VIROLOGY

177,699-709

(1990)

Genome

Structure

FRANK MEULEWAETER,* tflant

Genetic Systems Rijksuniversiteit Received

of Tobacco

Necrosis

JEF SEURINCK,t

AND

Virus Strain A’

JOHN VAN EMMELO+*

N. V., J. Plateaustraat 22, and *Laboratorium Gent, K. L. Ledeganckstraat 35, 9000 Gent, December

20, 1989; accepted

April

voor Genetica, Belgium

18, 1990

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5’proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV). red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5’ end no homology was found with the RNA of STNV (satellite of TNV). o 1990Academic PWSS. inc.

INTRODUCTION

virus particles (Salvato and Fraenkel-Conrat, 1977). Only minor amounts of 63-, 43-, and 22-kDa proteins were found. If there is no processing of the viral RNA in vitro this would mean that TNV RNA acts as a polycistronic messenger. In order to determine the genome expression strategy and relationship with other viruses, the RNA of TNV strain A was cloned and sequenced. We demonstrate that the proteins encoded by TNV RNA show significant sequence similarity to proteins from different icosahedral plant viruses, except for two small putative proteins which appear to be unique for TNV. Furthermore, we suggest that TNV is closely related to the carmoviruses with respect to its genomic organization.

Tobacco necrosis virus (TNV) is a small icosahedral, fungus-transmitted virus, which is often associated with satellite viruses (Kassanis, 1970; Uyemoto, 1981). Although the molecular biology of one satellite has been well studied (STNV, Ysebaert et a/., 1980; van Emmelo et al., 1987), very few data are available on the genome organization and expression of the helper virus (Fraenkel-Conrat, 1988). The single viral genomic RNA of TNV is 3.8-4.0 kb long (Condit and Fraenkel-Conrat, 1979; Bishop et a/., 1967) and shares less than 2% homology with STNV RNA (Shoulder eta/., 1974). It has no cap structure nor a VPg, the 5’.terminal sequence being ppApGptJp (Lesnaw and Reichmann, 1970). There is no poly(A) tail at the 3’terminus (Condit and Fraenkel-Conrat, 1979). Two double-stranded RNAs of less than full length were isolated from TNV-infected tobacco (1550 and 1380 bp, Condit and Fraenkel-Conrat, 1979; 1550 and 1250 bp, Ameloot, 1989). These could correspond to two 3’-terminal subgenomic mRNAs. The smallest one encodes the coat protein (30 kDa) (Condit and Fraenkel-Conrat, 1979), which suggests that the coat protein cistron is located at the 3’end of the genomic RNA. On the other hand, the coat protein was the major in vitro translation product of full-length TNV RNA purified from ’ Sequence EMBUGenBank ’ To whom

data from this article Data Libraries under reprint requests should

has been deposited with Accession No. M33002. be addressed.

MATERIALS

AND

METHODS

Materials AMV reverse transcriptase was obtained from Boehringer (Mannheim, FRG). The RNA sequencing kit was from Bethesda Research Laboratories (Life Technologies Inc., Gaithersburg, MD). All other enzymes and random primers were from Pharmacia (Uppsala, Sweden). pGEM3Z, a plasmid containing SP6 and T7 RNA polymerase promoters and the /acZ a-peptide coding sequence, was purchased from Promega-Biotee (Madison, WI). Satellite-free TNV (PV68, Kassanis strain A; Babos and Kassanis, 1963) was obtained from the American Type Culture Collection (Rockville, MD). 32P- and 35S-labeled products were from Amersham

the

699

0042.6822/90

$3.00

CopyrIght 0 1990 by Academic Press. Inc. All rtghts of reproduction in any form reserved

700

MEULEWAETER.

SEURINCK.

AND

VAN

EMMELO

(Buckinghamshire, England). Antiserum against TNV coat protein was a kind gift of Dr. P. Ameloot (State University, Gent, Belgium). Wheat germ was obtained from General Mills (Vallejo, CA). Methods cDNA cloning. TNV strain A was propagated in Phaseolus vulgaris L. Virus purification was essentially as described by Lesnaw and Reichmann (1969). RNA was obtained by extraction with phenol. The first strand cDNA was synthesized by random priming starting with 3 pg of viral RNA (Krug and Berger, 1987). The RNaseH method (Gubler and Hoffmann, 1983) was used to obtain dsDNA. cDNAs longer than 800 bp were purified from agarose gels and ligated into the Smal site of pGEM3Z. Recombinant clones were selected by color screening. Viral RNA was alkali-fractionated by incubation in a 60 mM Na,CO,, 40 mM NaHCO, solution for 40 min at 60”. This RNA was labeled at the 5’end with 32P byT4-polynucleotide kinase and used as probe to identify TNV-specific clones. Subsequent DNA manipulations were according to Maniatis et al. (1982). cDNA from the 5’ end of TNV RNA was obtained by reverse transcription from a 5’-32P-labeled oligodeoxynucleotide primer complementary to nt 233-250 of the genomic RNA. This ssDNA was used directly for sequencing. Sequencing. DNA sequencing was performed according to Maxam and Gilbert (1977). For direct RNA sequencing of the 5’ end, the viral RNA was labeled by guanylyltransferase with [cx-~‘P]GTP as substrate. In this way, only the intact 5’ end of the viral RNA was marked with a radioactive cap. RNA sequencing reactions were performed according to the supplier’s recommendations. The sequence data were analyzed using the lntelligenetics programs SEQ, PEP, and IFIND. Identification of in vitro translation products. In vitro RNA synthesis was done essentially as described by Krieg and Melton (1984). Wheat germ extract preparation and in vitro protein synthesis were according to March et a/. (1986) with final concentrations of 1 mM Mg2+ and 110 mM K+ for the protein synthesis. Immunoprecipitation was performed according to van Emmelo et al. (1984). Analysis of subgenomic RNAs. Single- and doublestranded RNA were prepared according to Diaz-Ruiz and Kaper (1978). Double-stranded RNA was further purified by CFl 1 -cellulose chromatography (Morris and Dodds, 1979). The RNA was denaturated with glyoxal (McMaster and Carmichael, 1977) and run on a formaldehyde 1.59/o agarose gel as recommended by Amersham. Blotting, hybridization, and probe synthesis were also according to Amersham’s guidelines, ex-

