Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Journal of General Virology (1992), 73, 2173-218l. Printed in Great Britain 2173 Sequence analysis of the 3'-terminal halves of RNA 1 of two strains of barley mild mosaic virus Satoshi Kashiwazaki, ~.I- Ken Nomura, 1 Hisao Kuroda, 2 Kazutoshi Ito 2 and Hiroyuki Hibino ~ 1National Agriculture Research Center, Tsukuba, lbaraki 305 and 2plant Bioengineering Research Laboratories, Sapporo Breweries Limited, Nitta, Gunma 370-03, Japan DNA complementary to the Y-terminal halves of R N A 1 of two strains of barley mild mosaic virus (BaMMV) from Japan, BaMMV-Kal and B a M M V - N a l , was cloned and sequenced. The sequences start within a single long open reading frame (ORF), and are followed by 337 and 338 3' non-coding nucleotides, for BaMMV-Ka 1 and B a M M V - N a l respectively. The two strains have 88 % nucleotide identity in the ORFs and 92 % identity in the non-coding regions. The putative O R F products contain the capsid proteins at the C termini, as indicated by amino acid sequence analysis, and two putative non-structural proteins are arranged in the same manner as in RNA 1 of barley yellow mosaic virus (BaYMV). The deduced capsid proteins of BaMMV-Kal and B a M M V - N a l each contain 251 amino acids and have 94 % sequence identity, which is compatible with their close serological relationship. Most of the sequence differences between the two capsid proteins are found in the N-terminal region, and might explain their serological differences. Significant sequence similarities of the capsid proteins of the two BaMMV strains (37 and 35 % respectively) with that of BaYMV, and their marginal similarities (21 to 26 %) to the capsid proteins of aphid-borne or mite-borne potyviruses support the classification of B a M M V and BaYMV as distinct members of the same virus group, which is separate from the group(s) containing aphidborne or mite-borne potyviruses. Introduction species of Y-polyadenylated ssRNA of 7.6 kb (RNA 1) and 3.5 to 3-7 kb (RNA 2) (Huth et al., 1984; Ehlers & Paul, 1986; Usugi et al., 1989; Kashiwazaki et al., 1989a, b; Anderson et al., 1990). Hybridization experiments using uncloned or cloned eDNA and restriction enzyme analysis of cloned cDNA have indicated that, in either virus, little sequence homology exists between R N A 1 and R N A 2 (Koenig & Huth, 1988; Batista et al., 1989; Kashiwazaki et al., 1989a; Pr61s et al., 1990). It has been shown for BaMMV that both RNA species are needed for infection (Koenig & Huth, 1988). BaYMV and BaMMV, however, differ in some biological and physical properties, and are serologically unrelated (Huth & Adams, 1990). Moreover hybridization, restriction enzyme and in vitro translation analyses suggest that their genomes have substantially different nucleotide sequences (Pr61s et al., 1990). Examination of the complete nucleotide sequence of the BaYMV RNAs has revealed close evolutionary relationships between BaYMV and aphid-borne potyviruses (Kashiwazaki et al., 1989a, 1990a, 1991 ; Davidson et al., 1991). However the limited similarities in their gene products, including the capsid proteins, support their assignment to separate taxonomic groups (Kashiwazaki et al., 1991 ; Barnett, 1991). No sequence data have been Yellow mosaic disease causes serious damage to barley crops in Japan, China and several European countries. Barley yellow mosaic virus (BaYMV) was first discovered in Japan and has long been implicated as the agent responsible for this disease (Inouye & Saito, 1975). However in Europe, another virus, barley mild mosaic virus (BaMMV), has been shown to be associated with yellow mosaic disease in combination with BaYMV (Huth & Adams, 1990). Recent studies also show that BaMMV occurs in Japan (Kashiwazaki et al., 1990a). Both BaYMV and BaMMV are transmitted in soil by the fungus Polymyxa graminis and their plant hosts are restricted to Hordeum spp. (Huth & Adams, 1990). Their particles are slightly flexuous filaments with two modal lengths, 500 to 600 nm and 250 to 300 nm (Inouye & Saito, 1975; Huth et al., 1984). These particles contain one species of capsid protein (Mr 31K to 33K) and two l Present address: Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U.K. The nucleotide sequencedata reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide databases under the accession numbers D10949 (BaMMV-Kal) and DI0947 (BaMMVNal). 0001-1056 O 1992SGM Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 2174 S. Kashiwazaki and others BallllV-Kal 13a~i~V-Ka] UUAU(;G1JU~C1JZACUCAUGUAUC UCGUUUUCUGGGUL~CU@CA~.MACA~;ACGACA UCACCA~GAO4W~;AMC~A(~AUACAAL~GUGACAA~AU(~GUACGACU L W L P P }; S C ] S F S (; F L P (; N R T T S P L 1¢ (; K A G I1 T T V T S A W (; T T 120 40 Ba~lilV Kal C~UAC6~6GL~GACGA~G~UC~A~AGAUCAACAAA~AGUUCAA~AC~C~CAGA~U~4GG~C~C~4UAACUC~t~C~AAACAA(~UCGAAAUACC~UCU~AA~L~GAA 240 Ba}tldV Kal P T R K T K F A T 11 S BaIIIflV Kal ~MI~V-Kal UUU{3UUA~CUUCUAUA~UCU~AACCGAUGC(~AACG~GCU~CAA~CCGUGUUCACU~C~'~U{;GAC{~G~GUC~U~C~CA~AL~G~UC~U(~GACAUUC~AUGUCAAU F V N F Y X L K T l} A N V L {~ ~, V F T A ~1 I) G A V L L Q 2' E X P }~ A O 1 (; 11 V N Bal~l~V-Kal BaMMV KaI AGAC~JGC~J~`~CAG{;ACCAC~UGAAGACAG~GAGA(`~AA~CUG~GCAUGACG~L~CAACACCAU~&~GU~AC~AAC~AUGAAAGAU~;AAGACAGU~GA~UCGACAUGG~ 480 N L L Q 11 I[ F E I) S T K S E S S K S A D L i¢ F S Q A V 1{ E (; L N T [ N N S K P E T S S K 'i I¢ N L K Q E 80 V K C II L T M K i) G R Q F E L D M E 180 BaM~IV-Kal CAACAC(;AOCCC(;A&4COGUCt;CC&£~.CUUG{;GGC-CGAG(;UUG(;1JUUCAGAA UGAACA(;AGAC{3ACUUACGUCAAGUU fA;At~CACACGAUACA~AA~AAGA~AC~CAUCBt~A Ba~l~V Nal ~ U l; A A ~ (; (;1£ U LC C t £ C Ba~I~V-Kal Q II D P E 1' V A K L t; (; E V (; F R M N R D D L ff Q V f; A T I~ Y I N P 1( A Q T S A Ba}ll~g Nal ~t I Y K Ba~lt]V Kal lk]]~]~V-Nal lk~lll~V Kal ~lll~V-Nal BaI~I~V Kal ~ t ~ V Nal ~Wl~V Kal ~/tllV-Nal 3B0 120 600 200 ~CGA~GCU~I;~(;GC~U(;ACCAUG~.