VIROLDGV 170, 270-377(1989)

Nucleotide Sequence of Barley Stripe Mosaic Virus RNAa: RNAa Encodes a Single

Polypeptide with Homology to Corresponding Proteins from Other Viruses

GARY GUSTAFSON,' S . L. ARMOUR,2 GARY C . GAMBOA, STANLEY G . BURGETT, AND JOHN W. SHEPHERD

Lilly Research Laboratories, A Division of Eli Lilly and Company, P .O. Box 708, Greenfield, Indiana 46140

Received November 22, 1988; accepted February 3, 1989

The complete nucleotide sequence of RNAa from the Type strain of barley stripe mosaic virus has been determined .The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139amino acids (mw 129,634) . The open reading frame is flanked by a 5'-terminal sequence of 91 nucleotides and a 3'-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure . The aminoacid sequence of the polypeptide (as) encoded by the open reading frame has homology with the TMV 126K proteinand with related polypeptides from other viruses . The carboxy-terminal portion of the as polypeptide also has limitedhomology with the 58K (fib) protein encoded by BSMV RNA6 and includes a consensus sequence found in mononucleo-tide-binding polypeptides. nc 1989 Academic Press, Inc.

INTRODUCTION

etch virus and tobacco vein mottling virus (Forster et

Barley stripe mosaic virus (BSMV) is a tripartite RNA

al., 1988) . BSMV RNAy (3164 bases in the Type strain)

virus with rod-shaped particles belonging to the hor-

codes for two polypeptides . The protein encoded by

deivirus group (Jackson and Lane, 1981 ; Atabekov and

the 5'-portion of the RNA (ya) has amino acid sequencehomology with putative replicase proteins encoded by

Dolja, 1986). The (+)-stranded genomic RNAs encapsi- many plant and animal viruses (Kamer and Argos,dated by BSMV have been designated a, #, and y in The second

of decreasing molecular weight (Jackson et al.,

is expressed from a subgenomicisRNA Osc q

son e at,

1983a). Each RNA has a 7-methylguanosine cap at its

expresse sno significant amino acid

c

e

ho-5'-end (Agranovsky et al., 1979) and a poly(A) tract of 8

molo983b) and has no nigni codeded

ami

sequences.

to 40 nucleotides (Agranovsky et al., 1983) followed by

BSMV RNAa has been estimatedurology with

encencoded by othercontain n

ni4000toa 236- to 238-nucleotide tRNA-like structure at its 3'-

4200

(Dogs etal

93to ,Jackson et al.,

(Gustafson and Armour, 1986 ; Kozlov et al ., 1984) .

1) andnucleotidesis

nn

to direct thein

vitroJacaynteh i of

The complete nucleotide sequences of RNA# from

single

weight fthe Type strain and RNAy from the Type and ND 18

a120,000

(Gustyw

hewith an

al.,apparent

oIJe emolecular weih

ha)strains of BSMV have recently been determined (Gus- Whether this is

a tonly protein

encoded.

and Armour, 1986 ; Gustafson etal., 1987) . Anal-clu

eeterm n . In o

be RNAa hvs

ysis of these sequences demonstrated that the num-

thenever

otune

tic or aniatii SM resolve

ber and organization of genes in the BSMV genome

andt co

of thehe n ucleotw

dse

obey sequeuenceB

ofSMV

p

ryeNAe

differed from that of other tripartite viruses which had

sSMV omplete

e ima

been sequenced. BSMV RNA# (3289 bases) codes for

structureeoof BSMV

theBS

hn RNA«

uclave

.noThewseqetuuenc

thnceei thndicatees that

coat protein (Ba) and potentially three other polypep-

BSMV RNAa codes for a single polypeptide . This pro-tides (#b, #c, and #d) . None of these proteins have

tein has homology with polypeptides encoded by otheramino acid sequence homology with polypeptides en-

viruses and with another protein encoded by BSMV .coded by othertripartite viruses. However, the #b (58K)and #c (14K) proteins do share homology with polypep-tides encoded by beet necrotic yellow vein virus (Bou-

MATERIALS AND METHODSzoubaa etal., 1987), white clover mosaic virus (Forsteret al., 1988), and potato virus X (Morozov et al., 1987 ;Huisman et al., 1988) . The BSMV #b protein is also re-

Virus isolation and RNA purificationlated to the cylindrical inclusion proteins of tobacco

The Type (ATCC-PV43) strain of BSMV was main-Sequence Data trom this article have been deposited with the

tained in and isolated from "Black Hulless" (C .I . 666)EMBVGenBank Data Libraries underAccession No . 104342 .

To whom reprint requests should be addressed .

barleyViral RNA was isolated from purified virus asPresent address : Ciba-Geigy Corp ., Research Triangle Park, NC .

previously described (Gustafson eta!, 1 981) .370

0042-6822/89 $3 .00Copyright © 1989 by Academic Press, Inc .All rights of reproduction in any form reserved .

S

E B X E G K P H KRNA a m7GLII

I

L I I

I -OH

pBSM400 I'

pBSM54

pBSM18

pBSM405

I

FIG. 1 . Restriction map and sequencing strategy for RNAa from theType strain of BSMV. cDNA clones pBSM1B, pBSM54, pBSM400,and pBSM405 contain inserts derived from various portions of RNAa .Each insert is flanked by Pstl sites (not labeled) indicated by thelonger vertical bars . The sequence of RNAa was determined by di-deoxynucleotide sequencing of restriction fragments isolated fromthe inserts of the above clones . Arrows indicate the approximate lo-cation, direction, and length of each fragment which was se-quenced . B, BamHl ; E, EcoRl ; G, Bglll ; H, Hindlll ; K, Kpnl ; L, Bgll ; P,

Pvull ; and X, Xhol .