FIG. 1. (A) cDNA clones used for sequencing and their location in the TNV genome. The cDNA inserts of pFM21 (nt 20-2619 of TNV RNA), pFM24 (nt 19-1632) pFM20 (nt 1763-3660) pFM17 (nt 2302-3202). and pFM23 (nt 2593-3510) were sequenced completely. From pFM22 only 334 nucleotides (nt 19-352) were sequenced (boxed region). The asterisks show the location of the T7 promoter of pGEM3Z in each clone. (B) Open reading frames in the (+) and (-) strands of the TNV genome. Open boxes represent ORFs larger than 100 nucleotides. ORFs start with an AUG and end with a stop codon, except from ORF 2 which starts at the amber codon of ORF 1. The numbers refer to the ORFs which are discussed in the text and potentially code for proteins of 23 kDa (ORF l), 82 kDa (ORF 1 + 2) 7.9 kDa (ORF 3) 6.2 kDa (ORF 4) 30 kDa (ORF 5) and 6.7 kDa (ORF 6). The dashed regions represent the 3’end which was not sequenced.

cept that hybridization was at 68” for RNA probes and at 50” for DNA probes. For DNA probes small DNA fragments were isolated from agarose gel, self-ligated, and labeled with [a-32P]dCTP by nick translation. RESULTS Nucleotide sequence analysis cDNA clones of TNV genomic RNA were obtained by random priming. Six of these clones, comprising 3642 nucleotides (nt) of the TNV genome, were used for sequencing (Fig. 1A). Ninety-four percent of the bases were determined by sequence analysis of at least two clones in such a way that for all nucleotides the information was obtained from both plus and minus strands. The sequence of the uncloned region at the 5’end of the RNA (18 nt) was determined by sequencing singlestranded DNA obtained by cDNA synthesis initiating from a labeled primer. Only the terminal nucleotide could not be deduced by this method. However, direct sequencing of the RNA confirmed the presence of an adenosine residue at the 5’ end (Lesnaw and Reichmann, 1970) and demonstrated that the 5 adjacent nucleotides were complementary to the terminal nucleotides of the cDNA obtained by primer extension. Com-

GENOME

STRUCTURE

parison of this single-stranded DNA with overlapping cDNA clones showed no sequence differences up to nt 70 from the 5’end of the RNA. Since polyadenylation or 3’ end labeling of the viral RNA was unsuccessful, we were not able to determine the extreme 3’ end of the TNV genome. So, the sequence presented here (Fig. 2) represents the first 3660 nt of TNV genomic RNA. Depending upon the exact length of the RNA(3.8-4.0 kb; Condit and FraenkelConrat, 1979; Bishop et a/., 1967) approximately 150 to 350 nucleotides are still missing. Open reading frames Figure 1 B shows the open reading frames longer than 100 nucleotides found on the TNV plus and minus strands. The amino acid sequences of the major open reading frames located on the genomic RNA are shown in Fig. 2. On the + strand three large open reading frames are found. The first AUG (nt 60-62) of the viral RNA is the start of the coding region of a 23-kDa protein. This open reading frame (ORF 1) ends with a UAG codon (nt 666-668) which is followed by a second open reading frame (ORF 2) which ends with a UGA codon at position 2232-2234. Thus, read-through of the amber termination codon would give rise to an 82-kDa protein. The third large open reading frame (ORF 5) begins at nt 2613 and ends at position 3443. This codes for a protein of 30 kDa which corresponds by amino acid composition and molecular weight to the coat protein (Lesnaw and Reichmann, 1969). This was confirmed by immunoprecipitation of in vitro synthesized 30-kDa protein (as in Fig. 3, lane 5) using antiserum against the viral coat protein (not shown). Two small open reading frames are located between ORFs 2 and 5. ORF 3, which extends from nt 2218 to 2436, encodes a 7.9-kDa protein while ORF 4 (nt 2440-2610) encodes a 6.2-kDa protein. A third small ORF (ORF 6: nt 3467-3646) with a coding capacity for a 6.7-kDa protein was found downstream from the coat protein cistron. On the - strand four ORFs for proteins between 10 and 18 kDa are found. Noncoding

regions

The 5’-untranslated region of TNV RNA is 59 nucleotides long. The first four nucleotides (AGUA) are identical to the corresponding STNV sequence (Ysebaert et a/., 1980) whereas the remainder of the untranslated leader shows no homology with STNV RNA. The TNV genome is very compact, the intercistronic regions being very short. There are 3 nucleotides between ORFs 3 and 4 and 2 nucleotides between ORFs 4 and 5 while ORFs 2 and 3 overlap (17 nucleotides). ORF 6 starts 23 nucleotides downstream from the coat

OF TNV

STRAIN

701

A

protein cistron. Because we only determined 14 nucleotides downstream of ORF 6 no information is available on the remaining 3’end structure. Sequence

heterogeneity

Sequence analysis of overlapping cDNA clones revealed 14 point mutations, of which 9 cause amino acid changes in the TNV gene products. Six of these amino acid differences are located in the coat protein, one in the 7.9-kDa protein and two in the 82-kDa protein. Although one cannot exclude that some of these point mutations originate from the cloning procedure, these data indicate that there is some heterogeneity in our virus isolate, especially in the coat protein gene. One nucleotide difference in pFM24 has not been included because it is located at the third nucleotidefrom the 3’ end and thus originates from the primer used to start first strand synthesis. Besides these base substitutions there is also one deletion; nt 301 is absent in pFM21, which would cause a frameshift in the 23-kDa protein resulting in the production of a truncated (10 kDa) protein. Identification of TNV RNA

of in vitro translation

products

TNV RNA was introduced into the wheat germ protein synthesis system in order to identify its in vitro translation products. Figure 3 (lane 1) shows that the major translation products are a 30-kDa and a 23-kDa protein. The 30-kDa protein was identified as the coat protein by immunoprecipitation (not shown). The coat protein is translated from ORF 5 (Fig. 3, lane 5). The 23kDa protein is the translation product of ORF 1 (Fig. 3, lane 4). So, the 22-kDa protein observed by Salvato and Fraenkel-Conrat (1977) probably corresponds to our 23-kDa protein and not to the coat protein of contaminating STNV RNA. While our wheat germ extract supports read-through of the amber codon of the coat protein cistron of beet necrotic yellow vein virus (F. Meulewaeter, unpublished observations) (Ziegler et al., 1985) we never observed a 82-kDa read-through protein which could be predicted from the primary sequence. Instead, the largest protein is about 50 kDa, which is also smaller than the 63-kDa protein observed with TNV strain AC36 (Salvato and Fraenkel-Conrat, 1977). In order to exclude the possibility that the coat protein is synthesized from contaminating small RNAs, an in vitro synthesized uncapped nearly full-size TNV RNA was introduced into the wheat germ system. This RNA was synthesized with T7 RNA polymerase using the hybrid plasmid pFM 1 19 as template. This is a recombinant of pFM22 and pFM20 at the unique Nsil site (nt 2048). The same pattern of protein bands is observed as with genomic TNV RNA (Fig. 3, lane 3). Moreover,

