Structure of the 3′ extremity of barley stripe mosaic virus RNA: Evidence for internal poly(A) and a 3′-terminal tRNA-like structure

VIROLOGY

119, 51-58 (1982)

Structure of the 3’ Extremity of Barley Stripe Mosaic Virus RNA: Evidence for Internal Poly(A) and a Y-Terminal tRNA-like Structure A. A. AGRANOVSKY, V. V. DOLJA, AND J. G. ATABEKOV’ Laboratory of Bioorganic Chemistry and Department of Virdogy of Moscow State University, Moscow 117234,USSR Received August 24, 1981;accepted December 16, 1981 The results of a previous study suggested that the poly(A) sequence in barley stripe mosaic virus (BSMV) RNA is intercalated between a 3’-terminal tyrosine-accepting structure and the 5’-terminal coding part of the BSMV genome. Here we show that poly(A)+ and poly(A)- fractions of BSMV RNA can be cleaved into two fragments specifically at the position of poly(A) or oligo(A) sequence with RNase H from Escherichiu cdi in the presence of oligo(dT)ic. The shorter fragment (Sh) retains the ability of intact viral RNA to be aminoacylated, i.e., it represents the 3’-terminal part of BSMV RNA. Electrophoretic analysis of Sh-RNA reveals three closely positioned subspecies with an average length of about 210 nucleotides. The long 5’-terminal RNA fragment (L) produced by RNase H treatment has electrophoretic mobility similar to that of intact BSMV RNA, but displays neither amino acid-accepting ability nor infectivity. Nevertheless, L-RNA possesses the same messenger activity as the intact viral RNA and codes for the same pattern of polypeptides in rabbit reticulocyte lysate in vitro translation assays. INTRODUCTION

Barley stripe mosaic virus has a multipartite genome (Jackson and Brakke, 1973; Lane, 1974). However, several lines of evidence show that BSMV differs from other viruses of this type in the basic principles of genome structure (for review see Atabekov and Morozov, 1979). Our studies of the r-terminus of BSMV RNAs indicate that the arrangement of the well-known terminal structures [poly(A) and tRNAlike structure] in BSMV RNA is unusual. Total RNA from various virus strains can be separated into fractions of unbound [poly(A)-] and bound [poly(A)+] RNA by means of chromatography on oligo(dT)cellulose or poly(U)-Sepharose (Agranovsky et al, 1978, 1981). The 3’ extremity of both the intact poly(A)+ and poly(A)RNA molecules end with a tyrosine-accepting (tRNA-like) structure. The length of the shortest poly(A)+ segments of BSMV

RNA capable of being aminoacylated, i.e., containing both tRNA-like structure and poly(A), was estimated at 150-200 nucleotides (Agranovsky et aL, 1981). These data indicate that the poly(A) sequence is localized internally within the 3’-terminal part of BSMV RNA. Recently the method of addressed fragmentation of RNA molecules has been developed in our laboratory (Stepanova et al, 1979) employing RNase H from Escherichia co& and short oligonucleotides complementary to a given site in an RNA chain. Using RNase H and oligo(dT),O complementary to internal poly(A) in BSMV RNA, we have cleaved the 3’-terminal tyrosine-accepting fragment from the BSMV genome and characterized it. Similar methods were applied by Rowlands et al. (1978) to cleave foot-and-mouth disease virus RNA at the site of a poly(C) tract, using RNase H in the presence of oligo(dG). MATERIALS

AND METHODS

-en& Ij5SjMethionine (1049 Ci/mmol), 14C-amino acid mixture (54 mCi/matom)

i To whom all correspondence and reprint requests should be addressed. 51

0042-6822/82/070051-08$02.00/O Copyright All rights

Q 19232 by Academic Preaa, Inc. of reproduction in any form reserved.

52

AGRANOVSKY.