£~C~CAUI;AGt~CUUUCACC~UUG~A~(~AAAGAUG(;~G~AGAAGA~(~£~GA~A~ACUC~U~AU~UUU~CG~GA~UC~ 720 a U A A T L E t; ~1 T ~t K P )t S (; S F T A I O S t; C U U U A U A K ~1 V (; F I K "r A K D r t, N c i U A U L '~" ¢; D W 240 ~UUAUAC~CUCCUGCGC~AGCAGGC~2GAC'{P~GC~;A~AUAA~U~UUC~M~A~UA~UUCAL~A~A~AA~UGA~GUUUA~UA(EUAU~A~UCGACAAUUC~AGGU 840 C I I A P C A U A A 1[ T Q Q G E (; l) I I T F I ,~.C (; U A U C C F Q I1 V Q F T T T T g 'r A R L A S C (; Y (; I G 1~ Q F C K (; 280 Ball~dV Kal ~U~GACCUUGL3JG~cMJccCGcC'LL`CUcAC~CA~AUAcC~C(~AI;UcAA6AA~A~AU(~G~UA~AU~UcGAcAcAcL~AC~GAAAUAcAA~U(~UcUAc~UGUccAcUAAA~GGU l',a}l]~V Nal U A C U C C U t; G C A A C C C l~a~ll~V-Kal L O L V V I R I{ P Q Q [ l{ A V K K I) M i~ ,~ S I L I1 T P T E I Q iI L Y L S T K (; l; L~)~V-Nal v 9UO 320 AA(;UACCAAGUGUCCAC(;UCCGCUGUGUGCUUUC~ACACUACAAUAACCGAUG(`~;GACACG~CA~E3JCAACAGCAGAAGC~C~U~UGCG(~UGUAUUGUA~CAAC~CA~AC(;AACCAC1080 I~)IMV Kal ~tlflV-Nal IM~I~V Kal Ba~II~V Nal U L U C U C K Y Q V S T S A V C F P I1 Y N N R W G II V [ I~MI~V Kal ~]~l~v Nal Ba~IIIV Kal l~a~lI~V Nal AUC(;UUG(;AAUCCACGUCUCCUACAACGACACGCGCAGAAGAAAlJ(;AGUlJUCAAC~CUUUCACAAGUGACGL~UGACAACGAUUAAUC~AC~CC~ACACGAGAUAC~UUUCA(~AUCG 1200 U G U U {; U A A C C 't A C U I V (; I II V S Y N I1 T I( R R N E F Q h F T S I) V L T T I N A P (; I1 E I P F 8 P ~ 400 Ba~V Kal l~llI~V-Nal ~)~ttV-Kal Ik~fiV .Nai (, U U (2 I F i1 ~/ K F C t; V S C T A g C G ~1 C (; C [ U A V F N P T T N II 3BB A~JCUUCG~CUGGA~G~U~UG~(;~G~JA~CAAC~A31C~ACG~A~tdJG42A.~IE~ACC~AC~ACA~A~ACU~A~CAAUt~A~(GAUUU~;UUU~A~UCG~U~A~ 1320 I; Y T T (; K P ~ N M Q~ S U A A P S (; U 3. 2" L E R L N I 12 N A T l; L F (; F A A K L I~ ,\ @ N 440 l ~ V Kal l~)tllV-Nal Ba~l}~V Kal L~II~V Nal (;AI;A~Jl~AA~L`CAGCCAUGClJUCI;UUCCACAGAGUC6UUClJCC~AGUUU~CA~A~ACACA~UAAACUCA~;I;GAA(;U~AAGAAA~ACUCA~ACAAACA~AC~U~ACAGI}A1440 l~lfilV Kal Ba~IMV Nal ~]~l~V-Kal l~ll~tV Nal GAGAAL~;CAUACUUCCUUGAA~CCUGAACACA~CAAACCAUACcAAU(;GGU~AGIL~CAU(~;ACGA(;UAC~ALL~UC~AUCC~UUAUGACC~GUAU~UC~(;A~UGAAG1560 C U C A A U C C G CU g S P Y F L E F L S 'r F K P Y Q W V Q A F }~ l) IS Y A P S I L A Y D A Y F K D L K 520 Ba~I~V-Kal I~l~l~V-Nal l~lll~V Kal Ba]dl~V Nal AAAUAUGAUAG(g~CACEACACGCA~AUGUUL~CUGUC~GGA~A~BA~L~2AAAE4~UA&h~AUAAGAUGAUCA~AU~GA~AA~A~AU~S~(~;AC~UAGUACGAAL~AL~ Ifi80 (; C (; (; (; (; U (; C (; A U A(; U A (; U C A K Y D R P P II ~ N V F C ~ D T L T K A K II K.~t I S I L E E A G ~1 1; R T L V R T T 5B0 S E K l~tl~V Kal M~l~V Nal ~llllV Kal GAI;~AGGUCUUGCUUGACAUC1;CI;~JG{;ACCACCUCCGGUGGACCCL~UGUACCAU~;UAAGA~AUCGA~AL~GU~AACAC~UG~I~ACG~C~A~UUGUACA~UUCAGU~A~U(~ 1800 l~l~V Kal I!allllV-Nal MllllV-Kal I~IIIIV Nal ~A~AGGCU~UUAUCA~UGGAA~GCU~;I;MJGG~UCUGGAA~AUC~CUCAAA(;CCGA(;CU~(;CU~UU~(~A~£~AAU~U~IGAAAA~G~U~A~UC~U~L~AUA C A C {; 1; U U {; C C Q Q A L I T {; T L D {; V ~ N G S L K A E L R S S Q K [ L E I~ K T R V F T A A P I ~llltV Kal Ba~llIV Nal P,a]iilV Kal I~]IItV-Nal A~AAGCCU~AUUI`'CGAUGAAAU~UAUI;UC~GAUG~UUU~AA~AG~U~UAA~A~A~U~A~AC~(~A~A~AGUCC~AU~AA~A~GU~AA~G~U~AGAAU~UG 2040 ~ItlIV Kal Ba}t~V-Nal L~tI~V Kal Ba)i~V Nal (; C A U E I K P A ] I; C L I~ S ,~A A 3" E S F T S l( E F A A C [ E Q V L L D II A W '1" T S G (; P T S U A L I t' A )1 K Y g (2 GA C (; P 1) 3" Q F K L N L Y II (; C A V D O F N K Q F C U K K I D l I C A (; E (; V K K (; a A V Q II L S C (; U A U Y K T 1I L K A P II T L ] U U (; D K lI T I U U (; a I) i) E L V Q F S U U V {; I N K F T (; 480 A E A C (10{} U C N ~ (; ~' Q N L 1920 640 tJ8(} UAU~AGAA~/dUCAACL~ACCC{;GAU~;ACC~cA~C~;AGUC~;{~ACC~;UUC~AG~;UUU~;ACA~;~A~UGAUGGAUUC~U~UC(;AUGUCAUCAA~;ACAUUCGA£~{~AUU~CAUGf;AC 2160 g t~ K L g N K P {; rd 7 Ii (; S t; U C I) {; S it F U {2 G 5 S S i i) ~; F C L F 1) V I A t' C IK 1} 1 t~ K l] b" g U P Ba~IMV-Kal AC(G ; AACA~ACAAAG~AC~]CG~CACAAUCUACGAAGMA ; UAGUCAACACGAAGAUAUGUCUGC~GAACGGCCUC~UC~CAGAA(~ ;A~`~GGAAC~C~GUC~C~AC~CAA(~ACU 0C {; [ II K E L P T {; A T EII A Ba~I~V-Kal Balll~V Nal lkal~llV Kal lk~ll~V Nal ~;UUGU~G~CAACACACUCI~ACUCAUGAC~C¢JUUUUCUEUACGCAU~U(;CL~I;(x2l~CA~UGGAGAUCAC~UU~GAACUCAU(~AC~;A~ACUUC6UCUUCGUCU(EAAUG~U~AUGAC 2400 {2 C U[ U U C A {; A U U C C C V V O N T L A L M T A F L Y A Y A R L T G D II A F E L t~ O E N F V F V C N (; D O 80(} S P T K I UA A U U A C A U A U C A (; C L A N (; L V I q K N C {; N N S 6 Q P S 228B Ba~[IV-Nal M~IMV-Kal Baill~V Nal l U A P U Y E E I V N T 72B T 760 lk~}~l~g-Kal AAC~A~UUUUCC~UGUC~CCU~GC~JU~UGGC{;A~{;UUUG{;AU{;C~AUUUCU~CCC~UUCCU~U~U~;~;CUC~{~UAA~UACGA~U~UGM{~CUGA~6AUAUCU{~G~AA~2520 BalllV-Nal C{; C U C (; U A (; C C C C C U Ba}~}~V Kal N K F S }fl S P S F ~ A K F (; C 1) F S P F L S E L {; L T Y E F 1) E A T G 1) I C E N 840 I~}~I~V-Nal A_ V Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 2175 Nucleotide sequence o f B a M M V R N A 1 BallldV Kal CCCUACA~GUCCUUGACGAUC4;UCCGCACCUCGUUUGGGAUUGGUUUCUCUCUCUCC~UUGAGC~A~C~AGU~;~G~GGUC~AGU~UC£AU(~CUA~ 2640 I~MMV Nal C U C C C C A C A C U U BalMV-Kal P Y M S L T M V R T S F (; 1 G F S L S I E R I V A 1 L Q W S l~ A (; G V L II A Y L 880 BaMMV Nal BaMMV-Kol ~C~3C~GCCGCUCUULqJ(~;A~UCUUUCAACACACCCA~AC~UIjC~UCUCGU(?~ACAC~U~UCUCCUUUGGCUCG~AC~(~ACGAGGA~;A~CUC~UCCAUGAU~C~2760 Bal~MV Nal U (; C C C C ~. G ~J ~J U G Ba~I~V Kal S (; I A A L F E S F N T P K L F N L '/ ]1 T Y L L W L V T E If E E E L V S M J8 E L 920 I~IMMVNal I~MV Kal AAAGACAUGU~£AUGCCCUUGCCCACUAGGGAACAGAUAGCCUU~CUGCACUACG~.