Synthesis and isolation of cDNA clones

Two independent sets of cDNA clones were pre-pared from unfractionated Type RNA using methodsdescribed previously (Gustafson et al ., 1987) . Cloneswere screened for insert size by digestion with Pstl andinserts containing sequences from RNAa were initiallyidentified as previously described (Gustafson et al .,1982). Clones pBSM405, pBSM 18, and pBSM54, con-taining the entire 3'-noncoding region and most of thecoding region of RNAa (Fig . 1), were selected for se-quencing in this manner . Clone pBSM400 (Fig . 1) wasisolated by probing a portion of our cDNA library with a32P-labeled synthetic 20-mer oligonucleotide, the se-quence of which was derived from the 5'-most regionof pBSM54. The insert in pBSM400 extends from theinternal poly(A) region up to and through the 5'-nucleo-tide of RNAa (see text) .

Nucleotide sequencing strategy

DNA fragments isolated from BSMV RNAa cDNAclones were sequenced by the dideoxyribonucleotidechain termination method (Sanger et at, 1977) . The se-quencing was facilitated by the convenient spacingwithin the RNAa cDNA clones of restriction sites thatare compatible with sites in the M 13 polylinker (Fig . 1) .The Pstl sites (not labled in Fig . 1) located at each endof the cloned inserts in pBSM400 and pBSM405 wereused to obtain DNA fragments containing the se-quences from the 3'- and 5'-ends of the RNA . The entireinternal sequence of RNAa was determined by sub-cloning specific restriction fragments from the pBSMclones into M13mp18 or M13mp19 . Restriction frag-ments prepared using the enzyme Bgll were blunt-ended at the Bgll site by treatment with T4 DNA poly-

II

SEQUENCE OF BSMV RNAa

371

merase before subcloning into M13 . None of the otherrestriction fragments used in sequencing RNAa wasmodified before subcloning .

The identity of the 5'-nucleotide of RNAa was deter-mined as previously described (Gustafson et al., 1987) .Briefly, a 32P-labeled synthetic oligonucleotide (5'-GGAAAGTGTTGTTTAGAG) was used to prime cDNAsynthesis near the 5'-end of RNAa . The cDNA synthe-sized was divided into two aliquots and terminal deoxy-nucleotidyl transferase was used to add a poly(dC) tailto one aliquot and a poly(dG) tail to the other . Both sam-ples were then sequenced by the method of Maxamand Gilbert (1980) . The 5'-nucleotide was identified bycomparing the two sequences at the point where thepolynucleotide tails initiated .

Analysis of sequence homologies

Nucleotide and amino acid sequences were ana-lyzed with the University of Wisconsin Genetics Com-puting Group programs (DevereuxetaL, 1984) . Nucleicacid sequences were compared and aligned with theprograms GAP and BESTFIT using a gap penalty of 5 .0and a gap length penalty of 0 .3 . Amino acid sequencehomologies were detected with the COMPARE andDOTPLOT program and homologous regions were

dGlelhd dGI.II .d

A T

A T

G G C C G G C c

GGG

~cCATACA

cCCcC

ATACATTCAACGGAAAcS

FIG. 2 . Autoradiograph showing the nucleotide sequence of acDNA fragment complementary to the 5'-end of RNAa from the Typestrain of BSMV. A "P-labeled oligonucleotide (5'-GGAAAGTGTTGTT-TAGAG) complementary to a region of RNAa near its 5'-terminus wasused to prime cDNA synthesis from the RNA . The cDNA was iso-lated, tailed with poly(dG) or poly(dC) using terminal deoxynucleotidyltransferase, and sequenced . The derived sequence is complemen-tary to that of RNAa . A portion of the sequence which encompassesthe 5'-terminus of the RNA is given . Arrows indicate the final nucleo-tide before the beginning of the polydeoxynucleotide tail .

372

GUSTAFSON ET AL .

20

40

60

80

100m7GPPPGW000MGWGCCWUGGGUGUMAAW =GCAUGCACAUAAUCWAAUCGAWCWCWGAUCUCUMACMCACUWCCCGUUAGCAWGWAGCGA

M A S 0120

140

160

180

200UGAGAWMCCGCMUMWUWCCCW W GGAGGUMUGG WM WUGAWAGCACAGCWCUAGCUCAGVMGGUCACCCUUACAUGACGW WMGW CGCACWE I V R N L I S R E E V M G N L I S T A S S S V R S P L K D V L C S H V

220

240

260

280

300MGGACCWCWCWWCCGUGGAUAAGAMGCGM GUCGCMGCAUGMWUMACGGCGCMCWCUCMCUGAGGMWGCAWUWUGAUAMUGCAUAUCCR T I V D S V D K K A V S R K H E D V R R N f S S E E L 0 N L I H A Y P

