Structural peculiarities of the S'-end segments of genomic RNA were studied in F potato virus (F-PV) and white clover mosaic virus (WCMV). The methods of affinity chromatography on oligo(dT) cellulose and oligonucleotide mapping revealed a prolonged (up to 210 nucleotides) polyadenyl sequence at the 3'-end of F-PV RNA. A polyadenyl sequence is missing at the 3'end of WCMV RNA. A study of the translation products of WCMV and F-PV RNAs in a oe11-free protein-synthesizing system derived from rabbit reticulocytes showed that polypeptides electrophoretically comigrating with a structural protein of either virus were synthesized alongside high-molecular-weight polypeptides (M$_r$$\approx$ 180-150 kdaltons)

Artyukova, E. V.

Atabekov, J. G.

Gorboulev, Valentin G.

Krylov, A. V.

Rodionova, N. P.

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COMPARATIVE STUDY OF STRUCTURAL PECULIARITIES AND TRANSLATION OF POTEXVIRUS RNAs E. V. Artyukova, V. G. Gorbulev, N. P. Rodionova, A. V. Krylov. and 1. G. A tabekov UDC 578.2 Structural peculiarities of the S'-end segments of genomic RNA were studied in F potato virus (F-PV) and white clover mosaic virus (WCMV). The methods of affinity chromatography on oligo(dT) cellulose and oligonucleotide mapping revealed a prolonged (up to 210 nucleotides) polyadenyl sequence at the 3'-end of F-PV RNA. A polyadenyl sequence is missing at the 3'-end of WCMV RNA. A study of the translation products of WCMV and F-PV RNAs in a oe11-free protein-synthesizing system derived from rabbit reticulocytes showed that polypeptides electrophoretically comigrating with a structural protein of either virus were synthesized alongside high-molecular-weight polypeptides (Mr . f':i 180-150 kdaltons). Potexviruses form a very numerous group of plant viruses that have flexible thread-like particles 480-580 nrn long [1]. The capsule of these viruses, of spiral symmetry, consists of identical protein subunits of molecular weight 24-28 kdaltons. The virion RNA is a continuous polynucleotide chain of molecular weight 2.1-2.6 Mdaltons. It has been established rather recently that the RNA of some representatives of this group carries at the 5'-end a "cap" of the m 1G(5')ppp(5'} type [2-41, while the RNA of X potato virus has a poly-adenyl sequence up to 200 nucleotide residues long at the 3' -end [51. This RNA is an active template in the cell-'-free protein-synthesizing system [6, 71. ' The criteria used for referring viruses to the potex-group are highly arbitrary and are mainly based on the size and form of the viral particles [81. However, it cannot be precluded that viruses characterized by identical virion morphology may differ essentially in genome structure and the strategy of its expression. It was accordingly deemed useful to compare and characterize the structural peculiarities of the 3' -end regions of RNAs in several different potexviruses and study the peculiarities of their RNA translation in a cell-free protein-synthesizing system. The viruses studied, besides X-PV, were two, F-PVand WCMV, also referred by Koenig and Leseman [8) to the potex-group on the basis of virion-morphology similarity, Although these viruses were described long ago [9], no data about the structure of the genome and peculiarities of its expression are available. MATERIALS AND METHODS Specimens of F-PV were isolated from stramonium (Datura stramonium L.) and a species of tobacco (Nicotiana glutinosa L.) as described previously [101; WCMV was isolated from beans (Phaseolus vulgaris L., variety Bountiful) by a modification of the method of Mlki and Knight [111. Frozen leaves were homogenized in 0.2 M phosphate buffer, pH 7.8, containing 0.1% sodium sulfate and 0.2% EDTA. The homogenate was cen-trifuged at 10,OOOg for 15 min. The supernate was brought to pH 5.0 with 10lb acetic acid, kept for 30 min at 4°C, and centrifuged at 