702

MEULEWAETER,

SEURINCK,

AND

VAN

EMMELO

10 20 30 40 50 60 70 80 A AGUAUUCAUACCMGMUACCAAAUAOCUGCAAG~C~ACUCA~~A~GAGUCUAAAAUGGAGCUACCAAACCAACACAAGCAAACOGCCGCCGAGGG MELPNQHKQTAAEG

100

110 120 130 140 150 160 170 180 UUUCGUAUC~~~~~~UC~~AUGC~~~CAUCGAGACGACA~GAA~AGUC~~G~~GCAG~G~G~~~~AAAAAGA~~~~~~~G~~AU~GAGGA~ FVSFLNWLCNPWRRQRTVNAA~AFQKDLLAIED

190

200

290

300

390

400

480

490

500

580

590

600

210

220

230

240

250

260

270

C

280

UCCGAGCAIRRlGGAUGACAUCAAUGAGUGUUUCGA~~CU~u~~ACAAUCUCA~G~CUAAGGUUGUC~CGACGGAGCAUAUGCCCCC~~ SEHLDDINECFEESAGAQSQRTKVVADGAYAPA A U 320 330 340 350 360 370 380 ~~~~~~AGGAC~~~~GA~C~~G~AGCAOAAQAAOCAC~~GUAA~UAUC~GUCAA~GAAG~UCGUGC~GAG~UGGAU~G~~~AAA~~~~ KSNRTRRVRKQKKHKFVKYLVNKARARFGLPKPT NPIGPAEFVSRRSTSL. 410

420

430

440

450

460

470

UGAGDCAAACAQACUUAUOCMCA~C~~UCAGAGUGU~M~~~~G~G~ACUGCCCACGUACACGGC~UG~GCACUA~~G EANRLMVQHFLLRVCKDWGVVTAHVHGNVALAL 510

520

530

540

550

560

570

CCACUGGU~LNCAUCCCAACOOAAOAUGAUCUOCUAUCAC~~A~GAUG~CACACAU~UACUAGA~CGCUGUACGAGGCAUGGAC~UGUCCMG PLVFIPTEDDLLSRALMNTHATRAAVRGMDNVQ 610

620

630

640

650

660

670

680

690

700

780

790

800

GGaAGOOClLlOaUOGMCAUA~GGG~~G~~cAG~CGQACUG~C~CCGGUCC~UAGGGGUGCC~G~GGAGGCCA~A~CUCCAc GEGWWNNRLGXGGQVGLAFRSK.GCLERRPGFST 710

720

730

740

750

760

770

OUCCGUUUCOCGUGOOGAACAUCCU~UCUGOUCOUCAUACCAUCAGG~GCCCUGAG~CAGCGUCAGUUG~ACGCUAUAGUGGUAUA~GGCCAU SVSRGEHPDLVVIPSGRPEKQRQLLRYSGIGGH 810 820 830 840 850 860 870 880 ~AWMUCOOCAUCCACAACAACUCUCUUUCCMCCU~~AG~C~GAU~AGAGUAUUCUAU~CGA~~CCAAUGGGC~CAAGACGCCC LLIGIHNNSLSNLRRGLMERVFYVEGPNGLQDA

890

900

910 920 930 940 950 960 970 980 CUMGCCCGUCAAGGGAGCICG~CCC~GAU~G~CGUGAUCUCUAUACU~UAG~GGCGUCAUACCCCUGUAACUAGUG~C~~CCU PKPVKGAFRTLDKFRDLYTKNSWRHTPVTSEQFL 1010 1020 1030 1040 1050 1060 1070 1080 ~~G~~A~A~GGCCAGGCAAACUGACUA~ACAGAQAG~G~GAUAG~G~CG~A~~AAC~C~~~AG~~~A~GAGA~G~GAAA~~~AGA~A~~ MNYTGRKLTIYREAVDSLSHQPLSSRDAKLKTF

990

1000

1090

1100

1110 1120 1130 1140 1150 1160 1170 1180 GUOAAGGCCGAAAAAWAAAUCUUUCUAA~AA~CCU~~~~~~~CAUC~MC~~~G~~C~~~~C~~U~~AA~~~~~~~~AG~~~CC VKAEKLNLSKKPDPAPRVIQPRSPRYNVCLGRY

1190

1200

1210 1220 1230 1240 UCCGACAUUAUGAGCAUCACGC~~CCAUUOCCAACCAU LRHYEHHAFKTIAKCFCEITVFKGFTLEQQGEIM

1280

1290

1300

1380

1390

1400

1250

1260

1270

1310 1320 1330 1340 1350 1360 1370 GCGCUCG~~~~~~A~G~~~~~~~~~A~~~A~~C~~~~AG~~G~~GA~~~~A~G~GU~~G~~GAAG~A~~~GAG~A~GAG~A~ RSKWNKYVNPVAVGLDASRFDQHVSVEALEYEH 1410

1420

1430

1440

1450

1460

1470

1480

1490

1500

1640

1650

1660

1670

1680

1690

1700

OAAUIIUUACCUCAOAGACUACCCAAAUGAUAAACAOCUAA EFYLRDYPNDKQLKWLLKQQLCNVGTAFASDGI

IKYKKKGCRMSGDMNTSLGNCILMCAMVYGLKEH 1610

1620

1630

CLR)AAACAUCMULRIGUCCC~~~UMU~U~CU~~CA~~CUGUGAG~~GGA~~GA~~GAC~~AGCAUCGA~CAUAU LNINLSLANNGDDCVIVCEKADLKKLTSSIEPY 1710

1720

1730

1740

1750

1810

1820

1830

1840

1850

1760

1770

1780

1790

1800

1860

1870

1880

1890

1900

UUCAAGCAGUUUGGAUUCMGAUGGAAGu GOAAAAA~~~~~UA~A~~~~A~AG~~G~~AAA~~~AA~~UG~G~CGA~GGA~~~~AG~ FKQFGFKMEVEKPVDIFERIEFCQTQPVFDGSQ ACAUCAUGOUACOCAAACCLNCUGUOGUAACAUCUAAAOAGU YIMVRKPSVVTSKDVTSLIPCQTKAQYAEWLQAV

C

FIG. 2. Nucleotide sequence of the 5’-proximal 3660 nucleotides of TNV RNA and amino acid sequences of the major open reading frames. Nucleotides l-l 8 were derived from primer extension and direct RNA sequencing; and nt 19-1600, nt 1601-2200, and nt 2201-3660, from DNA sequencing of pFM24, pFM21, and pFM20, respectively. Nucleotide differences obtained with other clones are given above the sequence and the resulting amino acid changes are shown below the sequence. - indicates a missing nucleotide in pFM21 (position 301). For the regions sequenced on more than two clones, the mutations found at nt 274 and 2431 are only present in pFM21; at nt 89 and 308. in both pFM21 and pFM22; at nt 2824 and 2982, only in pFM23; at nt 2956 and 3190, only in pFMl7; and at nt 2773, in both pFM23 and pFM17.

