylamide slab gel electrophoresis were processed as described before ( 40).
Immunoprecipitation Assay -The double antibody method (indirect method) employing goat anti-rabbit y-globulin was used.The equivalent point for the reaction of goat anti-rabbit y-globulin against rabbit y-globulin was determined routinely for each prepa-ration.In a typical immunoprecipitation assay, 30 ~1 of 0.5% (w/v) SDS, followed by 0.25 ml of BSM buffer (100 rnM H,,BO,, 135 mM NaCI, 100 rnM methionine, pH 8.0), was added to 20 ~1 of an in vitro translation reaction mixture in a glass tube.To the tubes incubated at 35" were added 5 ~1 of a mixture of purified y-globulin from immunized and nonimmunized rabbits (0.2 mg/ml immune ~ 1.88 mg/ml non immune).
The reaction mixtures were allowed to precipitate for 60 min.Subsequently, 10 yl of goat anti-rabbit y-globulin (26 mg/ml) was added, the reaction tubes incubated at 35" for 30 min, and finally left at 4" overnight.
The resulting immunoprecipitates were washed three times with BSM buffer using a clinical centrifuge at 4", and the final pellet was prepared for SDS-polyacrylamide gel electrophoresis by dissociating in SDS-gel electrophoresis sample buffer for 3 min in a boiling water bath.One and two-tenths milligrams of ["Slmethionine-labeled VLP protein or a 1300.~1aliquot of the i n vitro translation reaction mixture supplemented with 0.9 mg of unlabeled VLP protein were fractionated on 3 mm thick SDS-polyacrylamide slab gels, and the excised protein bands from the stained and dried gels were cut into small pieces and digested for 14 to 16 h at 37" with stirring, in 8 ml of 1% (w/v) NH,HCO,, containing 0.1 mg/ml of trypsin.The incubation of gel slices was then repeated with 5 ml of fresh NH,HCO, containing 0.05 mg/ml of trypsin.
After 12 h at 37" the supernatant from this second digest solution was combined with the first and the total volume was lyophilized three times and dissolved in 100 ~1 of solvent for paper ionophoresis.
Ionophoresis was carried out for 2.5 h at 45 V/cm in pyridine acetate buffer, pH 3.5 (41).Following ionophoresis the paper was cut at 5-mm intervals from the origin and the radioactivity in each segment was determined by counting in toluene-based scintillation fluid.dsRNA.The slower sedimenting band, representing about 5% of the A,,,,, material, contains no dsRNA, but consist of empty particles as judged by electron microscopy.

Purification
Analysis of VLP proteins on SDS-polyacrylamide gels (7.5 or 10%) consistently gave a single major polypeptide (Fig. 3) migrating just below phosphorylase A (92,000 daltons).This major VLP protein has previously been estimated to have a size of 75,000 daltons ( 14), but the calculated molecular weight from these gels is about 88,000.At least three other minor bands (at approximately 140,000, 82,000, and 78,000 daltonsl are always seen regardless of the conditions of VLP preparation and dissociation.
The prominent bands at 53,000 and 37,000 daltons, reported by Oliver et al. (261,have not been reproducibly detected in our preparations.
The amino acid content of purified ScV-Pl polypeptide is shown in Table I.
Zsolation of L-dsRNA -L-dsRNA was isolated for in vitro translation by three different methods.Initially, total cellular nucleic acids obtained from strain NK2 by phenol extraction were hydrolyzed with pancreatic DNase and pancreatic RNase, and the products fractionated on Sepharose 4B columns.The RNA was characterized as dsRNA on the basis of insensitivity to pancreatic RNase in 150 mM NaCl sensitivity to pancreatic RNase in 15 mM NaCl and sensitivity to RNase III in 150 M NaCl (11,12).Polyacrylamide gel electrophoretic molecular weight determination and staining with ethidium bromide, which is specific for ds nucleic acid, confirmed the double strandedness of the RNA, as described by Vodkin et al. (13).The yield of dsRNA from strain NK2 by this method was 0.33% of the total nucleic acid fraction.This value agrees well with previous values for this strain3 Alternatively, dsRNA from strains 3/Al and NK2 was separated from the total nucleic acid fraction by Whatman CFll chromatography (341, as previously described ( 21).Yields of dsRNA by this procedure were 0.4% of the total nucleic acid for both strains.Finally, L-dsRNA was extracted from purified VLPs by phenol extraction after hydrolysis with proteinase K.The RNA products derived from the VLPs were then fractionated by Whatman CFll chromatography.