10,000g for 10 min to remove cell proteins. Virus was sedimented from the supernate after adding polyethylene glycol (molecular weight 6 kdaltons) to a final concentration of 4% in the presence Soil Biology Institute, Far East Scientific Center. Academy of Sciences of the USSR. Vladivostok. Insti-tute of Molecular Biology, Academy of Sciences of the USSR, Moscow. A. N. Belozerskii Problem Research Laboratory of Molecular Biology and Bioorganic Chemistry. M. V. Lomonosov Moscow State University, ¥oscow. Translated from Molekulyarnaya Biologiya, Vol. 19, No. 4, pp. 1021-1028, July-August, 1985. 842    of 0.3 M NaC!. The sediment was spun down at 12,000 rpm for 15 min. Subsequent purification was by differ-ential centrifugation followed by centrifugation in a sucrose gradient (10-40%) in 0.01 M Tris-HCl buffer, pH 7.0, at 25,000 rpm for 3 h (rotor SW27, Spinco L2-75B centrifuge). The RNA of F-PVand WCMV was iso-lated from purified virus preparations by the method of phenol deproteinization in the presence of sodium dodecylsulfate. A specimen of X-PV RNA was kindly provided by V. K. Novikov. The nucleoside composition of RNA was determined, after exhaustive enzymatic hydrolysis, by the chromatographic method on a "Varian-8500" high-pressure liquid chromatograph, using hydrolysate of the RNA of a conventional TMV strain as standard. An amount of 20-25 J.l.g RNA was diluted in 20 j-<liter of buffer, pH 8.4, containing NH 4HCO s and MgCl2 of 0.05 and 0.02 M, respectively, 50 flg/ml pancreatic RNase, 50 U/ml RNase Tl, 50 J.4g/ml snake-venom phosphodiesterase, and 20:J.lg/ml of Escherichia coli alkaline phosphatase, and was incubated at 37 DC for 18 h. The hydrolysate was evaporated dry and analyzed on an Aminex A-7 col-umn. Affinity chromatography of RNA on oligo (dT) cellulose (Sigma, USA) was done on a 1-ml column by the method described previously [12, 13 J. RNA was fractionated by electrophoresis in 2.4% polyacrylamide gel containing 0.5% agarose and 7 M urea, at 200 V (direct current) for 2 h. Molecular weight was determined with the following markers: TMV RNA (Mr 2 Mdaltons) and BMV total RNA containing four species (1, 2,3, 4) of molecular weights 1.09, 0.99, 0.75, and 0.28 Mdaltons [14J. The oligonucleotide analysis of RNA was done by the method of Pedersen a:nd Haseltine [151. An amount of 1 J-I.g RNA in 10 mM Tris-HCI buffer containing 1 mM EDTA, pH 8.0, was treated with Tt RNase (1 U/J.!g RNA) and~. coli alkaline phosphatase(4X 10-4 u/j-<g RNA) for 90 min at 37°C. The oligonucleotide mixture obtained was labeled at the 51-end with [y - 32p]_ATP (Amersham, United Kingdom) and phage-T4 polynucleo-tide kinase. Oligonucleotides labeled with 32p were fractionated by two-dimensional electrophoresis in poly-acrylamide gel. Dimension one: 10% gel, 25 mM citric acid, pH 3.5, in the presence of 6 M urea, 1700 V, 2-2.5 h; dimension two: 22.8% gel, 50 mM Tris-borate buffer, pH 8.3, containing 1 mM EDTA, 1200 V, 5-6 h. Labeling of the 31-end of RNA with 32p was done using 5,-[a2p]cytldine diphosphate (Am er sham , UK) and phage-T4 RNA ligase by the method of England and Uhlenbeck [lS], with some modifications. After introduc-ing the 31-end label, F-PV RNA was hydrolyzed by T1 RNase (1 U/J.l.g RNA) (50 mM Tris HCI buffer, pH 7.5, in the presence of 10 mM EDTA for 1.5 h at 37°C), and the hydrolysis products were separated by two-dimen-sional electrophoresis in polyacrylamide gel as described above. One-dimensional fractionation of 51_[~2pr ... oligonucleotides was done by electrophoresis in a 10% poly-acrylamide gel in 50 mM Tris-borate buffer, pH 8.3, in the presence 1 mM EDTA and 7 M urea. Labeled oligonucleotides were extracted from the gel with a solution containing 1 mM EDTA and 200 f'g/ml tRNA for 3 h at 37°C. The oligonucleotides were then sedimented with ethanol in the presenoe of 0.3 N sodium acetate and 0.01 M magnesium