DOLJA,

and L-[2,3,5,6-3H]tyrosine (104 Ci/mmol) were from Amersham (England); oligo(dT)cellulose (Sigma); oligo(dT), (P-L Biochemicals). vimcs and RNA. Isolation of BSMV and of viral RNA as well as their spectrophotometric determinations were described previously (Agranovsky et al, 1978). In this work, the two-component derivative of the Norwich strain (N 11) was used, obtained by Dr. M. Leiser by repeated passages on wheat plants of the initial threecomponent Norwich strain (Lane, 1974). Enzymes. Preparation of amino acyltRNA synthetases from wheat embryo and aminoacylation of RNAs in vitro were detailed in our previous paper (Agranovsky et d, 1981). RNase H purified from E. co& MRE-600 cells in general accordance with the method of Darlix (1975) was kindly donated by Dr. T. Lanina. The quantity of enzyme that hydrolyses 1 nmol RNA in an E. coli DNA-[14C]RNA hybrid in 20 min at +20° in digestion buffer (10 mMTris-HCl, pH 7.9; 0.15 MNaCl; 0.2 mM dithiothreitol) was taken as a unit of enzyme activity (Stepanova et al, 1979). Spe@c cleavage of BSMV RNA with RNase H and separation of RNA fragments. From one to four microliters of oligo(dT)i,, solution (0.9 mg/ml) was added to 40-200 pg of BSMV RNA dissolved in triple-distilled water at a concentration of l-l.6 mg/ml. The incubation mixture was buffered to the final concentration of digestion buffer (see above), gradually cooled to +4’, and left for 5-10 min. The mixture was then supplemented with 2-8 ~1 of RNase H solution (7 units/ml) and incubated for 3-3.5 hr at +4“. The reaction was stopped by the addition of SDS and EDTA to 0.1 and 0.02%) respectively. Samples were heated at +60° for 5 min and applied to lo-40% (w/v) linear sucrose gradients in 20 mM Tris-acetate, pH 8.3; 10 mM EDTA; 0.1 M NaCl. Gradients were centrifuged in a SW 41 rotor on a Beckman L2-65 ultracentrifuge as described previously (Agranovsky et al, 1981). RNA was precipitated from the peak fractions by adding 2.5 vol of ethanol with 0.02 M sodium acetate, pH 4.5 (-20’). RNA pellets

AND ATABEKOV

were collected by centrifugation, washed once with 70% ethanol, and dissolved in triple-distilled water. Polyacrylamide gel electrophoresis (PAGE). Electrophoresis was performed in accordance with the method of DonisKeller et al (1977). One to two micrograms of RNA was applied to one slot of 4 or 8% slab gel (2 X 120 X 170 mm) prepared with 0.1 M Tris-borate buffer, pH 8.3, 20 mM EDTA, and 6 M urea, and electrophoresis was carried out for 3 hr at 200 V. Gels were stained for 20 min with ethidium bromide (10 pg/ml in 0.01 M Tris-EDTA, pH 7.8) and then photographed under a uv lamp. Iqfectivity assays. Infectivity assays were carried out as described previously (Lane, 1974; Agranovsky et al, 1978). RNA preparations were dissolved in sterile 0.05 M sodium phosphate buffer, pH 7.6, at a concentration of 50 pg/ml, and compared samples were inoculated to the opposite half-leaves of Chenopodium awuzranticolor. Twenty microliters of RNA solution was inoculated to each half-leaf which had been dusted with Carborundum. Local lesions were counted 7 days after inoculation. Translation of the 5’-hrminal L fragment in a rabbit reticuloc&e cell-free system. The rabbit reticulocyte lyzate was isolated and treated with micrococcal nuclease as described by Pelham and Jackson (1976). The cell-free protein-synthesizing system was optimized for BSMV RNA. The incubation mixture (20 ~1) contained 20 mM HEPES-KOH, pH 7.6, 4 mM magnesium acetate, 10 mM potassium acetate, 1 mM ATP, 0.2 mM GTP, 15 mM creatine phosphate, 60 pg/ml creatine phosphokinase, 150 pg/ml total tRNA from wheat embryo, 8 ~1 of nuclease-treated lysate, 100 rg/ml BSMV RNA, and 20 &i of 14C-amino acid mixture or 10 PM each of the 19 common amino acids except methionine and 20 &i of [?S]methionine. Analysis of the translation products was carried out in accordance with the method of Laemmli (1970) in linear gradient 820% polyacrylamide slab gels. Gels were dried and then autoradiographed on HSll ORWO X-ray film.