340

360

380

400

420WMUAUGCCGU AUCCUCAGCUUWWUCUGGUAWCAUAGCAUGGCGGWUGU CGAVWMGWWCAWAVAUCUCWAGAUAUGWMWUGMAGA•

Y A V S S S A C E S G T H S M A A C F R F L E T E Y L L D M V P M K E440

460

480

500

520GACIM UW WAUGACAWG WGGCMCW GW WMGVAVWAGUW CGUGCUGAVA W WMW CAlM1GW GMGUCCGAU WUAU W WQu W W CUGMAGT F V Y 0 1 G G N V F S H M K F R A D R E I H 0 0 0 P I L S N R 0 S E R

540

560

590

600

620

640AMGGWCACGCAUGAUGGCMUGCMAMUAUAIIGCGVGGWCGMAGACAMCCGWACGCWGWMGCCCWAUCAAAAUAUCWGCGUGMCMGCGGCGAG

L E T R M M A M 0 K Y M R G S K U K P L R L L S R Y 0 N I L R E Q A A R660

680

700

720

740AACMCUGCCWUAUGGCAGMGAGGUGMUGCGG WGW WCGAUGGAGAUGUGUWUGUGAGMCACW W CMGACUGUGUGAGACGAGUGCCCGMGGW W WT T A F M A G E V N A G V L D G O V F C E N T F a D C V R R V P E G F L

760

780

800

820

840GMGACAGCU WAGCAGUVCAUAGCW W ACCAUAWAMGUGGAGGAAVWGCGUCUGCAUUGMAAGAAMGMAUMGCAGGC WAVGGGUGW W CUGWUCC

K T A I A V H S I Y D I K V E E F A S A L K R K G I T 0 A Y G C F L f P880

900

920

940

960VCCUGCVGUAW GAUAGGUCAGAAGGMGGCAW WACCUVWMGGACGGUMUUAWUGMGGAGMUGGCAGGAVUMGW WVCUWGCGMUGIIUCCGMUGC•

A Y L I G 0 K E G I L P 5 V D G N Y L V E N G R I K F F F A N 0 P N A

980

1000

1020

1040

1060CGGWACUEUCAUGACCUVMGGAWAUCUGMGUAUGUGGAAMGIICCUAUWGGAUAUAGAGGAUGUGUGUUUGCUAVUGAGCUGAUGCAMUGCGAGGUGAUAC•

Y S H D L K D Y L K Y V E K T Y V 0 1 E D G V F A 1 E L M 0 M R G D T

1080

1100

1120

1140

1160

1180CAUGWCWUAA4W000W000CACCGCMCMUWWCWAUGAMUACAGAGWAUGAMCGCGAUGA CMUCMWGCAUUCCWUGCUMMMWCAUC•

F F K I T 0 V T A A M Y H M K Y R G M K R D E T F K C I P L L K N 5 5

1200

1220

1240

1260

1290

UGWGUCWACMWAWUUCGUGGGACMCCGWMWMAGAUCACMGUGGWVAWACCACGMCWUGGUCGAGCAGGGUGCGGCUUWAUUAUGAMMCM•

V V P L F S N D N R S L K I T S G L L P R T L V E 4 G A A F I M K N K

1300

1320

1340

1360

1380

GGAGAAG W CWGMCGW GCUGUGUUGMGMCUAUCUWCCGCUGVGMCAACUCAUACAW W CMCGGWCCCAGGUUAGAGAUGGCGUGAAMUUGCCCCGGAE K D L N V A V L K N Y L S A V N N S Y I F N G S O V R 0 C V K I A P D

1420

1440

1460

1480

1500WUAAWUCCAAGWGGCAGUGACUCUGMCWGAGAWAAAAGUCDADCGACMAGAGAAAAUU A EMM000AWUCUGCMGMAUGCWCACGWCCCM•

1 5 K L A V T L Y L R E K V Y R 0 R E N 5 f 1 5 Y F E 0 E M L H 0 P N1520

1540

1560

1580

1600CUUGAAAGCCAUGWUGGAGACUWCUGUGGWUGWCCAMUAMCUWCGAWGUCUOWAGMCAVGCGAAAAUCACUGAUGGMUGGUWGGWACGCAGMUU•

K A M F G D F L N F V P N T L S S V V K N M R K S L M E N F G Y A E F1620

1640

1660

1680

1700

1720UGACUUGACMA UGWAUWGCGAUCCCGWCUMAUGUAGAGAUAWGGAUCGGUAUMGWCAWCAAMAGGGCGMWCCACUWGUGAGUUCUIAJWWG

•

L T T F D I C D P V L Y V E I V 0 R Y K I I 0 K G R I P L G E F F B C

1740

1760

1780

1800

1820UCWGMGMUGCW WAUUACGMCUE000GAGMGGAGAMMCGACCUAGCGWGAAMWGCCCAGMGGUMCAGGGACGMaCCGAWGCQWGGACCU

•

E E C E N Y E L R E K E K M D L A V K M A 0 K V T G T V T E C E K D L

FIG . 3 . The complete nucleotide sequence of RNAa from the Type strain of RSMV . The deduced amino acid sequence of the single long openreading frame present in RNAa is given in one letter code . The termination codon for the long open reading frame is identified by an asterisk .Numbering refers to the nucleotide sequence .

aligned with the program BESTFIT . The quality scorefor BESTFIT matches was calculated as follows: (num-ber of matches) - (0 .33 x number of mismatches)- (gap penalty) (number of gaps) - (gap length penalty)(total length of gaps) . A gap penalty of 4.0 and a gaplength penalty of 0 .15 were used in all protein se-quence comparisons . The normalized quality score(NOS) was defined as 100 times the quality score di-vided by the length of the aligned region (Doolittle,1981). The statistical significance of any alignment

was tested empirically using the method suggested byDoolittle (1981) . The mean NQS of 36 alignments pre-pared using randomly generated sequences with thesame amino acid composition as the test sequenceswas compared to the NOS of the true alignment . Thedifference between these two scores divided by thestandard deviation from the mean NQS of the random-ized alignments is referred to as the n value . n valuesgreater than 3.0 are normally considered to indicatesignificant homology (Doolittle, 1981) .

