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Cricket Paralysis Virus RNA Has a 3' Terminal Poly(A)

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Cricket Paralysis Virus RNA Has a 3' Terminal Poly(A) J. gen. Virol. 098o), 50, I67-17t I67 Printed in Great Britain Cricket Paralysis Virus RNA has a 3' Terminal Poly(A) (Accepted 5 March I98o) SUMMARY The single-stranded (ss) virion RNA of cricket paralysis virus, an invertebrate picornavirus, has a tool. wt. of approx. 2.8 x io 8 and contains a 3' terminal poly(A) 2o to 7o nucleotides in length. Cricket paralysis virus (CrPV) was isolated in I97o from laboratory and field populations of crickets in Victoria, Australia (Reinganum et al. I97o). The virus has been isolated from and replicates in a wide variety of orthopteran and lepidopteran species (Scotti et al. I979). In tissue culture CrPV replicates in Drosophila melanogaster line I cells (Schneider, I972 ) causing a distinct c.p.e. (Scotti, I976). CrPV is icosahedral in structure with a particle diam. of 27 nm, a sedimentation coefficient of I67S and a buoyant density in CsCI of 1-34 g/ml. It contains a single-stranded (ss) 37S RNA genome and three major structural capsid proteins whose aggregate mass is approx. 94ooo (Scotti et al. I979). These properties and the fact that the virus is stable at pH 3 and replicates in the cytoplasm of infected cells has led to the inclusion of CrPV in the genus enterovirus of the family Picornaviridae (Fenner, I976; Cooper et al. I978). One of the major gaps in our knowledge of the structure of CrPV concerns the nature of the virion RNA. Is it, for example, polyadenylated like other picornavirus RNAs (Cooper et al. I978). We provide evidence here that CrPV RNA has a 3' terminal poly(A) sequence and use a direct method to show that the poly(A) sequence is 20 to 7 o nucleotides in length. CrPV was propagated in penultimate instar Galleria mellonella larvae and purified by zonal centrifugation in sucrose gradients and equilibrium centrifugation in CsCI gradients as described by Scotti (i976). Virus was plaque purified on D. melanogaster line J cells using a 1"5 ~ agarose overlay prepared by mixing 6 ~ agarose in water with Schneider's medium (Schneider, 1972 ) containing 2o ~o heat-inactivated foetal calf serum. The agarose was overlaid with 2 ml Schneider's medium and plaques were picked after 3 days incubation at 26 to 28 °C. Stock virus was produced either by passing the virus once in monolayers of D. melanogaster cells or inoculating it into G. mellonella larvae. In the latter case, CrPV was purified as described above and diluted in Schneider's medium before use. The titre of stock virus ranged from I x io 8 to 3 x lo 9 p.f.u./ml. CrPV replicates in D. melanogaster cells producing peak titres approx, lo h p.i. at 5 p.f.u./cell (D. Rohel, personal communication). Six x io ~ D. melanogaster cells were seeded into 25 cm 2 plastic tissue culture flasks and t2 to 24 h later infected with CrPV at approx, to p.f.u./cell. After a I h adsorption period the cells were incubated at 26 °C in Schneider's medium. The intracellular virus RNA species were labelled with 5o/~Ci/ml aH-uridine from 3 to 6 h p.i. in the presence of ~'5/~g/ml actinomycin D and 5/~g/m[ ethidium bromide to prevent actinomycin D-insensitive mitochondrial transcription (Spradling et al. I977). RNA was extracted from the cells with phenol in o.I M-tris (pH 9), o-2 ~ (w/v) sodium dodecyl sulphate (SDS) and precipitated with two vol. of ethanol. Two species were detected when the celt-associated RNA was analysed by electrophoresis in a 3 ~o polyacrylamide gel (Fig. I e). The slower migrating, minor species was resistant to digestion with pancreatic RNase (data not shown) and is therefore double-stranded. The O022-1317/80/0000-4111 $02.00 (~ I980 SGM I68 Short communications 70 -- 60 --50 40 (a) (b) (c) (d) (e) (f) (g) 42S--~. 30 28S 26S XC~ f ", 2 18S ~ -- 20 Fig. I. Fig. z. Fig. I. Polyacrylamide gel electrophoresis of CrPV RNA. RNA was extracted from CrPV-infected D. melanogaster cells after labelling from 3 to 6 h p.i. with 3H-uridine. The RNA was divided into two aliquots. (e) One was analysed by electrophoresis in a 3 ~ polyacrylamide gel (Eaton, I979) and the position of the RNA species detected by fluorography (Bonnet & Laskey, t974; Laskey & Mills, I975). RNA in the second aliquot was divided into oligo(dT)-cellulose binding (c) and non-binding (d) fractions prior to gel electrophoresis and fluorography, aH-uridine-labelled 42S and 26S Sindbis virus RNA species (a) and 14C-uridine-labelled 28S and 18S chick embryo