INTERNAL

53

POLY(A) IN VIRUS RNA A

RESULTS

Addressed Cleavage of BSMV RNA with RNase H in the Presence of Oligo(dT)lo The genome of the N II isolate of BSMV consists of two RNA components, RNA 1 and RNA 2, their molecular weights coincide with those of RNA 1 and 2 of the original three-component strain Norwich, estimated at 1.5 X lo6 and 1.35 X 106, respectively. As has been shown with the total virion RNA from other BSMV strains (Agranovsky et &, 1978,1981), RNA from N II can be separated into fractions tentatively named poly(A)+ and poly(A)- RNA on the basis of their behavior on oligo(dT)cellulose. In our experiments, the total BSMV RNA and its poly(A)+ and poly(A)- fractions were used as substrates for RNase H. After incubation of total viral RNA with RNase H in the presence of oligo(dT)10 an additional low molecular weight RNA fragment was revealed by sucrose density gradient centrifugation (Fig. 1A). Its size can be roughly estimated at 150-200 nucleotides (molecular weight 4-6 X 104)from the log molecular weight - mobility plot based on the mobilities of marker RNAs in sucrose gradients (Agranovsky et aL, 1981). Unexpectedly, no difference was found between the specific products of RNase H cleavage of either total, poly(A)+, or poly(A)- BSMV RNA, as judged by gradient centrifugation analysis (data not shown). In all these cases, incubation of RNA with both the enzyme and oligo(dT) resulted in the appearance of a “light” peak in gradient fraction 6 (see Fig. 1A). In controls, BSMV RNA was incubated under the same conditions (see Materials and Methods), but without either oligo(dT) or RNase H. It can be seen from Fig. 1B that treatment of viral RNA with the enzyme without oligo(dT) produced no 200nucleotide-long fragments, though there was a rather low level of nonspecific degradation produced by RNase H. The same result was obtained when BSMV RNA was incubated with oligo(dT) but without RNase H (data not shown).

BSMV

tRNA

RNA

0.8

0.E

0.4

0.;

5: a?

0.25

t 0.i

0.

5

10 FRACTION

15 N”MBEi”

FIG. 1. Detection of the short fragment (Sh) produced by cleavage of total BSMV RNA with RNase H. (A) RNA incubated with RNase H in the presence of oligo(dT)ic. (B) RNA incubated with the enzyme without oligo(dT) (mock-treated RNA). Samples were analyzed by sucrose density gradient (1040%) centrifugation. Arrows indicate the positions of markers, total tRNA from wheat embryos, and native BSMV RNA, centrifuged in sister gradients. Sedimentation from left to right. Sh- and L-RNA are in fractions 6 and 14, respectively (A).

The RNA material from fractions 6 and 14-15 (Fig. lA), designated as short (Sh) and long (L) fragments, respectively, was collected and used in subsequent experiments.

54

AGRANOVSKY,

DOLJA, AND ATABEKOV TABLE 1

AMINOACYLATION OF Sh AND L FRAGMENTS ISOLATED AFTER CLEAVAGE OF BSMV RNA WITH RNase H IN THE PRESENCE OF OLIGO(dT)l,,a

RNA preparation Intact BSMV N II RNA, total preparation Sh fragment isolated from total BSMV RNA L fragment isolated from total BSMV RNA Without RNA (control) BSMV N II RNA, poly(A)+ fraction BSMV N II RNA, poly(A)- fraction Sh-RNA isolated from poly(A)BSMV RNA L-RNA isolated from poly(A)BSMV RNA L-RNA isolated from poly(A)+ BSMV RNA Without RNA (control)

Experiment I 7,790b

Experiment II 8,710

14,420

6910 9080

540 560

640 720

12,570 13,920

15,190 13,990

520 510

660 530

23,120 1,990

1,920

2,200 1,190

2,250 1,200

“5 pg of RNA was aminoacylated for 30 min at 30” in a reaction mixture (100 ~1) containing 50 m&f HEPES-KOH, pH ‘7.6; 1 mM ATP, 2 m.kf dithiothreitol; 40 mM KCI; 5 mM magnesium acetate; 80-100 Ng synthetase preparation and 2 &i g[H]tyrosine. bEach figure represents TCA-insoluble radioactivity in cpm/lOO al.