SEQUENCE OF BSMV RNAa

1840

1860

1880

1900

1920

GGGACWCWGWCUCCMW UGAUAWGGWCAA WGUGAUGCCCMUWGWUUGCGWGUGUGACWCGUAGCCCMCGUWCCWUGGACWMA

G P L V 0 P I K E I L V D L V H P N L Y R A L C R P R S P T S P L U L K

1960

1980

2000

2020

2040

MGUkUCCCAGGGIICMCIICUUCACAWCMG AGAWCUGMCAWCMUUWGMGMGCUGCUGCACCAWGCGGWAGCGUACCMCAUGGGMAWGC

•

I P G S T P S H S 5 S D S E H S M T E E A S C T I A G S V P T N E I A

2060

206D

2100

2120

2140

GA W AGGMAGW WMCCWJCAGCGAAW UUUAGAUAU WCUCGACGMCW W AUGCWCCAAUCCMM WMCWCUAGW ACMCAUGMUGCCAGAGC

T R K O L T F 0 R I D E D M S R R T G M P P R P K V T S S Y N M N A R A

2160

2160

2200

2220

2240

2260

UGAGWUCUWALUUCMCUWWAGCGUGAWUGUGMAGGGC AMWWGAGUGUUUCUAGACLUICGUCAGAA

UAWCUCAGAUAAAGUGGCCGU

E F L Y Y 0 L C S V I C E R A 0 I L S V I E D F R 0 N L I F S D K V A V

2280

2300

2320

2340

2360

UCCAUWMCGWAGAWCUAUGWWCAGI AWGAGACCCGGAUGGGUGUUCAAMCMaUCGCAWGWMGUGGCUUGWAUGCAWACAWWGACUU

•

L N A R F Y S F 0 S L R P G W V F K T P S H S E V G H S Y A V H F O F

2380

2400

2420

2440

2460

CAAUUAUUGGMCCGAUW GGMGkaGC WAGCLAMIUGCCGMUWUACCGAUUUC WGGUUAMAGCGGCAMUACAUCGCUCMCUCCUCAW WCCCGA

•

T I G T 0 L E E S L A F C R M V P I S N 0 K 5 G K Y I A T T P H F P E

2500

2520

2540

2560

2580

GAGACWGGWAWACGUUUWGUGACMCACUAMUUGUGUMCMWGGCWMUUACMUAA J WUGACWCUACGCACUAGUGGCUGAUAGACCUCUUG

R H G Y Y V I C D N T K L C N N N L I Y N K L V D V Y A L V A D R P L R

2600

2620

2640

2660

2680

AUUCGAGWGAWGACGUGWCCWGCUGCGUMGUCAACUUGAWUUMACAGWWUUAUWUCGCG WGWGUUGWGMGGACMUWCMWGA

F E L I D G Y P G C G K S T M I L N S C D I R R E V V V G E G R N A T D

2700

2720

2740

2760

2780

2800

UGACUVMGGGAGAGC,IAICMGCWMUMAAWUGMUAWAAGAWGCUMUCACAGAGWCGMCGCWGACAGCWAUUCUUGCUGMGGACWUGUWACC

•

L R E R F K R K K N L N S K T A N H R Y R T L 0 5 L L L A E G P C V P

2820

2840

2860

2880

2900

GCAAGCUUUAGGWUCAIIUWGAUGMGWCUAMAWUCAWACGGCGCUUMUGWCUGUGCUUUMGCWGGUGCCUUUMUUCUCGCUCAGGGAGAUAG

•

A D R F H F 0 E A L K V H Y G A I M F C A D K L G A S E I L A 0 G D R

2920

2940

2960

2980

3000

GGWCMWGCUWGRUC11WCWOJAGMGWAWGMCWUAWUCAWCUCWGALUACACGMGACUUCAUMUCCUMGCUACGWCAUACCW WCCC

A 0 L P M I C R V E G I E L 0 F 0 S P D Y T K T I I M P K L R S Y R I P

3040

3060

3080

3100

3120

UGGAGPUGUUGCCWCUAWUUGUGCUAAGGAAUWUACAAAGWMAGGMUACCUCAMAGGWAUMWUWMCAGUGUUMCGWCCWGUACGCUAGAGG

•

D V A F Y L S A K E F Y K V K G I P 0 K V I T S N S V K R S L Y A R G

3140

3160

3180

3200

3220

CGAMCGAWCCGGMAGAWCWGAG"GCWGAUWUCCGGUUGMMGACACCCACUAUWMCCWCWACMGWUGMGGA GU GAUGAWCAWU

•

T T P E R F V S L L D V P V R K P T H Y L T F L 0 A E K E S L M S H L

3240

3260

3280

3300

3320

3340

GAWCCAMGGGUGMMGMAGAGUCUAU WCGAUUCAUGAGGCGCAGGWGGUACCUAUGMMUGUGAUUCM6UCCGWUGCMCGGACGCCCMUGMAU

I P K G V K K E 5 15 T I H E A 0 0 0 T Y E N V I L V R L 0 R T P N F 1

3360

3380

3400

3420

3440

WAUCCGGGUGGACCUAGGUCCGCCCCUUACAWGUGMGGGACWCMGGCAUACAAMACUUUCACWAWGMGUGWACGGACGAUMGWGCUUIUAGAUAU

Y P G G P R 5 A P Y I V V G T S R H T K T F T T C S V 7 D D K L L L D I

3460

3480

3500

3520

3540