GENOME

STRUCTURE

OF TNV

STRAIN

703

A 1980

1990

2000

2080

2090

2100

2180 2190 G 2160 2170 2110 2120 2130 2140 2150 ~~~UAU~A~A~~AGACCUCCAAOAAOCAUUOCACAUC~CUAUGAUACCCAC~GCUUGA~GGAUGAUG~~A~~~~~~~GAUA~~~A~~AA~G~~ FGITPDLQEALEIFYDTHKLRLDDVIPTDTYQVS R 2290 2260 2280 2280 2210 2220 2230 2240 2250 A~AGAOCAINUGAUCMUDAWACCAAACUGAUGUMC~A~AC~UGUGC~UAC~G~C~GCUAGGAGCGWGA~~~G~CAC~~~ GEHLINGLPN. NDYQTDVTEDNVQIRGRARSVRGKKHNG

2200

2360 2310 2320 2340 2370 2330 2350 ~CGGGAWMCUOOCOUUM~~CAC~~U~~G~CAUCUCAG~UCACAGC~~UACUG~AA~GG~~~A~GA~~~~A~A~~G~G~ SGLTGVKRHAVSETSQKSQQGTGNGTMTNIASE

1910 1920 1930 A~GA~~~AUGADCA~~~~~A~CCUCUC~A~~AAGA~~ GECGMSINGGIPVMQNFYQKLQTGIRRTKFTKT

1940

1950

1960

1970

2070 2030 2040 2050 2060 2010 2020 ~GA~CCAGACGAACO~~AUCACUCUAGAUAUAUGCAUAGAGUG~CC~~~~~~~~~GAAA~~~G~A~~~~~~A~~~A~~ GEFQTNGLGYHSRYMHRVARVPSPRTRLSFYLA

2300

2380

2390

2400

2480

2490

2500

CAGACCAwACCwGACAUACAACLRTUAACULRRlAAGWAUGGCUGCGUGUC~UGUUGUGAUACwCACCAGGUAwACACUAUUcccwAc~GcAA MAACRCCDTSPGITLFPYFA QTITVTYNFNF. L 2580 2510 2520 2540 2560 2590 2530 2550 2570 WCUCAUCCWAUAWGGCAAUACW~WA~GACUCCC~UC~CAAUAUCACCAWCUCC~GCACUUACGAGUACAAGACUCAACACAUUUCGAU ILILILAILVVGTPNQQYHHSPSTYEYKTQHISI

2600

2410

C

2420

2440

2450

2460

2470

2690 2640 2660 2670 2680 2610 2620 2630 2650 CGCAAAAUAGACAUOOCAOa~GMG~CMC~C~C~UCAGUAUAU~UACUGCGUACUCCAGAGCAACAGGUGGAGAUAGAccAGcGcAAc~cC MAGKKNNNNGQYIILRTPEQQVEIDQRNA A K.

2700

2780 2710 2720 2740 2760 2790 2730 2750 2770 A GUCGUGCUCAAAUOOGUCOCAUGAAGAAOOCUAGACAGCCC~CAGCGAUACWACAGC~CACGGGUUGCG~ACGGAWGUCCGGUAGAGGGG~UA RRAQMGRMKKARQPVQRYLQQHGLRNGLSGRGGY Q 2810 2820 c 2830 2840 2850 2860 2870 2880 2890 ~A~AG~~~U~~~A~~~~~GGOGGG~~~ACUCGACCCAUA~~CG~A~CUCC~CAGG~AGAWCCACUAUAGUCCGUAACACUGAGAUUUUG IVAPTSGGVVTRPIVPKFSNRGDSTIVRNTEIL A 2910 2920 2930 2940 C2960 2950 2980 G 2970 2990 ~C~CCA~~CWA~OGCGAGGCAIIUCAAUAC~C~CUCC~ACU~~GCAGCA~ACCAUCAUGGCUGGCUA~AUCGCUGAUCWUACA NNQILAALGAFNTTNSALIAAAPSWLASIADLY T G 3010 3020 3030 3040 3060 3080 3050 3070 3090 ~~~A~AGA~G~~~~~A~~GAGAU~A~~UACAWCC~UGCCCCACCACCACCA~~AUC~WGCCAUGGCUUUCACAUACGACAG~UGA SKYRMLSCSIIYIPKCPTTTSGSIAMAFTYDRND

2800

3110 3120 3140 3130 3160 3150 3170 ~G~U~A~~~A~~G~~~~~~AG~~~~A~MUC~ACM~CAUCM~CCACC~AU~~AUACGAC~A~A~AUA~G~~CGAA~ AAPTARAQLSQSYKAINFPPYAGYDGAAYLNSN 3210

3220

3180

C

2900

3000

3100

3200

S 3230

3240

3250

3260

3340

3350

3360

3270 ~A~A~U~~A~~AU~~~~~~~~GAUGWACCMG~GGACM~CAU~UACCCCACUAUCUCCUCUGCCG~UUCG~GC~~AG~G

3280

IJ

3290

G

QGAGSAIAVQLDVTKLDKPWYPTISSAGFGALS

3310

3320

3330

U~~U~GAU~AG~~~~WW~UOCCCCGCGU~~~UA~GAU~ACCC~UACU~UACUCCAGCA~GAC~~CAU~MGUA~GU VLDQNQFCPASLVVASDGGPATATPAGDLFIKYV

3410 3420 3430 3440 3460 3450 GAUU~A~~~~AW~~~~UCAACCCAACAAUGAACGUACUGU~CW~CUMUGCCUAAGGUGGAGUCACACCAW~A~~~A IEFIEPINPTMNV.

3370

3380

3390

3ioo

3470

3480

3490

3500

3590

3600

MPKVESHHWRR

3510 3520 3530 3540 3550 3560 GACOOAUCcU~~cA~w~c~~~U~~CCCCGACGAC~~~~AC~~~~~~~~~~~~~~~~~~~~~~~~~~UcuGcc~ RRILGNRLDGRGVVPPTTHHSGYQWYTTMAGS,,K 3610 3620 3640 3630 3650 ~U~WW~A~~~G~~~~~UGGAAACGGGGGGGAGGGGGGUAG~ACAUAUCAUCCAG

3570

3580

3660

VLCTKNPWKRGGGG.