acetate, pH 6.0, and the sediment was washed with ethanol. It was dried and dissolved in 1 0 ~liters of 2Q mM Tris-H Cl buffer, pH 7.5, containing 2 mM EDTA. Sequencing of 51_[ 32p]-oligonucleotideswas done by the method of Donis-Keller et al. [171 after their partial enzymatic hydrolysis followed by fractionation of the hydrolysis products by electrophoresis in a 25% polyacrylamide gel. RNA was translated in a cell-free protein-synthesizing system (rabbit reticulocyte lysates) by the method of Pelham and Jackson [18]. Rabbit reticulocyte lysate treated with micrococcus nuclease was obtained from O. N. Denisenko (Insti-tute of Protein, Pushchino). The cell-free system was optimized for the translation of F-PV and WCMV RNA. The incubation mixture (20 flliters) contained 8 flliters of lysate; 20 mM HEPES-KOH buffer, pH 7.6; 2.0 mM magnesium acetate; 140-200 mM potassium acetate; 1 mmole ATP; 0.2 mmole GTP; 15mniole creatine phos-phate; 19 nonlabeled amino acids (0.2 mmole of each); 14C-labeled amino acids (0.3 j-<Ci) or [s5S1methionine (1275 Ci/mmole, Amersham, UK) (1 j-<Ci); 3 J..tgwheattRNA. TheconcentrationofF-PVRNA variedfrom30to150 J.<g/ml. The mixtures were incubated at 30 DC for 60-120 min. Translation products were analyzed by electro-phoresis on plates of 10% polyacrylamide gel by the method of Laemmli [19], but the acrylamide-N,N'-methylene bis-acrilamide ratio was 150: 1 to prevent gel cracking during drying. The markers were a mix-ture of carbon-labeled proteins (Amersham, UK), viz: myosin (20,000), phosphorylase (92,500), bovlne-serum albumin (68,000), ovalbumin (45,000), carboanp.ydrase (30,000), and lysozyme (14,300). The gel was stained with Coomassie brilliant blue R-250, dried, and radioautographed on an ORWO HS-ll film. 843 'Fig. 1. ElectrophoreElis of virion HNA of F-PV (c} and WCMV (d) in 2.4'10 polyacryl-amide gel conta.ining 0.5% agar:ose and 7 M urea. Total BMV HNA {u) and TMV HNA (b) were used as markers. RESULTS AND DISCUSSION Cr.romacCJgraphy of F-PV and WCMV ,HNase on an Oligo (dT)-Cellulose Column, The specimens of \\CMvari(fF-PV virion-RNA oi;talned w-ere hom-ogeneOlJA to a flitTsfaeto-ry degree ~. 1, c and d L According to polyacrylamide-gel-electrophoresis data, the molecular weights of \\'Cl\'IV and F-PV HNA were <~.O and 2.5 l\lcia l tons, respect ively. The nucleoside composition of both RNAs (results not shown! was characterized by a high content of adenosine (3 1.7% for F-PV RNA and ao% for WCMV RNA l. which i.s typical of potexvirns H!'\As in gener.ai and 3ug'geSt8 the presence of polyadenyt blocks in their structu re . Affinity chromatography showed that in a buffer of high ionic strength 77% of F -PV HNA was sorbet! t.o the oligo (dT)-cellulosecolumn and was eluted from it with water, i.e •• it contained lengthy polyadenyi se-quences (a poly (A)· fractioll\. Under the same conditions, specimens of polyadenylic acid wore Borbed up to 9~'" wh i1 e specim ens of 'I'M V UNA, not containing a poly (A) sequencf!. to only 5-10%. As etedropho res is re-sults showed. the poly (A)· fraetion of F-PV HNA did not differ from uoofractionated RNA in migration veloc-i tv in polyacryl amide gel, whereas the fraction eluted from the COlumn with high-tonic-strength buffer (poiy (1\ j - fraction) ',vas a mixture of fra.gments heterogeneous in size. In cont rast to F-PV RNA, ~93'!o of WCMV HNA did not atta.ch to oltgo(dT)-cellulose in hlgh ,-ionic.