CGCCGAC W CGWGGU W GCACAMCA=AWC WACW WGMU WCAUAUAG WIIMAAAAAAMMAMMMAAU W W GAUCAUUCAUUCAAW W UUG

A D V G G I A H T P I R T F E S H I V

3580

3600

3620

3640

3660

GUGCCCAUCMCCAUWGWWGAGUGq CMWCUCUAUMUCGMCWUAMCGWGCWGMWGGAMCUUUAUCUUMCGGAUUCWGAGAGAAMU

3680

3700

3720

3740

3760

WAGUAWGGUAUGUMGCUCMWUCCGWAGCUGCWCAUCWUMUW WGCWACUUGCCGMGCUCAGCUUCGGUCCCCCMGGGM6ACCA

FIG. 3-Continued

RESULTS AND DISCUSSION

Construction of the sequence

The complete sequence of BSMV RNAa was ob-tained from the cDNA clones pBSM1S, pBSM54,pBSM400, and pBSM405. THe alignment of thoseclones with RNAa and the fragments used to constructthe sequence are shown in Fig . 1 . Greater than 90% ofthe sequence of RNAa was determined by sequencingboth corresponding DNA strands .

373

An oligonucleotide primer (5'-GGAAAGTGTTGTTTA-GAG) that anneals with RNAa downstream from theSphl site (Fig. 1) was used to determine the sequenceof the 5'-end of the RNA. The cDNA synthesized fromthis primer was tailed with poly(dC) or poly(dG) usingterminal transferase and sequenced by the chemicaldegradation method . The sequence obtained termi-nated 34 nucleotides upstream of the Sphl site with thesequence CTTACATACC-3' (Fig . 2) . This suggests thatthe sequence 5'-GGUAUGUAAG represents the 5'-endof RNAa. However, BSMV RNAa is known to be

37 4

GUSTAFSON ET AL .

GUAUGUAAGWGCCUUUGCGUGUAAAAUUUCUUGCAUGCACACAAUCGUAAUCGAUUCUUCUUGAUCUCUAAACAACACUUUCCCCUUAGC(A)

I

I

I

11

111

I

1

1

111

11

1

I

I

I

I

I

II

II

II

I GUAAAAGAAAAGGAACAACCCUGUUGWCUUCGACCCUAUACUAAAUAUAUAUUAUCUUACUAGUGCAUUUCWWACCGCUUCACACU

CUAUGUAAGUUGCCUWGGGU-GUAAAAUUUCUUGCAUGCACACAAUACUAAUCCAUUCUUCUUGAUCUCUAAACAACACUUUCCCCUUAGC(s)

IIII

II

II

II

II

IIII

I

I

I

II

I

I

I

I

I

II

I

I

IGUAUAGCWGAGCAUUACCGUCGUGUAAUUGCAACACUUGGCUUGCCAAAUAACGCUAAAGCGUUCACGAAACAAACAACAAUUCGGC

GUAAAAGAAAAGCAACAACCCUGUUGUUGUUCGACGCUAUACUAAAUAUAUAUUAUCUUACUACUGCAUUUCUWUA[CGCUUCACACU(C)

111

I

II

I

II

IIGUAUAGCUUGAGCAUUACCGUCGUGUAAWGCAACACUUGGCUUGCCAAAUA-ACCCUAAAGCGUUCACGAAACAAACAACAAUUCGGC

FIG . 4 . Pairwise alignments of the 5'-noncoding regions from BSMV RNAs a, p, and y . Alignments were computer-generated using the programGAP. (A) RNAa (top) versus RNA# ; (B) RNAy (top) versus RNA-y ; (C) RNA$ (top) versus RNA-y .

capped with 7-methylguanosine (Agranovsky et at,1979) and cDNA transcripts of capped RNAs often ex-tend 1 base beyond the 5'-end of the RNA (Ahlquist andJanda, 1984; Gupta and Kingsbury, 1984). It is there-fore likely that the sequence at the 5'-terminus of BSMVRNAa is actually m 7GpppGUAUGUAAG .The Pstl/Sphl fragment from the clone pBSM400

(Fig . 1) contains the entire 5'-end sequence of RNAaplus an additional 65 nucleotides located upstreamfrom the 5'-nucleotide of the RNA . Although there areregions within this 65-nucleotide fragment that sharehomology with various portions of the BSMV genomicRNAs or their reverse complements, the mechanismby which this extraneous sequence was generated isnot evident .