FIG. 2-Continued

the ratio between the different proteins is the same when initiation is blocked after different periods of protein synthesis (10 to 120 min, not shown), which suggests that the synthesis of these proteins is initiated on

the intact RNA. Furthermore, we can hardly detect any degradation of the RNA during protein synthesis in our wheat germ system when analyzed by Northern blotting (to be published elsewhere).

704

MEULEWAETER,

SEURINCK,

-----92.569.046.0-

652 1001

30.0-

1299

1722 14.3123456 FIG. 3. Translation products in wheat germ extract of TNV genomic RNA and of in vitro synthesized uncapped RNAs representing specific parts of the TNV genome. The DNA template used for RNA synthesis is indicated above lanes 2-6:Asel cleaves 140 nt downstream of the amber codon of ORF 1; OxaNI, 31 nt downstream of the stop codon of the coat protein gene; while the other restriction enzymes cleave the plasmids in the polylinker region 3’ of the cDNA insert. ln vitro translation was performed for 2 hr at 25” with about 1 pmol of RNA in a total volume of 25 /II. Proteins were labeled with [35S]methionine and analyzed on 12.5% polyacrylamide gels (Laemmli, 1970). Positions of marker proteins (in kilodaltons) are indicated at the left. Numbers at the right indicate the nucleotide positions in the TNV genome of the AUG codons where the synthesis of the specific proteins is presumably initiated.

AND

VAN

EMMELO

by ethidium bromide staining of agarose gels and by Northern blotting using RNA probes complementary to the coat protein coding region (Fig. 4A, lane 3). Moreover, subgenomic RNAs of the same size (1.6 and 1.3 kb) could be detected in single-stranded RNA preparations from the same plants (Fig. 4A, lane 2). Both RNAs hybridize with RNA probes complementary to nt 1227261 9 of TNV-RNA (Fig. 4B), indicating that their 5’ends are located upstream from the initiation codon of the coat protein gene. The largest subgenomic RNA hybridizes with DNA probes specific for nt 2317-2495 and 2139-2316 of the TNV genome (Figs. 4C and 4D). Therefore, we predict that the largest subgenomic RNA is the mRNAforthe 7.9-kDa protein (ORF 3) rather than for the 6.2-kDa protein (ORF 4). The fact that on this RNA at least three AUGs (two of them in a very good context; Joshi, 1987) are located 5’ of the initiation codon of ORF 4 strengthens this hypothesis. The smallest subgenomic RNA shows only very little hybridization with the DNA probe from nt 2317 to 2495 (Fig. 4C) while it does not hybridize with the DNA probe from nt 2139 to 2316 (Fig. 4D). So, this RNA is presumably the mRNA for the coat protein with an untranslated leader sequence of about 150 nt (without AUG codons). This is consistent with a difference of 300 nucleotides between the two subgenomic RNAs.

A 123L

In vitro synthesized uncapped RNA from the complete pFM22 insert, which contains ORFs l-4, directs the synthesis of the same proteins except for the coat protein (Fig. 3, lane 2). The minor protein species (of 50, 46, 36, 30, and 19 kDa) are also translated from RNA synthesized from pFM21 (Fig. 3, lane 6) which covers the same region as pFM22 but has a 1 -bp deletion in ORF 1 resulting in the formation of a premature terminated protein (10 kDa). This indicates that the synthesis of these proteins is initiated internally. In order to determine the location of these initiation sites, we digested pFM22 and pFM21, prior to RNA synthesis, with different enzymes, leading to the synthesis of gradually shorter RNAs. These RNAs direct the synthesis of proteins which become smaller in a way that would be expected for proteins which have the same C-terminus as the 82-kDa protein (not shown). Moreover, for almost every minor protein band (50, 46, 36, and 19 kDa) we found an AUG codon in ORF 2, which could be responsible for the initiation of this protein (Fig. 3). Analysis

of subgenomic

RNAs

In TNV-infected tobacco leaves we detected two double-stranded RNA species of less than full length

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- B 1 2

--

T SS DS H

-

C 1 2 3

--

H DS

DSSS H

-

D 123

---

DSSS H

FIG. 4. Analysis of subgenomic RNAs of TNV by Northern blotting. In the H, T, and DS lanes 2 pg of single-stranded RNA from healthy tobacco leaves was loaded. In the T lanes this RNA was supplemented with 50 ng of TNV RNA purified from virus particles, while in the DS lanes it was supplemented with 100 ng of TNV doublestranded RNA (extracted from tobacco leaves 3 days after infection). In the SS lanes 2 pg of single-stranded RNA from TNV-infected tobacco leaves (extracted 3 days after infection) was loaded. The probes used for hybridization are (A) RNA (complementary to nt 2600-3660 of TNV RNA) synthesized with SP6 RNA polymerase from pFM20. which was digested with Paul and made blunt-ended with T4 DNA polymerase; (B) RNA (complementary to nt 1227-2619) synthesized with SP6 RNA polymerase from pFM21 digested with Dral; (C) a Hgal-Fokl DNA fragment (nt 2317-2495 of TNV RNA); (D) a Bglll-Hgal DNA fragment (nt 2 139-2316 of TNV RNA). The positions of these probes in the TNV genome are also indicated at the bottom of Fig. 6.

GENOME

STRUCTURE

OF

TNV

STRAIN

A

Ca 128

107 113 91

722 FGITPWQIALEGEIR-SLTINTNVGPPCEAADSLWlLNRK. ,,,,#I II III III,II III I I I ' 681 FGITPDMEALEIPIDTHKLELDDVI~D~QVSG~~INGL~N. II III I III 919 FGYTPDEQRALEEYFKSWTPTFEWSI~KC~~-~~~.

Car"V

763 724

TNV 965

xv

cam

ca

"GNSLQLNP “----LNp

MDIESE"P""GKQMLAGNR---GKQKTR-------RSVAK"AI~K~ASDS~G-G~~V~"ADKIS"~I"~~~. II I I1 I: II III MDYQTD"TEDN"QIRGRARS"~GKKHNGSGLTGVKRHAVS~SQKSQQG~G~G~~~~IA~SQ~I~"~y~~~~. II IIII I I III; MDPERIPYNSLSDSDATGKRKKGGEKSAKKRLVAS"AASS"~~KK~NSGSAS~GG~VIVADK"~"SINF~~.

