leaf and stunting of plants. In leaf-dip prepara- tion the virus particles appeared polyhedral in shape. The virus transmitted readily by sap inoculation.
I'biit Ptitliohay (1996) 45, K23--828
Biological, serological and molecular characterization of a cucumber mosaic virus isolate from India Q.M.R. H.AQ. B.P. S I N G H * ami K.M. SRIVASTAVAv Plant Viru.\ Liihuruiory. Naiional Boumical Researvh Insiituw. Litikmnv 226 0(H. Imiia A \irus causing nK>saic iind leaf dcformaiion o\' Pliy.\alis inininia has hccii itlcniilicd as an isoUiic of cucumber mtisaic virus (CMV) on the basis of its iransmission by nphids in a non-pcrsislcni manner, pnlyhedral particles of 29 nm diameter, molecular weight of coat protein subunits us 24-5 kDa. scrologica! relationship with a CMV isolate and a tripartite single-stranded RNA genome with a subgenoniic RNA4- Furthermore. cDNA representiiit! coat protein gene was synthesized and cloned. Complete nucleoitdc sequences (8^0 ntt v\ere obtained which showed a coat protein gene open reading lV;mie ol' 657 residues. The nuclcotide sequences provided the 2IS amino acid sequences of the coat protein. Nueieotide as well as amino acid sequences revealed more than ^0% identity with the CMV subgroup 1 strains.
INTRODUCTION Cucumber mosaic virus (CMV). type member of the cucLimovirus group, is :m icosahcdral. nonpersisiently aphid-transmitted \irus with a tripartite plus-sense RNA genome (Paiukaitis *• / (//.. 1992). \n additional subgenomic RNA4 is synthesized from RNA3 and carries the gene for coat protein (Sehv\inghamer& Syinons 1475. 14771. CMV has ix'en reported from most of the countries of the world and identified as causal agent of several disease epidemics (Paiukaitis cr al.. 1992). Based on molecular hybridization studies. Owens & Paiukaitis (198X) categorized CMV strains under two subgroups. I and II. Similarly. Wah\uni c/ ul. (19^2) ckissilied fourteen strains ofCMV. These studies revealed that cDNAs prepared against RNAs of subgroup I strains do noi cross-react with subgroup II strains and yicc vcr.ui. Several strains of CMV. mainly differing in biological characteristics, have been reported from India. MostK these were characterized on host range, transmission by vectors and physical properties. In a few cases, however. the particle morphology was observed and serological relationships established (Sri\asta\a *To whom correspondence should be addressed. +Deceased. The nuL-kMtiik- sfqLICIK-C roporicd in iliis pLificr luis been stibmiticd willi Gen Bank under Accession Niv X89652 CMVCPGENiE,
Accepted 27 Jtiiw 1996.
ciai. 1991. 1992: Raj c/(//.. 1991). In the present communication we report the properties of an isolate of cucumber mosaic virus (CMV-P) causing severe mosaic in Phy.salis luininui based on host range, physical properties, mode of transmission, particle niorpholog\. serological relationship, coat protein and nueleie acid properties. Furthermore, cloning of cDNA representing the coat protein gene and nueieotide sequence studies were carried out. Based on nueieotide as well as amino acid sequence homologies. it is also proposed that the present isolate is a member of CMV subgroup I.
MATERIALS AND METHODS Transmission, host ranjic and purification Naturally infected plants of I'ln.-^ulis nuniimt served as the source of primary inoeulum. The virus was propagated in Siailiumi rii.sliia by mechanical iiu>cLilation using D l m o l L ' sodium phosphate bulTer (pH 7'2) containing U-r'o sodium sultitc. The aphid.s Myziis pcr.sicac. A gossypiiatid A. imccivora were used for testing non-persistent transmission of the virus as described by Srivastava vi al. (1991) with .V. ruMiia as both donor and recipient host. For host range suidies, inoculinn was prepared from infected ;V. rusnica plants and back inoculation tests were done on P. minima. In a limited host range stud\. live plants of each species.