~UGGAACUGAGCCCAUCG~GGAGGAAACA~U~AA(~A(;~;ACAC~;AAGA~A(;A~ 2880 Ball~V-Nal G U UC A U A C A A A U AA U U U U BalAMVKal K P ld F i~ P L P T I~ E (~ I A L L ]I Y V t; T E P I V E E T F L QZA (; II E E P I) P I 960 I~-~MI~VNal K M Y | S" K P D I~MMV Kal ~;UUCCCCCAG~J~J~UGACACCY;AUCU~A~(;&AC~UC'(~U(L`C(~&CC~(X)C(;AC&~AG/~JL~`~;UC(;C(~G~A~GU~;A~KG~CU~C~UG~CL~AUG 301}0 BalIMV-Nal U U U c A A (; (; AUU (; I~I~MV Kal ~ P P V P 1) T 1) L T N 1~ A A A P P O N 1~ K S R A V 1 P R G T S D W S L P E P K ]~ lOOO ~lIMV-Nal • S I~ V N BaMIdV Kal CGAACACUG(~UUUC/~(;UCCAAGAUCAACAUCGAAACACUIJ~;~x2AAUGUCC~CGACG(~UAUAUGAACACCUUUCd2~R2CGUUGCAACU(;AA~C(~AA~UA~AU~;A~AG(~ 312(I I~MMV Nal UG A A A (; C (; A U i; A A Ba~llflVKal l~ T L (; F K S K I N 1 E T L A N V P D (; Y M N T F A S V A T E S O R I~ K W E E A 1040 BaMI~VNal M 1) E T BaHMV Kal ~a2ACGUG(~(;AUUUC~;~AU~ACU(;ACGAUGAGA~AUG(;(;AGAA~UUGCUcAUUG~Uf7~l~GUAUAUAUUUC~A(;AC~UG~A~A~(;~;ACGA~AACUCA~UAUGGA~ 3240 ~MHV Nal A (; U U l~]dV Kal A I¢ (; 1) F (; I Ba}~MV-Nal T C AC (; A C T 1) l) E K W E K b L I N C C A A C I U A C U t; A Y F A O N (; T S P N F O E E L T M E lhtflflV Kal (;UUAA~(;(;C(;(;UCU~CCAU~A/~AG~(;UA~CCC(;UUCGCCCCUUU(;~`(;UU~GA(~A~AU~C~U(~A~UCU~(~U~U~A~A~UCAU~ I~MM¥ Nal C U C U O C (; C A ~; U (; h A [~MMV KaI V R (; (; L N S I K ~ Y P V R P F ¥ ¥ R A IK K I S "r L I~ 1~ I F 1~ C Y S I ~ T K L ~l l~l~lflV Nal 1080 3360 I120 BaMMVKal UU~(~GAAGCUGAGGC~7~GUG~CACACU~;G(;~C~UAAA(;~AC~;GAU(;CCU~GAUGAG~GUC~UG~U~CAU~AUC~GA~AGU~ACCUCUA(;(;ACU~(~UU(;~CACUGAAG 3480 BaMMV--Nal A A U (; U A C A U C A A C BaMMV Kal F V K L 1~ R V P 11 W A I K II {; C L O E l V F 1) F lfi I P O Q F T S 1{ T A L E 'f L K 1160 I~MMV-Nal Q BaI~MVKal ~MI~V Nal ~BI~V Kal ~lflflv Nal CAGACCAAACU(;~JUGC~AUUGGUGUU~(~CACAA(~CAAUUCUCUCCUCACCUC~;~(~AGAC~;AACAU~A(~;A~ACU~AC~GAC~A~(~U(;ACUACGA~(~;UCA~;A~UC 3600 (; U A A A C C U Q T ~ L A /~ I (; V l; 'r S N S L L "r S E (~ 'I N I~ t~ 7 7 E '1' R R 1~ N D '/ O (; II E A L 1200 BaMMVKal ~UcCGG~A~A~U~UC~GCA~UGC~UUUCCUUUUAAGCAAU6C~U~UCAA~AcGCUUUUAAUUAUAUCAA{~GU~AAGMU~cUc~UU~A~;~U~A~ ;GUW~AcU 3720 Ik~MMV-Nal (; A CA (; C C A C LE C (; (;U (; Ba~MV Kal L 1¢ * BaMMV-Nal l',ai~MV Kal I ~ V Nal UUGUG~AUGCAAU(~GAAAAUIJCAGUGAC(;CAAUCAACCAUCAUUC4;UU~;UGACA.ACGCAGUACU~OUC~qJ AUUUCACAUCAAGCi;ACCA(;CACAUC(~-.JJCC, CUUACAGCUAAUCCUA 3840 C (; h C UG CO U I~MMV Kal I~MMVNal C~UGAG(;(;U(;GC~CUCU~;UGUUAUGUA~U(~AAUGU(;CA~UCUCGCAALE~CCGUC~U~J(~;UUGAUG(;U(~G(~AACA~C(~C~C~G~AUACC(~A(~(A)n A A C Fig. I. T h e n u c l e o t i d e s e q u e n c e s a n d d e d u c e d a m i n o acid s e q u e n c e s of the Y - t e r m i n a l 3946 n u c l e o t i d e s from B a M M V - K a l R N A 1 a n d o f the T - t e r m i n a l 3464 n u c l e o t i d e s f r o m B a M M V - N a l R N A 1. T h e B a M M V - K a l s e q u e n c e is g i v e n in its entirety, w i t h o n l y the differences from B a M M V - K a l b e i n g g i v e n for the B a M M V - N a l sequence. # i n d i c a t e s the start of the B a M M V - N a l sequence. Possible c l e a v a g e sites for p o l y p r o t e i n p r o c e s s i n g are s h o w n by arrows. A possible p o l y a d e n y l a t i o n s i g n a l ( U A U G U ) is underlined. T h e N - t e r m i n a l a m i n o acid s e q u e n c e s d e t e r m i n e d by d i r e c t protein s e q u e n c i n g for the B a M M V - K a l a n d B a M M V - N a l c a p s i d p r o t e i n s are boxed. published for the RNAs of BaMMV. lmmunoelectron microscopic tests failed to detect any serological relationship between BaMMV and BaYMV, although relationships were found between BaMMV and aphid-borne potyviruses (Stanarius et al., 1989). It is therefore of great interest to compare the capsid protein amino acid sequence similarities between BaMMV and aphid-borne potyviruses or BaYMV. Although the use of resistant barley cultivars is the only practical means to avoid damage caused by yellow mosaic disease, resistance-breaking virus isolates have been reported in Japan, Germany and France (Kashiwazaki et al., 1989 b, 1990 b; Friedt et al., 1990; Hariri et al., 1990; Huth, 1990). For example, Kashiwazaki et al. (1990b) described two Japanese BaMMV strains, BaMMV-Kal and BaMMV-Nal, which infect barley cultivars having the BaYMV resistance gene Y m l , but differ both in pathogenicity towards different barley cultivars and serologically. Here we report the nucleotide sequences of the 3'terminal halves (which include the capsid protein gene) of RNA 1 of BaMMV-Kal and BaMMV-Nal, and we assess similarities at the nucleotide and amino acid levels among these BaMMV strains, BaYMV, and aphid-borne or mite-borne potyviruses. Methods Virus purification and R N A extraction. T w o B a M M V strains, B a M M V - K a l a n d B a M M V - N a l , were p r o p a g a t e d in barley by m e c h a n i c a l i n o c u l a t i o n a n d t h e i r p a r t i c l e s were purified as d e s c r i b e d by U s u g i & Saito (1976). V i r a l R N A w a s isolated f r o m purified v i r u s p a r t i c l e s as d e s c r i b e d by K a s h i w a z a k i et al. (1989a). Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 2176 S. Kashiwazaki and others cDNA cloning. Double-stranded cDNA was obtained from a mixture of RNA 1 and RNA 2 as described previously (Kashiwazaki et al., 1989a). The cDNA product of BaMMV-Kal was size-selected ( > approx. 3 kb) by 1% low melting point agarose gel electrophoresis and then ligated into SmaI-cut, dephosphorylated pBluescript II SK(+). The cDNA product of BaMMV-Nal was ligated to an EcoRI NotIBamHI adaptor (Takara Shuzo) and then size-selected ( > approx. 