The "dsRNA" fractions from CFll chromatography were determined to be dsRNA on the basis of insensitivity to pancreatic RNase in high salt, sensitivity to pancreatic RNase in low salt, insensitivity to pancreatic DNase.They were character-Translation of Yeast L-Species dsRNA ized as L-species by electrophoretic migration behavior and ethidium bromide staining following standard procedures (13, 21).

In Vitro
Translation-In vitro translation of these dsRNA preparations was carried out using the wheat cell-free translational system.Incorporation of L-[Wlmethionine into products was detected by SDS-polyacrylamide slab gel electrophoresis and autoradiography.
Undenatured L-dsRNA does not direct the in vitro synthesis of any ["Wmethionine-containing polypeptides detectable by long autoradiographic exposures (Fig. 4).Slight A (92,000), bovine serum albumin (67,000), and carbonic anhydrase (30,000).Times of heating at 100" in SDS sample buffer prior to application to the gel were 0 min (g), 0.5 min (f), 1 min (e), 2 min (b, d), and 5 min (c).All were dialyzed against phosphate/glycerol buffer prior to denaturation.For one sample (b), this was performed in the presence of 1.2 mM phenylmethylsulfonyl fluoride.dogenous (wheat embryo) mRNAs did occur, as revealed by comparison with the control (no added RNA).
In contrast to the lack of stimulation by dsRNA, prior denaturation of L-dsRNA by a l-or 2-min incubation at 100 in the presence of EDTA, followed by quick cooling, results in the in vitro synthesis of a single major and three or four minor molecular weight species of [Wlmethionine-containing polypeptides which do not co-migrate in SDS-gels with major endogenous polypeptides (Fig. 4), and are consequently judged to be encoded by L-dsRNA.The major band synthesized in vitro, L-Pl, has a migration rate corresponding to a polypeptide of about 88,000 daltons and co-migrates on SDS-gels with ScV-Pl (Fig. 4).The most prominent minor bands migrate faster, although a small amount of a component .6 x 30 cm) (175ml bed volume) of Sephadex G-25 coarse equilibrated with 20 m&r Hepes, pH 7.2, containing 40 mM KAc, 2 rnM magnesium acetate, 1 rnM dithiothreital.The void volume of the column was approximately 70 ml, and the flow rate was maintained at wheat germ lysate (A,,,, of 50 to 70).Conditions used for RNA derived from dsRNA and polysome preparations differed only with respect to the concentrations of KAc and RNA employed.
as described by Morrison and Lodish (411, with slight modification. FIG. 3. 7.5% SDS-polyacrylamide gel electrophoresis of the major peak of VLPs from the sucrose gradient fractionation shown in Fig. 1.The gel was stained with Coomassie blue.The markers (slot a) are phosphorylaseA (92,000), bovine serum albumin (67,000), and carbonic anhydrase(30,000).Times of heating at 100" in SDS sample buffer prior to application to the gel were 0 min (g), 0.5 min (f), 1 min (e), 2 min (b, d), and 5 min (c).All were dialyzed against phosphate/glycerol buffer prior to denaturation.For one sample (b), this was performed in the presence of 1.2 mM phenylmethylsulfonyl fluoride.
Vortex mixer in a 30.mlCorex tube for four 15-s bursts at 4".Total cellular RNA was isolated from whole cell homogenates and extracted with phenol, as described for the extraction of dsRNA from VLPs (see above).
distilled H,O, and finally resuspended i n 1.5 volumes of PKE buffer (30 rnM Na,HPO,/NaH,PO, 150 mM KCl, 10 rnM EDTA, pH 7.6).Cel l s were disrupted by shaking with glass beads for 15 min on an orbital shaker at 350 rpm at a ratio of 3 g of 0.45 pm glass beads/ml of cell suspension.Breakage was estimated to be 90 to 95% complete.Preparation of Wheat Germ Cell-free University of Massachusetts Medical Center/The Lamar Soutter Library on July 10, 2019 http://www.jbc.org/ at