Aminoac@ution of Sh and L Fragments of BSMV RNA The in vitro tyrosine-accepting activities of poly(A)+, poly(A)-, and total BSMV RNA were compared with the activities of SH and L fragments isolated after RNase H digestion of these RNA preparations. It can be seen from Table 1 that Sh-RNA derived from total and poly(A)- BSMV RNA retains the accepting activity of the intact viral genome. Theoretically, the aminoacylation of Sh-RNA should be about 20 times higher than that of the fulllength BSMV RNA because of the higher proportion of r-OH ends per unit weight of this fragment. However, the extent of aminoacylation of the Sh-RNA was only about twice that of the intact RNA (Table 1). This supposedly was due to a rather low activity of aminoacylating enzymes used in these experiments. Therefore, the results concerning the tyrosylation of the Sh fragment should be considered as a qualitative illustration of its accepting ability rather than as its quantitative es-

timate. Amino acid-accepting activity of the Sh fragment isolated from poly(A)+ BSMV RNA was not tested. L fragment derived from all RNA preparations was shown to possess no tyrosineaccepting activity (Table 1). This result indicates that specific cleavage of BSMV RNA with RNase H in our experiments was about complete. Samples of total BSMV RNA treated with RNase H without oligo(dT) and then subjected to sucrose density gradient centrifugation (mocktreated RNA, see Fig. lB), were used as an additional control. No detectable loss of accepting activity of viral RNA after this treatment was found (data not shown). Size Dete-rminatkm of Sh and L Fragments A more accurate size determination of the Sh fragment was performed by PAGE in 8% gels containing Tris-borate, pH 8.3, and 6 M urea; this method allows one to resolve fragments differing in length by as little as a single nucleotide residue (Donis-Keller et al., 1977). It can be seen

INTERNAL

55

POLY(A) IN VIRUS RNA

from Fig. 2 that the Sh fragment isolated from total BSMV RNA is represented by three subspecies, the major one having about 208 nucleotides and the minor ones having 195-199 and 213 nucleotides in length, respectively. The size of these fragments has been estimated from the log molecular weight vs mobility plot based on the mobilities of markers: 5.8 S RNA (n = 158) and 5 S RNA (n = 120) from yeast, and tRNAV”’ from E’. coli (n = 76); the latter is not seen in Fig. 2. The minor 195- to 199nucleotide fragment appeared to be less reproducible than the other two and even was absent from some Sh-RNA preparations; this fragment as well as some other additional “light” fragments seen on Fig. 2 are most likely nonspecific degradation products of the larger ShRNA species. A similar set of fragments was revealed when Sh fragments isolated from poly(A)+ and poly(A)- BSMV RNA were analyzed by PAGE (data not shown). Thus, the poly(A) sequence is mapped at a distance of about 210 nucleotides from the 3’-end of BSMV genome. These data also imply that poly(A)- RNAs of BSMV as well as poly(A)+ RNAs, respectively, contain the internal oligo(A) or poly(A) tracts at the same distance (210 nucleotides) from their r-end. The preparation of the L fragment isolated from fractions 15-16 of the sucrose gradient (see Fig. 1A) was found to consist of only two RNA species having the same mobilities as the intact virion RNAs 1 and 2 in total BSMV N II RNA when subjected to PAGE in 4% gels (data not shown). This result indicates that BSMV RNA contains no additional poly(A) sequences more distant from its 3’-end than 210 nucleotides; otherwise RNase H cleavage would have produced another set of fragments shorter than the L fragment observed. Translation of the L Fragment in a CellFree Rabbit Reticulocyte System When added to a rabbit reticulocyte lysate, the L fragment of total BSMV RNA stimulates [8SS]methionine incorporation into polypeptides with essentially the same effectiveness as does the intact total BSMV

B

A T

*

T”’

5.6 s (n=I58)

5

s+

(n=I20)

FIG. 2. Electrophoregram of Sh fragments produced by the RNase H cleavage of total BSMV RNA (B). Sh fragments were isolated from the gradient fraction 6 after treatment of viral RNA with RNase H in the presence of oligo(dT)iO with subsequent sucrose gradient fractionation of Sh and L fragments (see Fig. 1A and Materials and Methods). Sh fragments were analyzed by PAGE in 8% slab gels photographed after staining with ethidium bromide under uv light. The positions of marker 5.8 S RNA and 5 S ribosomal RNA, 15% and 126-nucleotide-long, respectively, in PAGE are indicated at the left track (A).

RNA. According to the results of a typical experiment, [%S]methionine incorporation into the acid-insoluble fraction was 63,960 cpm for total BSMV N II RNA and 59,280 cpm for the L fragment (per 2 ~1 of incubation mixture containing 0.2 pg RNA, see Materials and Methods), as compared to 5840 cpm for a sample with no RNA (endogenous synthesis).