3 kb) electrophoretically. The cDNA with the adaptor was phosphorylated by T4 polynucleotide kinase and ligated into EcoRI-cut, dephosphorylated pBluescript II SK(+). The cDNAs were used to transform competent NM522 cells. Screening ofcDNA clones. Colony hybridization was done using viral RNA 1 and RNA 2 probes isolated electrophoretically as described previously (Kashiwazaki et al., 1989a) and the ECL gene detection system (Amersham). Two large cDNA clones, KalSK11 (4 kb) and KalSK46 (4 kb) derived from BaMMV-Kal RNA 1, and two other clones, NalSK2 (3.5 kb) and NalSK15(3.5 kb) derived from BaMMVNal RNA 1, were selected for sequence analysis. DNA sequencing. A series of unidirectionally deleted cDNAs were produced by digestion from one end of a clone by exonuclease III and the ssDNAs were prepared for sequencing. The nucleotide sequences were determined by the dideoxynucleotide chain termination reaction (Sanger et al., 1977) using an automated DNA sequencer (377A, Applied Biosystems) as described previously (Kashiwazaki et al., 1990a). Protein sequencing. Capsid proteins from dissociated virus particles (approx. 15 ~tg)of BaMMV-Kal and BaMMV-Nal were separated by SDS-PAGE (Laemmli, 1970), and then blotted onto the Problot membrane (Applied Biosystems) in 10 mM-3-cyclohexylaminopropanesulphonic acid (pH 1 I) and 10% (v/v) methanol using the Mini-Transblot Electrophoretic Transfer Cell (Bio-Rad). The blots were stained with Coomassie blue R, and the capsid protein bands were excised. Amino acid sequences were analysed in a gas-phase protein sequencer (477A, Applied Biosystems). Computer analysis. Sequence data were compiled and analysed using DNASIS (Hitachi Software Engineering) or U W G C G (Devereux et al., 1984) software packages. Amino acid number 500 1000 (a) . . . . . I . . . . /" // / 2000 / / / 1000 / [ , , , i BaMMV-Kal 500 1000 (b) 3000 / J / > / / 2500 / / 2000 f r f f I , , , , BaMMV-Kal 150O (c) I 20O0 . . . . I 3000 ,/ / > e~ ( / 2500 / Results Clones KalSK11 and KalSK46 covered the T-terminal half of BaMMV-Kal RNA 1 in opposite orientations. Clones Nal SK2 and Nal SK 15 covered the corresponding region of BaMMV-Nal RNA 1 in the same manner. Sequence analysis of KalSK11 provided the T-terminal 3946 nucleotides, upstream of the poly(A) tail, of BaMMV-Kal RNA 1 in the 5' to 3' direction and these sequences were confirmed by the sequence obtained for KalSK46 in the reverse orientation. The Y-terminal 3464 nucleotides of BaMMV-Nal RNA 1 were sequenced using NalSK15 in the 3' to 5' direction and were then confirmed using Nal SK2 in the reverse orientation. The nucleotide sequences obtained are shown in Fig. 1. Both sequences start within a long open reading frame (ORF) which terminates with an UAG codon at position 3607, leaving 337 non-coding nucleotides for BaMMVKal and 338 for BaMMV Nal at the 3' terminus. These non-coding regions have 92% nucleotide sequence 2000 BaYMV Fig. 2. Dot plot comparisons of the partial polyproteins encoded by BaMMV-Kal RNA 1 and BaYMV RNA I (a), BaMMV-Kal RNA 1 and PVY RNA (b), and BaYMV RNA 1 and PVY RNA (c). Data were analysed using the U W G C G program DOT with a window of 21 and a matching number of 14. identity with only one gap in the middle (at position 3793). As observed with the BaYMV RNAs (Kashiwazaki et al., 1989a, 1991), a possible polyadenylation signal (UAUGU; Zaret & Sherman, 1982) was found 85 nucleotides upstream of the poly(A) tail. The partial ORFs found in BaMMV-Kal and BaMMV-Nal RNA 1 encode large polypeptides of 1202 and 1041 amino acids, respectively. The sequences of the two ORFs were aligned without a gap, showing 88% Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 Nucleotide sequence of B a M M V RNA 1 identity at the nucleotide level and 96~o identity at the a m i n o acid level. Direct sequencing o f the capsid proteins gave their N-terminal sequences, A G H E E P D P I V for B a M M V - K a l and S G K D D P D P I V for B a M M V - N a l , both of w h i c h are located in the putative O R F products at positions 952 to 961. T h e predicted capsid proteins o f the two B a M M V strains each contain 251 a m i n o acids, with calculated Mr values o f 28421 for B a M M V - K a l and 28510 for B a M M V - N a l . D o t plot comparisons (Fig. 2a) showed that the O R F products share extensive homologies with the C-terminal half o f the 270K polyprotein encoded by B a Y M V R N A 1 (Kashiwazaki et al., 1990a). Thus by analogy with B a Y M V , the O R F products o f the two B a M M V strains start within the N I a region, followed by the N I b and capsid protein regions (Kashiwazaki et al., 1990a). T h e N I b and capsid protein regions found in the B a M M V polyproteins are separated by respectively Q A (in B a M M V - K a l ) or QS (in B a M M V - N a l ) dipeptides which correspond to the Q A dipeptide at the equivalent position in the B a Y M V polyprotein (Kashiwazaki et al., 1990a). The QS dipeptide found in both B a M M V strains at position 417 corresponds to the Q A dipeptide at the putative b o u n d a r y between the B a Y M V N I a and N I b regions (Kashiwazaki et al., 1990a). D o t plot comparisons of the B a M M V O R F products and the potato virus Y (PVY) polyprotein (Robaglia et al., 1989) displayed significant but limited similarities in each region (Fig. 2b). These similarities were o f the same extent as those found between the B a Y M V and