--qtr'cngth buffer. The RNA fraction not unHing with the sorbent: was homogeneous in analysis In polracrylamid e gel. and did not differ from the starting WCMV RNA 8pecimml by its electrophoretic behavior. These results indieat(? thlt t lengthy blocks of poly (A) are missing I.n WCMV RNA, which distinguishes this virus substantially fl'(ml the other two representatives, X-PVand F-PV, of the potexgroup. The relatively high ooolent of adenine in \VCMV ENA is apparently due to the presence of short intercalary blocks in the viral HNA molecule. One cannot fu Uy preelude that the end bloek of poly (A) may have been lost as a result of d(.'gradatlon In the pro-cess of the isoln.tion, but this seems rather improbable, It should be pointed out that in their early studies Sonenberg et a1. (41 did not find any poly (A) sequenecs in X-PV HNA, in contrast to Morozov et at. r 51. Compa rative AnalysiS of X-PV, F-PV, and WCMV Genomtc HNAs by Oligonucleotide MappIng. Ollgonu-eleotlde rtHl[)S of genoml-c X--PV-;- F-PV. and-WCMV RNAs hydrolyzed by 'fl HNase were obtained by 'two-dimensional electrophoresis of the hydrolysates in polyacrylamide get. Large, Tl-RNase-resistant blocks of electrophoretic mohitlty characteris ti c for poly (A) -containing fragments (Fig. 28, b) were found among the oligonueleotides obtained from F-PV and X-PV. but were not present in WC1\1.V RNA (Fig. 20). One --dl.mcnsional el.ectrophoresis in 10;1(: polyaerylamtde gel in the presence of markers aUowed tenta-tive estimation of the size of the poly (A)-colltaining fl'agment..'{, They were 2)0 and 250 l1ucleotides long In F-PV llnd X-PV RNA, respectively. To prove that the poly (A )-containing blocks were localized at the a'-end o f the molecul e, specimens of F-PV wel'e labeled at, the 3'end with {5,-3't1»cytldlnediphosphateusingphilse-T4 844 •• - ... " " -... FIg. 2. Oligonc~leottde maps of RNA hl'droiysates: a) X-PV, b) F-PV. c) WCMV. Tl HNase was used. Left to right dimension 1; bottom to top. dimension 2. Cross€s denote position of strains: brompheno! btue (toP) and xvlene c','a nol FF (bottom). Arrows indicate poly (A )-conf1lining fr agmenl3. Hl\A ligatw. Subsequent hydrolysis of the Inbeled RNA wi.th '1' 1 HNAs(! and ana!.ysis of the hydro ly sa tes by lIVU-dimensIonal electrophoresis in polyacrylamide gel showed that all of the radi.oactive label was eoncent.rated in the region of the enriched poly(A) fragmeni. . 'l (Fig. :1). To determine the $oquence ofnuclcotides adjacent f',() the poly lA) bloek, F-PV RNA was cleaved with Tl HNase and a S'-elld label was introduced into tbe forming oligonuc! eotides u sf ng r y - 32p 1- ATI' and polynucleotide kin ase. Follow! ng el oetrophores is in polyacrylnm Ide gel , the poly(A )-cont:alning fragments were eluted and subjected to direct sequcncing (Fig. 4) . In thei.r pri -mary stru c ture the segments adja.cent to the poly lA) blocl, in the F-PV and X-}::lV RNA proved closely re-lutedr51: FB-K 5' . .. AAL'AAU- p\~ ly \ .\) ::r X ·ElK 5' . . . AA Ai\ .. :\UAUAAAL ·-- p" ly (A) J ' '11m3, rhe presence of lengthy poly (A)-containing bLoeks at the a'-end of the F-PV H?~A molecul e reo, lates it with the RNA of X-PV (a. typical represenuitiv8 of pote,"{viruses) _ On the other hand, a ~J' -end-pol.:y(A :1 s(;.'quence was absent from the RNA struoture of \:I,;'CMV. which is also classified with the potexvlruses and, accol'dlng to [91. is tlntigenicaUy related wUh X-PV. Translation Peculiarities of F-PV and WGMV IWA in a Cell-Free System from Rabbit Reticulocytes . The cell-free system was opUmized :for F-PV and WC1fV RNA translation': Maximum stimulation"of the in-clUSion ()f I~C-Iabeled amino a.cids into polypepiides was observed at a 140-200 mM potassium aeetate and a R45 Fig. 'I Fig. 3. ,';nalys is ofF'-PV RNA laheled at the 3'-end. r :12Pl_RNA was treated with Tl RNase and the hydrolysis products were fraction-ated by two-dimensional electrophoresis in polyacrylamide geL Cross denotes position of stain xylene eyanoJ. FF. Fig. 4. Determination of primary structure of segment adjacent to the 3'-end poly (A) sequence in F-PV HNA. Lane OH-. alkallne hydrolysis; lanes U2, A, and Ph, rlbonucieases tJ2, A, and HNase from Phy~~rlunl e91ycephalum, respectively (different concentra-tions). night: mwieotide sequence of the F-PV RNA segment, read from the