fibroblast ribosomal RNA species (b) were prepared as described (Eaton, I979). CrPV virion RNA was prepared as described in the text and divided into oligo(dT) binding (f) and non-binding (g) fractions. Fig. 2. Size of the poly(A) in CrPV RNA. CrPV virion RNA was labelled with 32pCp and T4 RNA ligase and the 3~P-labelled poly(A) isolated and purified as described in the text. The poly(A) was sized by electrophoresis in a 2o ~ polyacry!amide gel containing 7 }a-urea using 0"o5 M-tris-borate, pH 83, buffer. Preliminary experiments in which 32P-labelled poly(A) was partially digested in 0"05 M-NaHCO3, o'ool M-EDTA, pH 9, at 90 °C for 30 rain and subjected to electrophoresis indicated that xylene cyanol (XC) migrated at the same positions as an oligonucleotide with 25 residues. Short communications I69 major, faster migrating form was RNase-sensitive and migrated at the same rate as ssRNA extracted from the virus (see Fig. ~f, g). Based on its electrophoretic mobility compared with that of chicken embryo fibroblast 28S and ~8S ribosomal RNA (Fig. I b) and 42S and 26S ss Sindbis virus RNA species (Fig. 1 a), a mol. wt. of 2"8 × IOn was estimated for the ss CrPV virus RNA. Intracellular CrPV RNA in o'3 M-NaCI, o.I M-tris, pH 7"2, was added to a small column containing approx. 15° mg oligo(dT)-celtulose (type 7, P-L Biochemicals, Milwaukee, Wis., U.S.A.) and divided into binding and non-binding fractions. Binding species were eluted from the column in o.oI M-tris, pH 7.2. Both binding and non-binding species were precipitated with ethanol, analysed by gel electrophoresis and fluorography (Fig. Ic, d) and the proportion of binding and non-binding species determined by densitometric scanning of the fluorograms; 93 ~ of the ssRNA and approx. IO ~ of the dsRNA bound to oligo(dT). These results suggest that the intracellular CrPV ssRNA contained p01y(A). The next experiment was done to determine whether virion RNA contained a poly(A) sequence. D. meianogaster cells were infected at an approx, multiplicity of IOO p.f.u./cell for I h. Cells were incubated in Schneider's medium and 3 h later I/zg/ml actinomycin D and 75/zCi/ml 3H-uridine were added. At IO h p.i. the culture medium was removed and cellular debris removed by centrifugation at IOOOO g for lO min. The labelled virus was pelleted and purified as described by Scotti (I976). The virus banded in CsC1 at a buoyant density of I'34 g/ml. CrPV was dialysed against TNE (o.oi M-tris, o.oI M-NaC1, o.ooI r~-EDTA, pH 7-2) and the RNA extracted with phenol at pH 9. CrPV virion RNA migrated in polyacrylamide gels with a mobility similar to that of intracellular ss CrPV RNA; 69 ~ of 1o5oo ct/min of CrPV virion RNA bound to oligo(dT)-cellulose. The integrity of both binding and non-binding species was checked by gel electrophoresis and fluorography (Fig. I f, g). To show that the poly(A) sequence was covalently bound to the CrPV RNA, ZH-uridine- labelled virion RNA (roooo ct/min) was suspended in o.oi M,tris~ pH 7-2, containing 9o ~ (v/v) dimethyl sulphoxide (DMSO) and incubated at 37 °C for 2o min. The RNA was then precipitated with ethanol; 94 ~ of the RNA was able to bind to oligo(dT)-cellulose after this treatment, indicating not only that the poly(A) was covalently bound to the virion RNA, but also that most of the molecules contained poly(A). The increase in oligo(dT)-binding capacity after DMSO treatment may be due to poly(A) in the RNA being more accessible after denaturation. An attempt was made to size the poly(A) sequence by determining the proportion of radioactive counts in 3H-adenosine-labelled CrPV RNA which was capable of binding to oligo(dT)-cellulose after treatment of the RNA with pancreatic and TI RNases. Unfortunately it proved impossible to obtain enough 3H-adenosine-labelled CrPV to examine virion RNA. Labelling of intracellular CrPV RNA with ~H-adenosine was also difficult but by using ~oo/zCi/ml 3H-adenosine in serum-free Schneider's medium it was possible to isolate and purify, by zonal centrifugation in a sucrose gradient, 4I 5oo ct/min of intracellular ss CrPV RNA. RNA was treated at 37 °C for 30 min with Io/zg/ml pancreatic RNase and 40 units/ml T I RNase in o'3 M-NaC1, o'o2 M-tris, pH 7.2. Resistant oligonucleotides were precipitated with ethanol. Of this intracellular CrPV RNA 1.47 bound to oligo(dT)-cellulose after RNase treatment. Taking a value of 3r.6 ~ for the adenyfic acid content of CrPV RNA (Longworth, I978), this yields' a figure of approx. 35 A residues in poly(A), assuming every molecule to be polyadenylated.
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