P V Y polyproteins (Fig. 2c). Table 1 presents the percentage of identical a m i n o acids found in the alignment of three protein regions from B a M M V - K a l , B a M M V - N a l , B a Y M V , six aphid- Table 1. Percentage of identical amino acids* found in three regions from viral polyproteins (a) Capsid protein region BaMMV-Kal BaMMV-Nal BaYMV PVY PPV TEV TVMV BYMV TuMV WSMV BaMMV-Kal BaMMV-Nal BaYMV PVY PPV TEV TVMV BYMV TuMV 94.0 37.2 24.9 25-0 24.6 23-2 23.6 26-3 24-2 35.2 24.2 23.3 25.4 22.4 21.4 25-8 24.1 24.5 22.7 25.3 26.5 20.1 23.2 20.3 58.9 62.7 53-6 61.1 62.9 25-8 60.1 49.8 57-5 59-7 26-4 57.8 64.3 62.8 26.6 58.2 52.5 25-8 51.1 26-9 25.7 BaMMV-Kal BaMMV-Nal BaYMV PVY PPV TEV 96.6 57-7 33-2 32-9 34.7 32.3 57.9 33.0 34.1 35.3 33.1 32.8 33.3 35-2 31.8 62.2 58.5 61.4 62.7 61.0 62.5 BaMMV-Kalt BaMMV-Nal t BaYMV PVY PPV TEV 96.9 31.7 20.9 22.3 18.9 20.8 37.8 22.6 23.0 20.0 20.5 19.9 20.4 19.8 20.8 54.0 48.1 46.4 51.6 50.5 48.8 (b) Nlb region BaMMV-Kal BaMMV-Nal BaYMV PVY PPV TEV TVMV (c) NIa region BaMMV-Kalt BaMMV-Nalt BaYMV PVY PPV TEV TVMV 2177 * Percentage identity was calculated with the UWGCG program GAP which made an optimal alignment of two sequences. t Only partial sequences were available for comparison. Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 2178 S. Kashiwazaki and others (a) BaYMV PVY TEV TuMV BYMV PPV TVMV WSMV AADPLTDAQKEDARIAAADGARFELADADRRRKVEADRVEAARVK ANDTIDA GGTV AGETLDADLTEEQKQAEKEKKEREKAEK SDQEQLNAGEEKKD ADEREDEEEVDAGKPIVVTAPAATSPILQPPPVIQPAPRTTAPMLN~IFTPATTQPATKPVSQVPGPQLQ SDTVD QSNNVSVMAGLDTGGAKTG BaMMV-Kal BaMMV-Nal BaYMV PVY TEV TuMV BYMV PPV TVMV WSMV DASADVGKKKDQKD~KVAEQASKDRDV~AGTSGT~PRMKGEVVV~LNH~LGYKPQQ ERERQKQLAFKKGK~VAQEEGKRDKE~GTSGT~PRYEKRVAL~LDH~ILYTPEQ BaMMV-Kal BaMMV-Nal BaYMV PVY TEV TuMV BYMV PPV TVMV WSMV BaMMV Kal BaMMV-Nal BaYMV PVY TEV TuMV BYMV PPV TVMV WSMV BaMMV-Kal BaMMV Nal BaYMV PVY TEV TuMV BYMV PPV TVMV WSMV TFGTYGNEDAS~SNSNA~V~TNRDRDVDAG~IGT~KLSLPKVKG~AIF~LNH~YSPAQ ~KDKARDQKLADKP~LAIDRTKDKDV~TGTSGT~MNM~LPKV~GS~VV~LD}~LTYKPAQ Q~SGSKGTGGSFTSNPVR~GGR~TDVQDQTPGLM~TVRDKI~PEM~NNMIKYQPRT . • ~E~N.MP~VM~LNV~CIE ~NVgTA~N~EEQ.SKZ~aFMVWC~ ~LS~S~~F~V~Y~L~.MQZ~N~LaV~CIE D D I S N V I ~ Q E ~ F E A ~ Y N G V K Q A Y E V E ~ S R . MGI I~NGLMVWCIE VDLSNTR~_PQ~CFQT~Y~GVKR~YD~.MSII~NGLMVWCIE EFVVNTR~J}~FKA~HTNVMAELELNEEQ.MKIV~/~NGF~WCIE ELIDNRY~T~NT~IKE~SEGLDV~E~VFINT~PGWVYHCII . . . ~ W g V ....... NNMM~C~I~ ~ g~ g~ L~ G T . . . . . . . NNMMDeDE ~ e ~ ....... ~SDe~D ~LQG~ . . . . . . . W~MD~EE ~ M ' ' I ..... W~MMDGET ~ 9 ~ I S G V . . ' ..... WTMMDGDE ~NRALG~WRVVNNAGKDNEQ ~ g ~ K E P Y M P R Y G N I R N L R D V G L A R Y ~ Y E V T I EMRN~. ERPYMPRYG~QRN ITDMSLSRY ~ Y E L T • ~~AYIE~NQ.DRPYMPRYG~QRNLTDMSLARY~YEMT P~F~MSHF~FVAEAYIE~NA.TERYMPRYE~QRNLTDYGLARYA~95{~YELT P~F~VARF~VAEACVE~NY.EKAyMPRYG!QRNLTDYSLARYA~YEMT P~S~~NRNS.EQVY~PRYGNQRGLVDRNLAPFA~FEVN ~~EESV~IGKPI~PR~DFKAGVLSINNIVAAC~IMRG QQVS~/I~LT~: " P~F~MTHF~DLAEAY QL~FKI~YKA~. ~ E N ~ E ~ H T ~ T A E E D ~ H ~ T T V E N ~ H ~ G T D E E N ~ ~ V G T Q K Q I ~ ~ H ~ y N ADDTPNFVQVQNSVA T < ~ V S P S M H T L L G V K N M T A H ~ V N R N M H T L L G V R Q T T E ~ V I R N M H N L L G V Q G L H T A G ~ Y N R D M H T M L G V R I ~ H T ~ V N R N M H T F L G V R G V A Q M H H L L G V K G V VR~NTHSFNGVNALA (b) BaMMV-Ka I BaMMV-Na I BaYMV PVY PPV TEV TVMV PD~TIVF,~FR . . . . PI~IVK~F , PIf6~TImK~H , P D ~ T ~ BaMMV-Kal BaMMV Nal BaYMV PVY PPV TEV TVMV G ~ Y ~ V ~ H ~ L F ' g ~ I L ~ K D F P V F P Q K L H . . . F G F ~ L ~ V T N Q ~ L F ~ I ~ L ' ~ K D F P P F P R R L Q . . . F G F ~ P F ~ ] T N K ~ L F ~ ~ D F P P F ~ K L K . . . F GF~PY~NQ~LFRRNN MAKDFPPFPQKLK...F BaMMV-Kal BaMMV Nal BaYMV PVY PPV TEV TVMV R~JQNERIC.~VGTNFQENYA~~F~N~LP~C]VSTADGC~SLA R ~ T E D R V C ~ I G S N P Q ~ S I ~ ~ L P I V S T R D G S ~ S L A R ~ R E E R I ~ V T T N F ~ S M ~ ~ P L V S T R D ~ S A S R Q ~ I K D R V C M V S T N F Q ~ S V ~ ~ S P L V S I I D G N ~ S L T • S A S~MVV~LIKECVE..~[EINSTC~F~LLIAVN~EKES VIL~T~SLLK~GHH..P~DTH~CICRYFV~LVLAVH~AYES ~ MVII~TCEK..CG..INKEEIV..YYV~LLIAIH~DKAE MVVL~M~LSK~GV~..INSQEDVCK~FA~LIIAI~ELEH illll Fig. 3. Alignment of amino acid sequences of the capsid (a), NIb (b) and NIa (c) proteins of BaMMV-Kal and BaMMV-Nal with the corresponding proteins of BaYMV, PVY, PPV, TEV, TVMV, TuMV, BYMV and WSMV. The capsid protein sequences are shown in their entirety. The partial NIb region (at positions 731 to 811 in Fig. 1) and NIa region (at positions 236 to 368) of BaMMV-Kal are aligned with the equivalent regions of other viruses. Amino acids identical to the BaMMV-Kal sequences are boxed. Amino acids of specific motifs are indicated by asterisks (see text). Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 Nucleotide sequence o f B a M M V borne potyviruses [PVY, plum pox virus (PPV; Maiss et al., 1989), tobacco etch virus (TEV; Allison et al., 1986), tobacco vein mottling virus (TVMV; Domier et al., 1986), bean yellow mosaic virus (BYMV; Hammond & Hammond, 1989) and turnip mosaic virus (TuMV; Tremblay et al., 1990)], and a mite-borne potyvirus [wheat streak mosaic virus (WSMV; Niblett et al., 1991)]. Amino acid sequences of the ORF products of the two BaMMV strains are highly conserved but the similarities between the two BaMMV strains and BaYMV are much lower in all three regions. Amino acid sequence comparisons between BaMMV and aphidborne or mite-borne potyviruses and between BaYMV and these viruses reveal only a low level of identity in all the regions examined. The sequences in the NIb regions are more conserved between