autoradiogram. ~~ rn,\] magnesium ace ta te concentra tion; the optimal concentrati on of the virus HNA template under these Gon-diti,ons was 65 f"g 'm!. Analys is of the F-PV-HNA translation products showed heavy polypeptldes of mol ecular wei gh t '~ 180, 1 GO, and 150 kdait.ons as the basic material. A minor amount of a component of moleeular wei.ght ,., 190 kdalLOns was also synthesized (Fig. 5b). Among the low-molecular-weight products of F-PV HNA trans-la,ti on a poly peptide of 26 kdalton was found that in electrophoretic: mobility coincided with the structural pro-tein of the virus. The translation of WC.MV HNA (Fig. Sc) resulted in a rather heterogeneous group .of products, among which polypepti.cl es of molecular weight 170 and ]55 kdaltons were detected. It had previously been shQwn that in a cell- free sy stem de rived from wheat germs, WCMV RNA directs the synthesis of proteins of molecular weight 170 and 140 kdaltons [20 ]. TIle system of retioulocyte lysates also contained among the basic WCMV-RNA translation products a low-molecular-weight polypeptide, of 24 kdaltons, that coincided with the stru c-tural p r otein of the virus in mobUity . 'Il1 8 UNA of X-PV controlled the synthesis of high-molecular-weight polypeptides, among which products of molecul a r weight 180 and 145-150 kdaltons were prorninent. In contrast to WCMV and F-PV RNAs, however, the genomic HNA of X-PV did not, In the ,in vitro translation. code for a protein identical in molecular weight to the envelQpe protein of this virus (Fig, Sa); this is 1n a.greement with other authors' results £6.71. If the polypeptides of molecular wetght 26 and 24 kdaltons encoded ,in vitro by F-PV and WCMV RNAs 846 Fig, 5. Electrophoresis of in vitro translation products of X-PV HNA (a), F-PV rrNA (bi:-and \VCMV HNA (c) in an 8-20'10 polyaerylamide-gel gradient contlllning 0.1% sodium dodecylsulfate. Arrows indicate position of F-PV (1). WCMV (2). and X-PV (3) structural proteins. The numbers correspond to the molecular weight of protein markers, eorrcspond to the structural proteins of these viruses, it is very probable that specimens of J1onfractionated viral HNA inc l.llde a subgenomic RNA for structural protein. According to the electrophort,sis data, the po!v-acnla rni de gels with F-PV and WCMV RNA specimens did not, under the denaturing condition,«, Gontain RNAs tbat cOlTesponded in s ize to the subgenomlc UNAs of the envelope proteins of th.ese virusNl. which. mwer the-l ess, dues not fully preclude the p<Jssi.hll1ty that suhgenornic HNAs were pl'esent tn small quantities. }'lecenUy th e presence of 311 Gh an HNA has heel) deteeted in another potexvtrus, the nareissus-lnosaic virus 12 11. LITEHATl:RE erTED 1. H. E. F. Matthews, [ntervi.rology. 17, 1-19 (1982). 2. 1\1. ... AbouHaidar and I .. B. ,Banc:roft, ~T~ C;en .. Viro,t1 39, 559-5sa (1978). :3, M. AbouHaidar, J. Gen. Viroi,,57. 199-203(1981). 4. N. Sonenberg, A. J. Shatkin, R. P. Rlcciardi, M. Rubln, and R. M. Goodman, Nucl. Acids TIes .• j!. 2 ~i Ol-2512 (1978). 5, S. Yu. Mol'ozov. V. G. Gorbulev, V. K. Novikov, A. A. Agranovskii, Yu. V. Kozlov, I. G. Atabeko\', and A, A . Baev, Dokl. Akad. Nauk S8SB. 259, 723-725 (1981). 6. A. 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Acids Res .• 1. 2527-2539 (1977). 18. H. R B. Pelham and R. J. Jacltf.!on.l<-;ut'. J. Biochem. 67, 247-256 (1970). 847 19. U. K. Laemmli, Nature. 227, 680-685 (1970). 20. U. Szybiak and A. B. Legocki, Acta Biochim. Polon., 28, 99-104 (1981). 21. M. N. Short and J. W. Daves, Biosci. Rep •• ~, 837-846 (1983).                                              

Comparative study of structural peculiarities and translation of potexvirus RNAs

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Comparative study of structural peculiarities and translation of potexvirus RNAs

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