FIG. 5. (A) Alignment of the amino acid sequences from the read-through parts of the putative repllcases from TNV. CarMV. and MCMV. Numbers indicate the amino actd residues, - represent gaps to allow maximal alignment, 1 indicate identical amino acid residues, asterisks represent the conserved GDD motif of RNA replicases, and dots are stop codons. (B) Alignment of the amino acid sequences of the coat proteins of TBSV, TCV, TNV, and SBMV. Amino acid residues marked as white letters on a black background are identical to the corresponding residues of TNV coat protein. Asterisks indicate the residues that are also conserved in the coat proteins of CarMV, CNV, MNSV, and RCNMV. The different domains and the Ca’+ bindlng sites (Ca) of TBSV and SBMV coat protein are indicated respectively above and below the sequences. The secondary structural elements of SBMV (o( helices A-E and p strands B-I) are also indicated. (C)Alignment of the 7.9.kDa protein of TNV, the p8 protein of TCV, and the p7 protein of CarMV.

Sequence

similarity

with other viruses

The read-through part of the 82-kDa protein contains the GXXXTXXXNX,8m5,,GDD sequence motif typical for RNA polymerases (Kamer and Argos, 1984; Haseloff et al., 1984). This protein also shows extensive sequence similarity with the putative RNA polymerases of carnation mottle virus (CarMV; Guilley et al., 1985), turnip crinkle virus (TCV; Carrington et a/., 1989), cucumber necrosis virus (CNV; Rochon and Tremaine, 1989), red clover necrotic mosaic virus (RCNMV; Xiong and Lommel, 1989), maize chlorotic mottle virus (MCMV; Nutter eta/., 1989), and barley yellow dwarf virus (BYDV; Miller et al., 1988). The percentage of identical amino acid residues for the read-through part varies between 44% (CarMV and MCMV) and 33% (BYDV). These homologies are clustered in regions which are the same for each pair of viruses (Fig. 5A). The 23-kDa protein also shows some sequence similarity with its counterpart in CarMV in the central part of the sequence (not shown). Moreover, the coat protein of TNV also shows sequence similarity with the structural proteins of several

other small icosahedral plant viruses, like southern bean mosaic virus (SBMV; Hermodson et a/., 1982), tomato bushy stunt virus (TBSV; Hopper et a/., 1984), TCV (Carrington e2 al., 1987), CNV, CarMV, RCNMV, and melon necrotic spot virus (MNSV; Riviere et al., 1989). Figure 5B shows the alignment of the amino acid sequence of TNV coat protein with those of the tombusvirus TBSV, the carmovirus TCV, and the sobemovirus SBMV (cowpea strain). For these viruses the three-dimensional structure of their coat protein subunits has been determined at high resolution (Olson et al., 1983; Hogle et al., 1986; Abad-Zapatero et a/., 1980) and appeared to be similar. Essentially, the subunits are organized into four distinct domains: R (random, N-terminal), a (arm, connecting R and S), S (shell), and P (projecting, C-terminal). Although there is some sequence similarity in the arm and the R domain, the highly structured S domain is more conserved. For TNV coat protein the highest number of identical amino acid residues in the S domain was found with SBMV coat protein (63, i.e., 34% of paired residues) whereas with

MEULEWAETER,

706

SEURINCK, AND VAN EMMELO

TCV and TBSV only 22 and 26% similarity, respectively, was found. Similar values were found for CarMV (24%) CNV (269/o),and MNSV (27%). Moreover, the total number of residues in the S domain of TNV (186) is closer to that of SBMV (191) than to TBSV (167) and TCV (162). Only a few small gaps are needed for the alignment of TNV and SBMV coat protein. So, TNV coat protein could be aligned against the a-helices C and D of SBMV which are lacking in TCV and TBSV. Figure 5B also shows that at only two of the six Ca*+ binding sites of TBSV (Hopper eTal., 1984) TNV has the same amino acid residues. On the other hand, TNV has identical amino acids at three of the four Ca2+ binding sites of SBMV (Rossmann et al., 1983). At the fourth site a glutamic acid residue is replaced by a lysine as is also the case in the bean strain of SBMV (Mang et al., 1982). Furthermore, both SBMV and TNV coat protein lack the P domain. The 7.9-kDa protein shows, besides an identical Cterminal sequence (FNF), only limited sequence similarity with the small proteins of CarMV (~7) TCV (~8) (Fig. 5C), and MCMV (p9). The putative 6.2-kDa protein does not show any similarity with any other viral protein. The most striking feature of this protein is a stretch of hydrophobic residues (FAILILILAILVV) for which some similarity (9 out of 13 residues) is found with the transmembrane segments of other proteins (e.g., the P-subunit precursor of Torpedo californica acetylcholine receptor; Noda eta/., 1983). We could not find any sequence similarity between the 6.7-kDa protein and any other viral protein. DISCUSSION Genome organization and expression The nucleotide sequence of TNV RNA reveals that this virus has a genome organization similar to that of CarMV and TCV (Fig. 6) viruses which also have small RNA genomes (4.0 kb) (Guilley et a/., 1985; Carrington et a/., 1989). This was already suggested by Morris and Carrington (1988) on the basis of the number and sizes of subgenomic RNAs. ORF 1 of TNV is somewhat shorter, but as for these other two it is separated from coding region 2 by an amber termination codon. The context around this amber codon (CCAAAWGGG) is identical for TNV and CarMV. In contrast with CarMV, the TNV and TCV polymerase cistrons are not followed by an in-frame ORF. Although the precise map position of the 5’ end of the subgenomic RNAs has not been determined the most likely translation product of the largest subgenomic RNA of TNV is a small protein (7.9 kDa). As for CarMV and TCV the coding region of this protein partially overlaps with the polymerase gene. A second small ORF has a coding capacity for a 6.2-kDa protein,