Sequence analysis

The complete nucleotide sequence of RNAa fromthe Type strain of BSMV is presented in Fig . 3 . Assum-ing that the internal poly(A) region has an averagelength of 21 adenine residues, the length of the (+)-stranded (virion polarity) RNA is 3768 nucleotides . The91-nucleotide 5'-noncoding region of the RNA is fol-lowed by a single long open reading frame (ORF) whichterminates at positions 3509-3511 . The long ORFcodes for a polypeptide comprising 1139 amino acidswith a predicted molecular weight of 129,634 . If trans-lated, the next largest ORFs on the (+)RNA-strandwould code for proteins of 72 (nucleotides 1156 to1371) and 36 (nucleotides 3336 to 3443) amino acidswhile the largest ORFs on the (-)RNA-strand would en-code polypeptides comprising 74, 56, 49, and 47amino acids. The long RNAa ORF is followed by apoly(A) tract and a 238-nucleotide region which can bearranged into a tRNA-like structure (Kozlovetat, 1984) .As in BSMV RNA/3 (Gustafson and Armour, 1986) andRNAy (Gustafson et at, 1987), the first two adenineresidues of the RNAa poly(A) tract are used to form thestop codon for the 3'-proximal ORE The completepoly(A) tract in clone pBSM405 consists of 21 adenineresidues .

There are two potential translation initiation codonswhich precede the putative initiation codon (positions

92-94) for the RNAa long ORF . The first of these islocated at positions 3-5 and is followed immediatelyby a termination codon . The second is located at posi-tions 35-37 and codes for only eight amino acids be-fore encountering a stop codon . Such "nonfunctional"AUG codons have also been found in the 5'-noncoding

ABSMV 130K

(1139 amino acids)

Arg-54 to Val-31DTMV 126K

(1116 amino acids) : Arg-45 to Phe-286

RKHEDVRRNISSEELQMLINAYPEYAVSSSACESGTHSMAACFRFLETEYLLDM:

II

I :

:

1111 : :

:

II :I :

:I

I1

111 :RPKVNFSKVISEEQTLIATRAYPEFQITFYNTQNAVHSLACCLRSLELEYLMMQ

VPMKETFVYDICCNWFSHMKFRADREIHCCCPILSMRDSERLETRMMAMQKYMR:I

:

:

HIM :

11 :

1 : :

:111

1

1

:11

1

1

: :

I :IPY-CSLTYDICCNFASHL-FKCRAYVHCCMPNLOVRDIMRHEGQKDSIELYL-

CSKDKPIRLLSRYQNILREQAARTTAFMAGEVNAGVLDGDVFCENTFQDCVRRV:1

:

I

: :I

11111SRLERCCKTVPNFQKEAFDRYAEIPEDAVCHNTFQTMRHQP

PEGFLKT-AIAVHSIYOIKVEEFASALKRKGITQAYGCFLFPPAVLIGQtECIL111 :111111

:11 :

11

11

:

I :

1

1

:I : : :

IMQQSCRVYAIALHSIYDIPADEFCAALLRKNVHTCYAAFHFSENLLLEDSYVNL

oSVDCHY!.VENCRIKFFFAVDPNAGYSHDLKOYLKYVEKTYV: :

1

II

:

I :1

:

1111

IIIDEINACFSROGDKLTFSFASESTLNYCHSYSNILKYVCKTYF

BSMV 130K

(1139 amino acids)

Pro-630 to Vat-1139TMV 126K (1116 amino acids) 5er-825 to Ch-1116

8

PLRFELIDCVPCCCKSTWILNSCDIRREVVVCEGRNATDDLRERFKRKKNLNSKI :SS1111

li

I

: :I

ISAKVVLVDGVPCCGKCKTKEILSRVNFDEDLILVPGKCKQAAEMIRRRANSSCIIVAT

TANHFVRTLDSLLLAECPCVP-QADRFHFDEALKVHYCAIMFCADKLGASEILA

KDN--VKTDVSFMMNFCKSTRCQFKRLFIDECLMLHTCCVNFLVAMSLCEIAYV

QOORAQIPMICRVEGIELQFQSPDYTKTIINPKLASYRIPGOVAFYLSAKEFYKII

I :1

I

II

I :

:

:

: .

:

I

III

IIYGDTQQIPYINRVSGFPYPAHFAKLEVDEVETRRTTLRCPAOVTHYLN

VKGIPQKVITSNSVKRSLYARGETTPERFVSLLDVPVRKDTHYLTFLQAEKESLI :

III I :

:

:

:

III

:II

I-RRYEGFVMSTSSVKKSV---SQEMVCCAAVINPISKPLHGKILTFTQSDKEAL

MSHLIPKGVKKESISTIHEAQCGTYENVILVRLQRTPNEIYPCCPRSAPYIVVG:I :

:1 :11 :11 :11

:1

IIII

II

1

:I :

I

: :1 :LSRCYSDVHTVHEVQCETYSDVSLVRLTPTPVSIIAGD---SPHVLVA

TSRHTKTFTYCSVTDDKLLLDIADVGCIAHTPIRTFESHIVIlls

:

I :

I

I

I :

I

I : :LSRHTCSLKYYTVVMDPLVSIIRDLEKLSSYLLDMYKVDAGTQ

FIG . 5. Alignment of the amino-terminal (A) and carboxy-terminal(B) portions of the as (130K) polypeptide encoded by BSMV RNAawith the tobacco mosaic virus (TMV) 126K protein . Alignments wereprepared using the program BESTFIT . Identical amino acid residues(I) and favored substitutions ( :) are identified within the aligned aminoacid sequences .