56

AGRANOVSKY, DOLJA, AND ATABEKOV

A comparative analysis of the ?S-labeled in vitro translation products of the L fragment and of total BSMV RNA by PAGE reveals similar patterns of polypeptides (Fig. 3). Polypeptides with molecular weights of 120,000, 85,000, and 25,000 indicated in Fig. 3 are the major products translated from BSMV N II RNA 1 (~120) and RNA 2 (~85 and ~25) in vitro (Dolja et al., 1979, 1981). The BSMV coat protein coded for by RNA 2 (Dolja et al., 1979, 1981) does not contain methionine and is not seen therefore among the %Slabeled translation products. Some additional zones observable in the figure result mostly from abortive translation and/or the presence of fragments of genomic RNAs in the total BSMV RNA preparations, which is characteristic of rod-shaped viruses (Atabekov, 197’7).Labeling with a 14C-amino acid mixture yielded the same products for both messengers, with the addition of the BSMV coat protein (mol wt 23,000) (data not shown).

A

B

C

pI20

P85

~25

Nonirlfectivity

of the L Fragment

The Norwich strain of BSMV produces local lesions on Ch. awmranticolor. The lesions induced upon inoculation with BSMV RNA contain infectious virus which can be isolated and purified by a standard technique (unpublished data). The infectivity of total N II RNA treated with RNase H without oligo (dT) (mock-treated RNA, see Fig. 1B) was compared with that of viral RNA digested with RNase H in the presence of oligo(dT)10 (L fragment, see Fig. 1A). The L fragment derived from total BSMV RNA was found to possess very low residual infectivity, if any. Indeed, the total number of lesions produced on eight individual half-leaves was 859 for the mock-treated BSMV RNA and 37 for the L fragment, which corresponds to a 96% loss of infectivity of the latter. Mixing with a Sh fragment (1 pg per 20 ,ug of LRNA) does not restore the infectivity of the L fragment (data not shown). DISCUSSION

The data of the present paper taken together with the results of our previous

FIG. 3. Autoradiographs of SDS-PAGE (8-2056 slab gels) of the [86S]methionine-labeled translation products synthesized in the cell-free reticulocyte system under the direction of either native total BSMV RNA (C) or its L fragment (A, B). L fragment was isolated from the sucrose gradient fractions 14-15 after centrifugation of the RNase H cleavage products of BSMV RNA (see Fig. 1A and Materials and Methods). Products in lanes A and B were translated from L-RNA preparations obtained independently.

works (Agranovsky et al., 1978, 1981; Negruk et al., 1979) clearly demonstrate the presence of a poly(A) tract of 8 to 30 nucleotides at an internal position near the r-end of the viral RNA. The poly(A) sequence divides the BSMV RNA molecule into two unequal parts-the 5’-terminal coding L fragment and the b-terminal nontranslated part (Sh fragment) containing a tyrosine-accepting (tRNA-like) structure (see Table 1). These fragments were isolated after site-specific cleavage of BSMV RNA with RNase H in the presence of oligo (dT),o. It was demonstrated by Metelev et al. (1980) that RNase H from E. coZi splits a molecule of RNA at the position corresponding to the r-end of het-

INTERNAL

POLY(A)

eroduplex, i.e., at the position of the 5’-end of complementary oligodeoxyribonucleotide. Therefore the size of the Sh fragment which is about 210 nucleotides (Fig. 2) gives a measure of the distance of the poly(A) tract from the r-end of BSMV genome. This scheme has been confirmed by T1 fingerprint analysis of Sh-RNA as compared to individual BSMV RNAs. The Sh fragment was shown to be represented only by universal T1-oligonucleotides localized at the r-region of all genomic components (RNA l-4) of all BSMV strains tested (S. N. Belzhelarskaya, J. M. Adyshev, and Yu. V. Kozlov, unpublished results). The existence of poly(A) sequences covalently bound to the r-end of RNA is a common feature of many eukaryotic and viral messengers (for review see Lewin, 1974; Atabekov and Morozov, 1979). Internal locations in polynucleotide chains have heretofore only been reported for intercistronic poly(A) in eukaryotic heterogeneous nuclear RNA species and in promRNA transcripts of some negative-strand viruses (Nakazato et al., 1973; Herman et al., 1978; Robertson et al., 1981). Recently such intercistronic tracts were found in brome mosaic virus RNA 3, which can be aminoacylated with tyrosine (Ahlquist et al., 1981; Fowlks and Lee, 1981; Hall, 1979). Thus, one can see that BSMV and BMV share a number of common features-they infect the same hosts, they both have multipartite genomes, their RNAs contain the 3’-terminal tyrosine-accepting structure and an internal poly(A). However, the situation with polyadenylates inserted between coding and noncoding parts of the BSMV genome seems to be different from that with intercistronic poly(A) in other viral RNAs. The 3’-end of the BSMV genome is marked by the presence of a tandem of poly(A) plus the tRNA-like structure. The total preparation of BSMV virion RNA represents a population consisting of poly(A)+ and poly(A)- molecules in terms of binding of RNA to poly(A)-specific sorbents (Agranovsky et al., 1978, 1981). It is important to note, however, that poly(A)- RNA of BSMV was cleaved