which is unique for TNV. However, it is not clear if this protein is expressed in viva since there is apparently no subgenomic RNA for this ORF. Although it is still possible that such an RNA is synthesized only at low levels early in virus infection in analogy with the I2 subgenomic RNA of tobacco mosaic virus (Watanabe et a/., 1984). The possibility of read-through of the stop codon of ORF 3, as happens with the corresponding protein of MCMV (Nutter et al., 1989) is very unlikely as ORF 3 ends with a UAA codon. If this ORF is not expressed during virus infection there would be a long noncoding region (180 nt) between ORF 3 and 5 as for TCV, where 170 nucleotides are present between the p8 protein coding region and the coat protein cistron. All three viruses express the coat protein gene from the smallest subgenomic RNA. For TNV, this RNA has presumably a long untranslated leader sequence comparable with those of TCV and CarMV (both 137 nt) (Carrington et al., 1987; Carrington and Morris, 1986). In TNV this gene is followed by at least one small ORF which is unique for this virus. Although we never found a subgenomic RNA that could be responsible for the expression of this ORF, one cannot exclude the presence of such an RNA since it is not sure that such a small RNA will be precipitated by LiCl (Diaz-Ruiz and Kaper, 1978). If no additional ORFs were to be found in the remaining 150 to 350 nucleotides, TNV would have a long untranslated trailer sequence, comparable with those of TCV and CarMV. While in vivo the coat protein is presumably expressed from a subgenomic RNA, the coat protein is the main in vitro translation product of TNV genomic RNA and in vitro synthesized, uncapped nearly fulllength TNV RNA. The data presented here suggest that in vitro TNV RNA really acts as a polycistronic messenger. Also the minor protein products are initiated internally, mainly on AUG codons of ORF 2. It has been shown that incorrect internal initiation occurs in some in vitro protein synthesis systems (Dasso and Jackson, 1989). It is in fact not surprising that this also happens with TNV RNA since long, viral, and uncapped RNAs showed the highest degree of internal initiation. So, the minor protein products are most likely typical in vitro translation products. As the AUG codon of the coat protein is a very active internal initiation site in vitro, the coat protein gene is the best candidate to investigate the role of internal initiation in TNV genome expression in viva. Relationship with other viruses Based upon its overall genomic organization, tobacco necrosis virus is closely related with CarMV and TCV. However, the different proteins encoded by TNV show sequence similarity with those of various other

GENOME

STRUCTURE

26.8

OF TNV

85.8

STRAIN

707

A

97.7

I 6.7 CarMV

I

27.7

37.8

87.7

I

I

I

I

I

+

TCV

.

82.1

22.7

---

0

1

2

3

4kb

D--p FIG. 6. Comparison of the genome structures blocks) and on the subgenomic RNAs which which there are apparently no subgenomic encoded by these ORFs. The dashed lines positions of the different probes used for the

of CarMV, TCV, and TNV. Corresponding ORFs are drawn in the same way (white, black or gray are thought to be responsible for their expression. The stippled boxes represent ORFs of TNV for RNAs. Numbers above the ORFs indicate the molecular weight (in kilodaltons) of the proteins represent the 3’ end of TNV RNA which has not been sequenced. The bars below represent the analysis of the subgenomic RNAs of TNV (cf. Fig. 4).

small icosahedral viruses. First, the putative RNA polymerase of TNV is also homologous to those of CNV, BYDV, MCMV, and RCNMV. Second, the coat protein sequence is similar to that of SBMV, although the RNA polymerase of the latter (Wu et a/., 1987) shows only limited sequence similarity with that of TNV (around the GDD motif). Less sequence similarity was found with the capsid proteins of other icosahedral viruses, like TCV, CarMV, CNV, RCNMV, TBSV, and MNSV. Moreover, both TNV and SBMV coat proteins lack a P domain and are also more similar at the Ca2+ binding sites and in the regions of the cu-helices C and D. So, all these coat proteins have probably evolved from a common ancestral protein, with the TNV and SBMV structural proteins clearly more related to each other than to the carmovirus and tombusvirus coat proteins (Fig. 7). This is comparable with MNSV which has also a CarMV-like genome organization but with the coat protein being more similar to those of the tombusviruses (Riviere et al., 1989). All these relationships, together with those previously described (Mayo et al., 1989; Veidt et a/., 1988) (Fig. 7) support the concept of modular evolution (Zimmern, 1987) by which genes or parts of genes can be exchanged between viruses, permitting a more or less independent evolution of different genes. It also dem-

onstrates that all these viruses are in some way related to one another and, as more sequence data become available, these relationships will become more and more complex. For these small icosahedral viruses there basically seem to be two types of polymerases and at least two main types of coat proteins. Each virus is characterized by its combination of these two proteins, by the location and the nature of the other ORFs, and by its expression strategy. TNV RNA is characterized by the presence of three small ORFs, two of which have a coding capacity for proteins which have no known counterpart in other viral sequences. The 7.9-kDa protein on the other hand is common to TNV, CarMV, TCV, and MCMV. The limited region of homology of the 6.7-kDa protein with some membrane-bound proteins is believed to be a functional rather than a structural homology. The significance of these ORFs needs however further investigation. As suggested earlier (Morris and Carrington, 1988) TNV is clearly related to the carmoviruses, but there are also some major differences, as in the absence of a cap structure at the 5’end of TNV RNA. Furthermore, the coat protein of TNV is more related to the structural protein of SBMV. The presence of two additional ORFs is also a potential difference. These arguments still

708

MEULEWAETER,

SEURINCK.

FIG. 7. Relationships of TNV with other small icosahedral viruses on the basis of amino acid sequence similarities of their coat proteins and their putative RNA polymerases. Viruses placed within the same open oval have similar coat proteins and those within one shaded oval have similar replicases. The coat protein groups of TNV and CarMV are placed in one dashed oval to stress their common origin, as far as their coat proteins are concerned. The RNA polymerases of TBSV and MNSV could not be classified because the sequences were not yet available.

support the classification of TNV in a monotypic virus group. Another interesting feature of TNV is its transmission by the fungus Olpidium brassicae. Riviere et a/. (1989) found for two viruses transmitted by Olpidium radicale, the tombusvirus CNV and the carmovirus MNSV, a region of sequence similarity in the P domain of their coat proteins. It was speculated that this region was involved in fungal transmission or in the infection of cucumber. However, because of the lack of a similar sequence in the structural protein of TNV, it seems that this region is not essential for the transmission by Olpidium. The TNV RNA sequence presented here does not show significant homology with STNV RNA. However, it is possible that significant homology is present at the 3’ end of these RNAs, as is the case for TMV and its satellite virus (Mirkov et al., 1989). ACKNOWLEDGMENTS We thank Dr. P. Ameloot for the gift of the TNV antiserum and J. Decock for DNA preparations. We also thank Dr. C. Bowler and Dr. M. Cornelissen for critical reading of the manuscript and Dr. A. Depicker, Dr. L. Herman, G. Angenon, I, Ingelbrecht, and X. Danthinne for stimulating discussions. F. Meulewaeter is a Research Assistant of the National Fund for Scientific Research (Belgium).