FIG . 6. Alignment of the putative nucleotide-binding motifs of BSMV as and #b with related regions of proteins encoded by other RNA virusesand with two replication-associated proteins from Escherichia coli (uvrD and recB) . The viral proteins compared with BSMV as (BSMV1) and #b(BSMV2) include the brome mosaic virus 1 a protein (BMV1) ; the tobacco mosaic virus 126K protein (TMV) ; the Sindbis virus nsP2 protein (SV) ;the white clover mosaic virus 147K (WCIMV1 a) and 26K (WCIMV1 b) proteins ; the potato virus X 166K (PVX1a) and 25K (PVX1b) proteins ; andthe beet microtic yellow vein virus 237K (BNYVV1) and 42K (BNWV2) proteins . The first number in each line denotes the position within theprotein of the initial amino acid in the aligned region . All other numbers indicate gaps introduced during alignment . Locations of amino acidsthat are completely conserved (a) or biochemically related (+) in all proteins are noted . The classification scheme of Allison et al. (1986) wasused to determine biochemically related amino acids. All sequences except BSMV1, WCIMV1 a, WCIMV1 b, PVX1 a, and PVXtb were previouslyaligned by Hodgman (1988) . Amino acid sequences of the WCIMV and PVX proteins were obtained from Forster et al. (1988) and Huisman etal. (1988), respectively .

regions of cucumber mosaic virus RNA2 (Rezaian etat, 1984), alfalfa mosaic virus RNA1 (Cornelissen et al .,1983), and cowpea mosaic virus M-RNA (van Wezen-

beek et at, 1983). Of the five AUG codons located

within the first 150 nucleotides of RNAa, only the AUGcodon immediately preceding the long ORF has theA_31G+4 motif that is optimal for initiation of translationin eukaryotes (Kozak, 1986) and the G+4/C+5 motif sug-gested to be optimal for initiation of translation in plants(Lutcke et al., 1987). A second AUG codon (positions140-142) that is in-frame with the long ORE also has afavorable motif (G_ 3/G+4)for eukaryotic translation initi-

ation . The remaining three AUG codons have unfavor-able motifs and translation is therefore unlikely to initi-ate at those positions .

Sequence homology among BSMV RNA species

Sequence homology between BSMV RNAy and the

other BSMV genomic RNAs (,3 and y) is primarily limitedto the poly(A) tract and 238-nucleotide tRNA-like struc-ture found at the 3'-end of each RNA . The sequence ofthe coding region of each RNA is unique and the 5'-

noncoding regions, although similar in length (88-91nucleotides), have limited sequence homology . Pair-wise alignments of the 5'-noncoding regions of RNAs

a, R, and y are presented in Fig . 4. In the aligned se-quences, there are no blocks of homology greater thanfour nucleotides in length and the maximum number ofnucleotide matches between any pair of sequences is35. A common 6-nucleotide sequence (AACAAC) isfound in the 5'-noncoding region of each RNA, but thelocation of that sequence in RNAS (positions 14-19) ismuch different than in RNA" (positions 72-77) or RNAy(positions 75-80) .

SEQUENCE OF BSMV RNAa

375

Amino acid sequence homology

Domains of amino acid sequence homology havebeen detected within replication-associated, nonstruc-tural proteins encoded by a number of plant and animalRNA viruses (Haseloff et al., 1984 ; Kamer and Argos,

1984 ; Cornelissen and Bol, 1984 ; Ahlquist et al ., 1985 ;Rezaian et at, 1984, 1985 ; Allison et al., 1986; Domieret al., 1984; Gustafson et al., 1987; Bouzoubaa et al.,1987 ; Goldbach, 1987). The polypeptide encoded byBSMV RNAa (aa) shares amino acid sequence homol-ogy with a group of proteins that includes, among oth-

ers, the brome mosaic virus (BMV) la protein, the al-falfa mosaic virus (AIMV) la protein, and the tobacco

mosaic virus (TMV) 126K protein. We have aligned theregions of greatest homology between the BSMV as

RNA. m7 .'G -4 - lAA)A7. pa nt on

da

aarcnPNAd RY)

m

AM 2 M OH

RNA, IM nr19~ TA.aL4

(NN7

m,G-mw=RollF~ANgn 2M n' OH

FIG . 7. Structure and genetic organization of the BSMV genome .RNAa from the Type (TY) strain of BSMV codes for a single polypep-tide (as), while RNA# codes for four proteins (#a-#d) including theBSMV coat protein (CP) . RNAy encodes two polypeptides (ya andyb), one of which (-yb) is expressed from a subgenomic RNA . Themolecular weight (mw) of each polypeptide encoded by the BSMVgenome is given . The Type RNAy is larger than RNAy from the N D 18(ND) strain of BSMV due to a 366-nucleotide direct tandem repeat(RP1 . RP2) located near the 5'-terminus of the ya gene . Each of theBSMV genomic RNAs is capped at the 5'-end with 7-methylguano-sine and terminates with a short heterogeneous poly(A) tract fol-lowed by a 238-nucleotide (nt) tRNA-like structure .