IN VIRUS

RNA

57

with RNase H in the presence of oligo(dT) as well as poly(A)+ viral RNA. This observation implies that the poly(A)- fraction of BSMV RNA contains an oligo(A) tract in the same position as that of poly(A) in the poly(A)+ fraction. The length of these oligo(A) tracts might be sufficient for base pairing with oligo(dT)l,, but they are apparently too short for poly(A)- RNA to be adsorbed on oligo(dT)-cellulose. While the polyadenylates from total BSMV Norwich RNA were found to be up to 30 nucleotides long (Negruk et al., 1979), oligo(A) tracts from the poly(A)- fraction supposedly were 8-10 nucleotides in length. Therefore the difference between the poly(A)- and poly(A)+ fractions of BSMV RNA seems to be quantitative rather than qualitative, and poly(A)- RNA can now be characterized as oligo(A)+ RNA. A similar situation, but for r-terminal poly(A), was recently described for RNA of the foot-and-mouth disease virus. Unbound [poly(A)-] RNA of this virus was shown to contain a 3’-oligo(A) tract of less than 10 nucleotides while its bound fraction [poly(A)+RNA] had a 40-nucleotide-long poly(A) tract (Grubman et al., 1979). It is of interest that the L fragment is translated similarly to intact BSMV RNA (Fig. 3B), in spite of the fact that it is practically devoid of both tyrosine-accepting ability and infectivity. The artificial mixture of L- and Sh-RNA was also noninfectious suggesting that integrity of the viral genome is essential for its infectivity. To date a number of hypotheses concerning the role of the S-terminal tRNA-like structures in plant viral RNAs have been considered (for review see Hall, 1979). Our results provide experimental evidence that such a structure in BSMV RNA is involved in replication rather than in translation of the viral genome. The most plausible explanation would be that the amino acidaccepting sequence can serve as a signal for viral replicase (Litvak et al., 1973; Hall, 1979). The synthesis of internal poly(A) sequences, heterogeneous in length, is hardly consistent with the orthodox template-copying scheme for the replication of a positive-stranded viral RNA. This may indicate the synthesis of polydisperse poly(A) by “slippage” polymerization or

58

AGRANOVSKY,

DOLJA, AND ATABEKOV

reiterative copying of oligo(U) in (-) strand of RNA (Herman et al., 1978), or the existence of some novel mechanisms operating in the replication of BSMV genome. ACKNOWLEDGMENTS We express our appreciation to Dr. M. Leiser for the N II isolate of BSMV. The authors are thankful to Mr. A. V. Galkin for the assistance in translating the text. REFERENCES ACRANOVSKY,A. A., DOLJA, V. V., KAVSAN, V. M., and ATABEKOV, J. G. (1978). Detection of polyadenylate sequences in RNA components of barley stripe mosaic virus. Virology 91.95-105. AGRANOVSKY,A. A., DOLJA, V. V., GORBULEV,V. G., Koz~ov, Yu. V., and ATABEKOV,J. G. (1981). Aminoacylation of barley stripe mosaic virus RNA: Polyadenylate-containing RNA has a 3’-terminal tyrosine-accepting structure. fiw 113, 174187. AHLQUIST, P., DASGUPTA, R., and KAESBERG, P. (1981). Near identity of 3’ RNA secondary structures in bromoviruses and cucumber mosaic virus. Cell 23,183-189.