BaMMV or BaYMV and the potyviruses than those in the NIa or capsid protein regions. Amino acid sequence alignment of the capsid proteins of BaMMV-Kal and BaMMV-Nal (Fig. 3a) shows that their N-terminal regions are more variable than the remainder of the sequences; four of the first five amino acids differ between the two strains. The two conserved blocks NGTS and AFDF found in the core regions of the BaYMV and potyvirus capsid proteins (Kashiwazaki et al., 1989a) are also present in the corresponding regions of the two BaMMV strains at positions 1067 to 1070 and 1141 to 1144 with a replacement of the A by V in the second block. Alignment of the BaMMV and BaYMV capsid protein sequences also shows many differences in their N-terminal regions in contrast to extensive homologies in their core and C-terminal regions. In the core regions of the capsid proteins, only limited similarities exist between either BaMMV or BaYMV and aphidborne or mite-borne potyviruses. The two conserved stretches (T/S)GXXXTXXXN(T/S) and GDD, thought to form the core of RNAdependent RNA polymerases of positive-strand R N A viruses (Kamer & Argos, 1984; Domier et al., 1987), are present in the NIb regions of BaMMV-Kal and BaMMV-Nal at positions 755 to 765 and 798 to 800 (Fig. 3b), as found also in BaYMV and aphid-borne potyviruses (Kashiwazaki et al., 1990a; Koonin, 1991). The triad of catalytic H, D/E and C/S residues found for cysteine proteinases of comoviruses, nepoviruses, aphid-borne potyviruses and BaYMV (Gorbalenya et al., 1989a; Dougherty et al., 1989; Kashiwazaki et al., 1990a) are also present in the NIa regions of the two BaMMV strains at positions 246, 282 and 349 (Fig. 3c). Discussion This study suggests that the T-terminal half of BaMMV RNA 1 is translated into the C-terminal part of a RNA 1 2179 polyprotein from which the NIa, NIb and capsid proteins are derived by proteolytic cleavage in a manner similar to that for the translation products of BaYMV RNA 1 (Kashiwazaki et al., 1989a, 1990a) and aphidborne potyvirus R N A (Dougherty & Carrington, 1988). As established for aphid-borne potyviruses (Dougherty & Carrington, 1988; Riechmann et al., 1992), the putative NIa proteinase found in BaMMV would be responsible for the proteolytic activity. The putative cleavage site between BaMMV NIb and capsid proteins is QA or QS, compared with QA, QS, QG or QV in BaYMV and aphid-borne or mite-borne potyviruses (Shukla et al., 1991). The QS dipeptide found between the BaMMV NIa and NIb regions is probably the cleavage site because of its position, although comparisons of the amino acids surrounding this dipeptide with those at the site between the NIb and capsid proteins show no identical amino acids or significant chemical similarities. Analysis of peptides released by mild proteolysis from the aphid-borne potyvirus and BaYMV particles has demonstrated that the N- and C-terminal regions of their capsid proteins are located on the surface of the virus particles (Shukla et al., 1988; Kashiwazaki et al., 1989 a). The degradation observed in the BaMMV capsid protein (Ehlers & Paul, 1986; Kashiwazaki et al., 1989b) as well as the relatively hydrophilic nature of the terminal regions of the BaMMV capsid protein sequence (data not shown) may also indicate the surface location of the terminal regions. It has been shown for aphid-borne potyviruses that the long surface-located N-terminal region of the capsid protein is the immunodominant part of the protein and is so variable among viruses that it contains virus-specific epitopes (Shukla et al., 1988, 1989). The large amino acid sequence differences between BaYMV and BaMMV in the N-terminal regions of their capsid proteins fit well with the absence of serological relationship between them. The difference observed in the stabilities of their capsid proteins (Ehlers & Paul, 1986; Kashiwazaki et al., 1989b) may be attributable to differences in the occurrence of cleavage sites for the protease(s) that probably contaminate purified virus preparations (Ehlers & Paul, 1986). The extent of the capsid protein sequence similarities between BaYMV and the two BaMMV strains (35 or 37%) is comparable to the lowest value among the identities reported for distinct aphid-borne potyviruses (38 to 71%) (Shukla & Ward, 1988). Relationships between BaMMV or BaYMV and aphid-borne potyviruses are even more distant. Thus, although Stanarius et al. (1989) reported that BaMMV reacted with antisera to the aphid-borne potyviruses BYMV and TuMV, it has only slight capsid protein sequence similarities (21 to 26%) with these two and other aphid-borne or mite- Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 2180 S. Kashiwazaki and others borne potyviruses. Likewise, the BaYMV capsid protein has only weak similarity (20 to 27~) with those of aphid-borne or mite-borne potyviruses. Furthermore, the (I/V)DAG sequence implicated in aphid transmission of potyviruses (Harrison & Robinson, 1988; Atreya et al., 1990, 1991) is not present in the N-terminal regions of the capsid proteins of BaMMV or BaYMV, which are fungus-borne. The sequences of the non-structural proteins of BaMMV and BaYMV also slightly resemble those for aphid-borne or mite-borne potyviruses. These results support the current proposal that BaMMV and BaYMV should be distinct members of the genus Baymovirus, which is placed together with the genera Potyvirus (aphid-borne) and Ryemovirus (mite-borne) in the family Potyviridae (Barnett, 1991). The considerable similarities between BaMMV-Kal