. . . . . . . . . . . . a w w . . .

arD 26 VLAGAGSGKTRVLV 174 NILVDEFQNTN 16 VMIVGDODQSIY 26 QNYRSTSNI 267 LMTLHSAKGLEFPQVFIVG 23 LAYVGVTRAracB 20 IEASAGTGKIFTIA 345 VAMIDEFQDTD 18 LLLIGDPKQAIY 24 TNWRSAPGM 288 IVTIHKSKGLEYPLVWLPF 44 LLYVALTRS8MV1 687 VDGVAGCGKTTAIK 54 RLLVDEAGLLH 15 VLAFGDTEQISF 22 KTYRCPQDV 78 IKTVHEAQGISVDNVTLVR 13 YCLVALTRHTMV 829 VDGVPGCGKTKEIL 57 RLFIDEGLMLH 15 AYVYGOTQQIPY 24 TTLRCPADV 82 VHTVHEVQGETYSDVSLVR 14 HVLVALSRHSV 183 VIGTPGSGKSAIIK 50 VLYVDEAFACH 16 VVLCGDPMQCGF 24 ISRRCTQPV 58 VMTAAASQGLTRKGVYAVR 14 HVNVLLTRTWCIMV1a 567 IHGAGGSGKSHAIQ 51 IIVFDDYSKLP 16 AILTGDSKQSFH 20 PFCRVYLNI 63 SMTYAGCQGLTTKAVQILL 10 VIVTALSRAWCIMVlb 26 VHAIAGSGKSTVIR 37 LDILDEYGQLP 8 EFIFTDPYQAPT 11 TTYRFGPNT 62 FFKVSDVIGYQWPTVTLVL 13 LLFGILTRHPVX1a 732 IHGAGGSGKSHAIQ 50 IVIFDDYSKLP 16 IILTGDSRQSVY 20 KYCRYYLNA 64 TFTYAGCQGLTKPKVQIVL 10 VMYTALSRAPVX1b 26 VHAVAGAGKSTALR 37 FAILDEYTLDN 6 QAIFADPYQAPE 10 IFSRVPRKV 52 FVKPCQVTGLELKVVTIVS 11 AFYNAITRSBNYVVI 893 VKGGPGTGKSFLIR 48 IIFVDEFTAYD 11 IYLVGDEQQTGI 26 MNFRNPVHD 72 KTTVRANQGSTYDNVVLPV 12 LNLVALSRHBNYVV2 121 VLGAPGVGKSTSIK 49 TMLVDEVTRVH 11 VICFGDPAQGLN 18 ASRRFGKAT 67 SILYSDAHGQTYDVVTIIL 13 VRAVLLTRABSMVI 835 IDGVPGCGKSTMIL 58 RFHFDEALKVH 16 ILAQGDRAQLPM 25 RSYRIPGDV 78 ISTIHEAQGGTYENVILVR 17 YIVVGTSRHBSMV2 267 ISGVPGSGKSTIVR 41 LLIIDEYTLAE 11 VLLVGDVAQGKA 18 TTYRLGQET 62 CALAIDVQGKEFDSVTLFL 12 LRLVALSRH

376

GUSTAFSON ET AL .

(130K) protein and the TMV 126K protein in Fig . 5 .

REFERENCESThese regions are located in the amino-terminal (Fig .5A) and carboxy-terminal (Fig . 513) portions of the pro-teins. There are weaker regions of homology through-out the remainder of the proteins and the overall iden-tity between the two proteins is 21% . The homologybetween the BSMV as and the TMV 126K proteins isstatistically significant (n = 20 .3) indicating that theseproteins are related and may have diverged from acommon ancestor .

The carboxy-terminal portion of as contains a seriesof six motifs (Fig . 6) commonly conserved in nonstruc-tural polypeptides encoded by many viruses and in agroup of proteins from Escherichia coil and other or-ganisms that are involved in RNA and DNA replicationand recombination (Hodgman, 1988) . The first motif inthis series includes a short amino acid sequence (GXX-XXGK) that is commonly found in the nucleotide-bind-ing pocket of ATP- or GTP-utilizing enzymes (Mollerand Amons, 1985 ; Higgins et al., 1986). Althoughmany viral genomes apparently encode only a singlepolypeptide containing this nucleotide-binding region(Goldbach, 1987), this series of motifs is highly con-served in the beet necrotic yellow vein virus 237K and42K proteins (Hodgman, 1988), the BSMV as and fibproteins, the white clover mosaic virus 147K and 26Kpolypeptides, and the potato virus X 166K and 25K pro-teins (Fig. 6). Viral polypeptides containing this motifmay function in the unwinding of double-stranded repli-cation forms during viral RNA replication or in the viralRNA recombination process (Gorbalenya et at, 1988),or in some other aspect of RNA metabolism, such assystemic movement .

With this report, the nucleotide sequence of barleystripe mosaic virus has been completed . A geneticmap of the complete BSMV genome is presented inFig . 7 . BSMV can be clearly differentiated from othertripartite viruses based on the number and organizationof its genes . Some of the polypeptides encoded byBSMV (as and ya) have homology with proteins en-coded by many other viruses including members of thetricorniviridae . Other BSMV-encoded proteins (01D and,6d) have no recognizable counterpart in the tricorniviri-dae, but have homology with polypeptides encoded bymono- and bipartite viruses . No homology has beenfound between the BSMV go and yb proteins and poly-peptides encoded by other viruses . Although manyBSMV genes encode proteins which are related to pro-teins from otherviruses, the way in which BSMV genesare coupled is completely distinct . This suggests thatboth divergent evolution and RNA recombination havecontributed to the structure of the BSMV genome .

ACKNOWLEDGMENTSWe thank Dr . Rama Belagaje and Mr. Stan Ly (Lilly Research Labo-

ratories) for synthesis of the oligonucleotides used in this study andLisa VanDyke for typing the manuscript .

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