ATABEKOV,J. G. (1977). Defective and satellite plant viruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 11, pp. 143-216. Plenum, New York. ATABEKOV,J. G., and MOROZOV,S. Yu. (1979). Translation of plant virus messenger RNAs. Advan Virus Rea 25,1-91. DARLIX, J. L. (1975). Simultaneous purification of E. coli termination factor RHO, RNase III and RNase H. Eur. J. Biochem 51,369-376. DOLJA, V. V., LUNINA, N. A., LEISER, R.-M., STANARIUS,T., BELZHELARSKAYA,S. N., KO~LOV,Yu. V., and ATABEKOV, J. G. (1981). A comparative study on the in vitro translation products of individual RNAs from two-, three-, four-component strains of barley stripe mosaic virus: Translation control and overlapping of polypeptides encoded in the pseudomultipartite genome. Submitted for publication. DOLJA, V. V., SOKOLOVA,N. A., TJULKINA, L. G., and ATABEKOV, J. G. (1979). A study of barley stripe mosaic virus (BSMV) genome. II. Translation of individual RNA species in a homologous cell-free system. Mel GGTLGen& 175,93-97. DONIS-KELLER,H., MAXAM, A. M., and GILBERT, W. (1977). Mapping adenines, guanines and pyrimidines in RNA. Nucleic Acids Ran 4.2527-2538. FO~LKS, E. R., and LEE, Y. F. (1981). Detection and sequence of an internal A-rich T1-oligonucleotide

series in brome mosaic virus RNA 3. FEBS Letk 130,32-38.

GRUBMAN, M. J., BAXT, B., and BACHRACH, H. L. (1979). Foot-and-mouth disease virion RNA: Studies on the relation between the length of its 3’poly(A) segment and infectivity. virdogy 97, 2231. HALL, T. C. (1979). Transfer RNA-like structures in viral genomes. Int. Rev. Q&L 60,1-26. HERMAN, R. C., ADLER, S., LAZZARINI, R. A., CoLONNO,R. J., BANERJEE,A. K., and WESTPHALL,H. (1978). Intervening polyadenylate sequences in RNA transcripts of vesicular stomatitis virus. CeU 15,587-596. JACKSON,A. O., and BRAKKE, M. K. (1973). Multicomponent properties of barley stripe mosaic virus RNA. virdogy 55,483-494. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lmwkm) 227,680-685. LANE, L. C. (1974). The components of barley stripe mosaic and related viruses. lrirology 58,323-333. LEWIN, B. (1975). Units of transcription and translation: The relationship between heterogeneous nuclear RNA and messenger RNA. CeU 4,11-20. LITVAK, S., TARRAGO,A., TARRAGO-LITVAK,L., and ALLENDE, J. E. (1973). Elongation factor-Viral genome interaction dependent on the aminoacylation of TYMV and TMV RNAs. Nature New Bid 241.88-90. METELEV, V. G., RODIONOVA,N. P., CHICHKOVA, N. V., ATABEKOV,J. G., BOGDANOVA,A. A., SHABAROVA, 2. A., BERZIN, V. JANSONE,I., and GREN, E. J. (1980). Addressed fragmentation of RNA molecules. FEBS L&t. 120,17-20. NEGRUK,V. I., AGRANOVSKY,A. A., SKRYABIN,K. G., and ATABEKOV,J. G. (1979). Size determination of poly(A) sequences in barley stripe mosaic virus RNA. AnaL Bioch.tm 99,450-453. NAKAZATO,H., KOPP,D. W., and EDMONDS,M. (1973). Localization of the polyadenylate sequences in messenger ribonucleic acid and in heterogeneous nuclear ribonucleic acid in HeLa cells. J. Bid Chem. 248,1472-1476. PELHAM, H. R. B., and JACKSON,R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem 67,247-256. ROBERTSON,J. C., SCHUBERT,M., and LAZZARINI, R. A. (1981). Polyadenylation sites of influenza virus mRNA. J. viral 38, 157-163. ROWLANDS,D. J., HARRIS, I. J. R., and BROWN,F. (1978). More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA. J. ViroL 26,335~343.

STEPANOVA, 0. B., METELEV, V. G., CHICHKOVA, N. V., SYIRNOVA, V. D., RODIONOVA,N. P., ATABEKOV,J. G., BOGDANOV,A. A., and SHABAROVA, Z. A. (1979) Addressed fragmentation of RNA molecules. FEBS I,.& 103,197-199.