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EMMELO

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KRUG, M. S., and BERGER, S. L. (1987). First-strand cDNA synthesis primed with oligo(dT). In “Methods in Enzymology” (S. L. Berger and A. R. Kimmel, Eds.), Vol. 152, pp. 316-325. Academic Press, London. LAEMMLI, V. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LESNAW, I. A., and REICHMANN, M. E. (1969). The structure of tobacco necrosis virus. I. The protein subunit and the nature of the nucleic acid. Virology39, 729-737. LESNAW, J. A., and REICHMANN, M. E. (1970). Identity of the 5’.terminal RNA nucleotide sequence of the satellite tobacco necrosis virus and its helper virus: Possible role of the %terminus in the recognition by vlrus-specific RNA replicase. Proc. Nat/. Acad. Sci. USA 66, 140-145. MANG, K. Q., GHOSH, A., and KAESBERG, P. (1982). A comparative study of the cowpea and bean strains of southern bean mosaic virus. Virology 116, 264-274. MANIATIS, T., FRITSCH. E. F., and SAMBROOK, I. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MAXAM, A. M., and GILBERT, W. (1977). A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74, 560-564. MAYO, M. A., ROBINSON, D. J., JOLLY, C. A., and HYMAN, L. (1989). Nucleotide sequence of potato leafroll luteovirus RNA. /. Gen. Viral. 70, 1037-1051. MCMASTER, G. K., and CARMICHAEL, G. G. (1977). Analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acndine orange. Proc. Nat/. Acad. Sci. USA 74,4835-4838. MILLER, W. A., WATERHOUSE, P. M.. and GERLACH, W. L. (1988). Sequence and organization of barley yellow dwarf virus genomic RNA. Nucleic Acids Res. 16,6097-6111. MIRKOV, T. E., MATHEWS, D. M., Du PLESSIS, H. D., and DODDS, J. A. (1989). Nucleotide sequence and translation of satellite tobacco mosaic virus RNA. Virology 170, 139-l 46. MORCH, M. D., DRUGEON, G., ZAGORSKI, W., and HAENNI, A. L. (1986). The synthesis of high-molecular-weight proteins in the wheat germ translation system. ln “Methods in Enzymology” (A. Weissbath and H. Weissbach, Eds.), Vol. 118, pp. 154-l 64. Academic Press, San Diego. MORRIS, T. J., and CARRINGTON, J. C. (1988). Carnation mottle virus and viruses with similar properties. ln “The Plant Viruses,” Part Ill, “Polyhedral Viruses with Monopartite RNA Genomes,” (R. Koenig, Ed.), pp. 73-1 12. Plenum, New York. MORRIS, T. J., and DODDS, J. A. (1979). Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology69,854-858. NODA, M., TAKAHASHI, H., TANABE. T., TOYOSA~O, M., KIKYOTANI. S., HIROSE, T., ASAI, M.. TAKASHIMA, H., INAYAMA, S., MIYATA, T., and NUMA, S. (1983). Primary structures of p- and &subunit precursors of Torpedo californlca acetylcholine receptor deduced from cDNA sequences. Nature (London) 301, 251-254.

OF TNV

STRAIN

A

709

NUT~ER, R. C., SCHEETS, K.. PANGANIBAN, L. C., and LOMMEL, S. A. (1989). The complete nucleotide sequence of the maize chlorotic mottle virus genome. Nucleic Acids Res. 17, 3163-3177. OLSON, A. J., BRICOGNE, G., and HARRISON, S. C. (1983). Structure of tomato bushy stunt virus. IV. The virus particle at 2.9A resolution. J. Mol. Biol. 171, 61-93. RIVIERE, C. J., POT, J., TREMAINE, J. H.. and ROCHON, D. M. (1989). Coat protein of melon necrotic spot carmovirus is more similar to those of tombusviruses than those of carmoviruses. J. Gen. Viral. 70, 3033-3042. ROCHON, D., and TREMAINE, J. H. (1989). Complete nucleotide sequence of the cucumber necrosis virus genome. virology 169, 251-259. ROSSMANN, M. G., ABAD-ZAPATERO, C., HERMODSON, M. A., and ERICKSON, J. W. (1983). Subunit interactions in southern bean mosaic virus. J. Mol. Biol. 166, 37-83. SALVA~O, M. S., and FRAENKEL-CONRAT, H. (1977). Translation of tobacco necrosis virus and Its satellite in a cell-free wheat germ system. Proc. Natl. Acad. Sci. USA 74, 2288-2292. SHOULDER, A., DARBY, G., and MINSON, T. (1974). RNA-RNA hybridisatton using ‘z51-labelled RNA from tobacco necrosis virus and its satellite. Nature (London) 251,733-735. UYEMO~O, J. K. (1981). Tobacco necrosis and satellite viruses. In “Handbook of Plant Virus Infections and Comparative Diagnosis” (E. Kurstak, Ed.), pp. 123-147. Elsevier/North-Holland, Amsterdam. VAN EMMELO, J., AMELOOT, P., PLAETINCK, G., and FIERS. W. (1984). Controlled synthesis of the coat protein of satellite tobacco necrosis virus in Escherichia coli. Virology 136, 32-40. VAN EMMELO, J., AMELOOT, P., and FIERS. W. (1987). Expression in plants of the cloned satellite tobacco necrosis virus genome and of derived insertion mutants. Virology 157, 480-487. VEIDT, I., LOT, H., LEISER, M., SCHEIDECKER, D., GUILLEY, H., RICHARDS, K., and JONARD, G. (1988). Nucleotide sequence of beet western yellows virus RNA. NucleicAcids Rex 16, 9917-9932. WATANABE, Y., EMORI, Y., OOSHIKA, I., MESHI, T., OHNO, T., and OKADA, Y. (1984). Synthesis of TMV specific RNAs and proteins at the early stage of infection in tobacco protoplasts: Transient expression of the 30K protein and its mRNA. virology 133, 18-24. Wu, S., RINEHART, C. A., and KAESBERG, P. (1987). Sequence and organization of southern bean mosaic virus genomic RNA. Virology 161,73-80. XIONG. Z., and LOMMEL, S. A. (1989). The complete nucleotlde sequence and genome organization of red clover necrotic mosaic virus RNA-1 Virology 171, 543-554. YSEBAERT, M., VAN EMMELO. J., and FIERS, W. (1980). Total nucleotide sequence of a nearly full-size DNA copy of satellite tobacco necrosis virus RNA. J. Mol. Biol. 143, 273-287. ZIEGLER, V., RICHARDS, K., GUILLEY, H., JONARD, G., and PUTZ, C. (1985). Cell-free translation of beet necrotic yellow vein virus: Read-through of the coat protein cistron. /. Gen. Viral. 66, 20792087. ZIMMERN, D. (1987). Evolution of RNA viruses. ln “RNA Genetics” (J. Holland, E. Domingo, and P. Ahlquist, Eds.). CRC Press, Boca Raton, FL.