and BaMMV-Nal, in their capsid and non-structural protein sequences and in their 3' non-coding nucleotide sequences, are comparable to those reported for strains of individual aphid-borne potyviruses (Shukla & Ward, 1988; Ward & Shukla, 1991). This supports the classification of BaMMV-Kal and BaMMV-Nal as strains of the same virus (Kashiwazaki et aL, 1990b). The several amino acid differences found between the Nterminal regions of their capsid protein sequences could cause their serological differences (Kashiwazaki et al., 1990b), suggesting the presence of strain-specific epitopes in this region. However, because polyclonal antibodies raised against the two BaMMV strains are cross-reactive (Kashiwazaki et al., 1990b), monoclonal antibodies seem to be required for virus strain identification. Alternatively, it would seem possible to identify the two strains by hybridization techniques using oligonucleotide probes for strain-specific blocks of nucleotide sequence. The relation between sequence differences between the two strains and their differences in biological properties, such as pathogenicity, remains to be determined. The authors wish to thank Professor B. D. Harrison, University of Dundee, for critical reading of the manuscript. ATREYA, P. L., ATREYA, C. D. & PIRONE, T. P. (1991). Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proceedings of the National Academy of Sciences, U.S.A. 88, 7887-7891. BARNETI, O. W. (1991). Potyviridae, a proposed family of plant viruses. Archives of Virology 115, 139-141. BATISTA, i . F., ANTONIW, J. F., SWABY, A. G., JONES, P. • ADAMS, M. J. (1989). RNA/cDNA hybridization studies of UK isolates of barley yellow mosaic virus. Plant Pathology 38, 226-229. DAVIDSON,A. D., PROLS, M., SCHELL, J. & STEINBISS,H.-H. (I 991 ). The nucleotide sequence of RNA 2 of barley yellow mosaic virus. Journal of General Virology 72, 989-993. DEVEREUX, J., HAEBERLI, P. & SMITHIES, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387-395. DOMIER, L. L., FRANKLIN, K. M., SHAHABUDDIN,M., HELLMAN,G. M., OVERMEYER, J. H., HIREMATH, S. T., SLAW,M. F. E., LOMONOSSOFF, G. P., SHAW, J. G. & RHOADS, R. E. (1986). The nucleotide sequence of tobacco vein mottling virus RNA. Nucleic Acids Research 14, 5417-5430. DOMIER, L. L., SHAW, J. G. & RHOADS, R. E. (1987). Potyviral proteins share amino acid sequence homology with picorna-, como-, and caulimoviral proteins. Virology 158, 20-27. DOUGHERTY, W. G. & CARRINGTON, J. C. (1988). Expression and function of potyviral gene products. Annual Review of Phytopathology 26, 123-143. DOUGHERTY, W. G., PARKS, T. D., CARY, S. M., BAZAN, J. F. & FLETTER1CK, R. J. (1989). Characterization of the catalytic residues of tobacco etch virus 49-KDa proteinase. Virology 172, 302 310. EHLERS, U. & PAUL, H.-L. (1986). Characterization of the coat proteins of differenf types of barley yellow mosaic virus by polyacrylamide electrophoresis and electro-blot immunoassay. Journalof Phytopathology 115, 294-304. FRIEDT, W., ORDON, F. & GOTZ, R. (1990). Genetics of resistance to the 'barley yellow mosaic virus complex' and status of breeding. Schriftenreihe der Deutschen Phytomedizinischen Gesellschaft, Bd I. Proceedings of the First Symposium of the International WorkingGroup on Plant Viruses with Fungal Vectors, Braunschweig,Germany, August 21-24, 1990, pp. 117-120. Stuttgart: Ulmer Verlag GORBALENYA, A. E., DONCHENKO, A. P., BLINOV, V. M. & KOONIN, E. V. (1989). Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Letters 243, 103-114. HAMMOND, J. & HAMMOND, R. W. (1989). Molecular cloning, sequencing and expression in Echerichia coli of the bean yellow mosaic virus coat protein gene. Journal of General Virology70, 1961 1974. HARIRI, D., FOUCHARD, M. & LAPIERRE, H. (1990). Resistance to barley yellow mosaic virus and to barley mild mosaic virus in barley. Schriftenreihe der Deutschen Phytomedizinischen Gesellschaft, Bd I. Proceedings of the First Symposium of the International WorkingGroup on Plant Viruses with Fungul Vectors, Braunschweig, Germany, August 21-24, 1990, pp. 109-112. Stuttgart: Ulmer Verlag. HARRISON, B. D. & ROBINSON, n . J. (1988). Molecular variations in vector-borne plant viruses: epidemiological significance. Philosophical Transactions of the Royal Society of London B321, 447-462. HUTH, W. (1990). The yellow mosaic inducing viruses of barley in Germany. Schriftenreihe der Deutschen Phytomedizinischen Gesells- References ALLISON, R., JOHNSTON, R. E. & DOUGHERTY, W. G. (1986). The nucleotide sequence of the coding region of tobacco etch virus genomic RNA; evidence for the synthesis of a single polyprotein. Virology 154, 9-20. ANDERSON, J. F., COUTTS, R. H. A. & DAVIES, J. W. (1990). Molecular characterization of a UK isolate of barley mild mosaic virus. Schriftenreihe der Deutschen Phytomedizinischen Gesellschaft, Bd L Proceedingsof the First Symposium of the International WorkingGroup on Plant Viruses with Fungal Vectors, Braunschweig, Germany, August 21-24, 1990, pp. 127-130. Stuttgart: Ulmer Verlag. ATREYA, C. D., RACCAH, B. & PIRONE, T. P. (1990). A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus. Virology 178, 161-165. chaft, Bd I. Proceedings of the First Symposium of the International Working Group on Plant Viruses with Fungal Vectors, Braunschweig, Germany, August 21-24, 1990, pp. 113-115. Stuttgart: Ulmer Verlag. HUTH, W. & ADAMS, M. J. (1990). Barley yellow mosaic virus (BaYMV) and BaYMV-M: two different viruses. Intervirology 31, 38-42. HUTH, W., LESEMANN, D.-E. & PAUL, H.-L. (1984). Barley yellow mosaic virus: purification, electron microscopy, serology, and other properties of two types of the virus. Phytopathologisehe Zeitschrift l U , 37-54. INOUYE, T. & SAITO, Y. (1975). Barley yellow mosaic virus. CMI/AAB Descriptions of Plant Viruses, no. 143. Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56 Nucleotide sequence of B a M M V RNA 1 KAMER, G. & ARGOS, P. (1984). Primary structural comparison of RNA-dependent RNA polymerases from plant, animal and bacterial viruses. Nucleic Acids Research 12, 7269-7282. KASHIWAZAKI,S., HAYANO,Y., MINOBE,Y., OMURA,T., HIBINO,H. & TSUCHIZAKI,T. (1989a). Nucleotide sequence of the capsid protein gene of barley yellow mosaic virus. Journal of General Virology 70, 3015-3023. KASHIWAZAKI,S., OGAWA,K., USUGI,T., OMURA,T. & TSUCHIZAKI,T. (1989 b). Characterization of several strains of barley yellow mosaic virus. Annals of the Phytopathological Society of Japan 55, 16-25. KASHIWAZAKI, S., MINOBE, Y., OMURA, T. & HIBINO, H. (1990a). Nucleotide sequence of barley yellow mosaic virus RNA 1 : a close evolutionary relationship with potyviruses. Journal of General Virology 71, 2781 2790. KASHIWAZAKI,S., NOMURA,K., WATANABE,K., TOSHIMA,1., hDA, Y., USUGI,T., OGAWA,K., HIEINO,H. & TSUCHIZAKI,T. (1990b). Barley yellow mosaic virus and barley mild mosaic virus: strains and host resistance. Schriftenreihe der Deutschen Phytomedizinischen Gesellschart, Bd I. Proceedings of the First Symposium of the International Working Group on Plant Viruses with Fungal Vectors, Braunschweig, Germany, August 21-24, 1990, pp. 105-108. Stuttgart: Ulmer Verlag. KASHIWAZAKI, S., MINOBE, Y. & HIBINO, H. (1991). Nucleotide sequence of barley yellow mosaic virus RNA 2. Journal of General Virology 72, 995-999. KOENIG, R. & HUTH, W. (1988). RNA/cDNA hybridization and infectivity tests suggest that barley yellow mosaic virus isolate M has a bipartite genome. Journal of Phytopathology 121, 370-372. KOONIN, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. Journal of General Virology 72, 2197 2206. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680685. MAISS, E., TIMPE, U., BRISSKE, A., JELKMANN, W., CASPER, R., HIMMLER,G., MATTANOVICH,D. & KATINGER,H. W. D. (1989). The complete nucleotide sequence of plum pox virus RNA. Journal of General Virology 70, 513 524. NIBLETT, C. L., ZAGULA. K. R., CALVERT, L. A., KENDALL, T. L., STARK,D. M., SMITH,C. E., BEACHY,R. N. & LOMMEL,S. A. (1991). cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene. Journal of General Virology 72, 499-504. PROLS,M., DAVIDSON,A., SCHELL,J. & STEINBISS,H.-H. (1990). In vitro translation studies with cDNA clones corresponding to the RNAs of barley yellow mosaic virus and barley mild mosaic virus. Journal of Phytopathology 130, 249 259. RIECHMANN, J. L., LAIN, S. & GARCiA, J. A. (1992). Highlights and prospects of potyvirus molecular biology. Journalof General Virology 73, 1-16. 2181 ROBAGLIA, C., DURAND-TARDIF,M., TRONCHET, M., BOUDAZIN,G., ASTIER-MANIFACIER,S. & CASSE-DELBART,F. (1989). Nucleotide sequence of potato virus Y (N strain) genomic RNA. Journal of General Virology 70, 935-947. SANGER,F., NICKLEN,S. & COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. SHUKLA,n. D. & WARD, C. W. (1988). Amino acid sequence homology of coat proteins as a basis for identification and classification of the potyvirus group. Journal of General Virology 69, 2703-2710. SHUKLA,D. O., STRIKE,P. M., TRACY,S. L., GOUGH,K. H. & WARD, C. W. (1988). The N and C termini of the coat proteins of potyviruses are surface-located and the N terminus contains the major virusspecific epitopes. Journal of General Virology 69, 1497-1508. SHUKLA,D. D., TRIBBICK,G., MASON,T. J., HEWISH,D. R., GEYSEN, H. M. • WARD, C. W. (1989). Localization of virus-specific and group-specific epitopes of plant potyviruses by systematic immunochemical analysis of overlapping peptide fragments. Proceedings of the Notional Academy of Sciences, U.S.A. 86, 8192-8196. SHUKLA,D. n., FRENKEL,M. J. & WARD, C. W. (1991). Structure and function of the potyvirus genome with special reference to the coat protein coding region. Canadian Journal of Plant Pathology 13, 178191. STANARIUS,A., PROESELER,G. & RICHTER,J. (1989). Immunelektronenmikroskopische Untersuchungen zur serologischen Verwandtschaft des Gerstengelbmosaik-Virus (barley yellow mosaic virus) und des Milden Gerstenmosaik-Virus (barley mild mosaic virus) mit anderen gestreckten Viren. Archiv3~r Phytopathologie und Pflanzenshutz, Berlin 4, 303-307. TREMBLAY, M.-F., NICOLAS, O., SINHA, R. C., LAZURE, C. & LALIBERTI~,J.-F. (1990). Sequence of the 3'-terminal region of turnip mosaic virus RNA and the capsid protein gene. Journal of General Virology 71, 2769-2772. USUGI, T. & SAITO,Y. (1976). Purification and serological properties of barley yellow mosaic virus and wheat yellow mosaic virus. Annals of the Phytopathotogieal Society of Japan 42, 12-20. USUGI, T., KASHIWAZAKI,S., OMURA, T. & TSUCHIZAKI,T. (1989). Some properties of nucleic acids and coat proteins of soil-borne filamentous viruses. Annals of the Phytopathological Society of Japan 55, 26-31. WARD, C. W. & SHUKLA, D. D. (1991). Taxonomy of potyviruses: current problems and some solutions. Intervirology 32, 269-296. ZARET, K. S. & SHERMAN, F. (1982). DNA sequence required for efficient transcription termination in yeast. Cell 28, 563 573. (Received 23 April 1992; Accepted 19 May 1992) Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Fri, 14 Oct 2016 11:34:56