824
Q. M. R. Hail ct al.
niimcly Chenopodiuni ainaranticolor. C. quinoa. C. murale, Nicotiana tahacum var. White Burlcy. N. tahaciun var. Samsun NN. S. nistica. Lycopersicon csculcntnni. Capsicum annuum. Datura stramonium. Solatntm nigriim. S. mclongi'na. LagviHuia ciecraria. .Momordica charantia. Vigna nmngo. Vicia faha. Catlmrantlms roseus. Chrysantfietnum morifolitun. Tagctcs crccta and Zinnia elegans were inoculalcd. The virus was purified IVom .\\ rusiica as deiailed by Lot ct (//. (1972) and Raj ct al. (1993). Purity of the virus was established by UV absorption and electron microscopy. The yield of virus was calctilalcd by assuming the extinction coefficient at 260 nm lo be 5 (Francki ct al. 1979). Electron microscopy und serolo};y Virus preparations were stained with 2% aqueous uranyi acetate. pH 4 2 on a carbonstabilized, fornivar-coated grid and examined under a Philips CM 10 transmission electron microscope. Ouchtcrlony double dillusion tests were carried out to establish ihe serological relalionship of the virus with an antiserum to CMV-CR (Raj ct ai. 1993). The antiserum was raised by injecting formaldehyde tixed virus (Fnincki & Habili (1972) in New Zealand male albino rabbits. Coat protein and nucleic acid analysis SDS PAGE of the coat protein was done essentially as described by Maizel (1971). Viral nucleic acid was isolated by disrupting the particles wiih l"o SDS and an eijual volume of phenol (Srivastava et al.. 1992). Electrophoresis of the nucleic acid was carried out using Escherichia coH ribosomal RNAs as markers (23S and I6S) in I'Va formaldehyde denaturing agarose gel (Sambrook et al.. I9S9).
('/ (//.. 1989) Lising n—P-labelled homologous probe pCMVR3. prepared by random primer extension method (Sambrook ct al.. 1989). The probe pCMVR3 was a partial clone of coal protein gene of CMV-P isolate, confirmed by hybridization studies and sequence alignment (data not included). Unidirectional nested deletions were generated using an E.\o 111 Mungbean Nuclease deletion kit (Stratagene). The nudeotide sequences were determined from both cDNA strands by dideoxynudeotide chain-termination reactions (Sanger et ai. 1977) using a T7 sequencing kit (Pharmacia). Sequence data were collected and analysed using PC Gene (release 6 80 Intelligenetics Inc.) sequence analysis software package and EMBL Database CDEM33VO. RESULTS Naturally infected Physatis minima plants exhibited severe mosaic followed by delormaiion of leaf and stunting of plants. In leaf-dip preparation the virus particles appeared polyhedral in shape. The virus transmitted readily by sap inoculation. Myztis pcrsicae. .Aphis go.ssypii and A. craccivora transmitted the virus lo 10 10. 8 10 and 8 10 N. rustica plants, respectively. Limited host range studies indicated Chcnopodittm amaranticolor. C. ipiinoa and C. tnuralc as local lesion hosts. The isolaie induced kDa
M
1
66-0
cDNA synthesis, cloning and sequencing Viral RNA (5y;g) was polyadenvlated with E. coH Poly A polymerase (Pharmacia) and subsequently used for lirst-strand cDNA synthesis with oligo (dT) primer and avian myelobiastosis virus (AMV) reverse transcriptase. The second strand was synthesized according lo the method of Gubier & HolTman (l9S3t. Double-stranded cDNA was then ligaled inlo the .S'»(ivl sile of pUC 18 (Pharmacia) and subsequently transformed into compeieni E. coli, XL I-blue cells. cDNA clones containing the coat protein gene were screened b\ colon\ hybridization (Sambrook
24 0
18-4 14-3
1 SDS-PAGE nC \iriil cixil protein: hitics M molecular weight markers, lanes 1 and .'' viral protein (difVcrcnl amounts).
825
Molecular characterization of CM V isolate from India
systemic symptoms in Nicotiatia tahacum var. White Burley (mosaic). A', tahacum var. Samsun NN (mosaic and stunting of plants), A', rustica (severe mosaic and leaf deformation), and Lycopersicon eseiiientuin (mosaic and shoe siring of leaf lamina). However, no symptoms were observed on other lest plants. Back inoculation tests to P. ndnima also indicated the absence of virus.
RNA-1&2
23 S
RNA-3
16 S
RNA-4
Virus yield was 200mgk.g"' fresh tissue from 6-7 days posl-inoctilated plants. The virus preparations were infectious when inoculated back to P. mimima and produced the similar symptoms. The virus particles lost their infectivity on treatment with RNase and degraded in l-5molL"' KCl solution, Electronmicroscopy of the purified preparation stained with uranyl acetate revealed the presence of polyhedral particles of 29 nm diameter.
Fig. 2 Viral RNA eleclroplioiesed along wilh 16 S ;ind 23 S Eicherichia roll ribosomol RN.A markers in 1% agiirose gel containing formaldehyde. Liiiies 1-3 represenls different amounts of RNA.
The molecular weight of the coat protein subunits was 24-5 kDa as determined by SDS~ polyacrylamide gel electrophoresis (Fig. I) In Ouchterlony gel double-dilTusion tests. crude antigen as well as purified preparations
10
20
30
40
50
I
I
I
I
I
1 TCCGCCCATT CATTACCAGC GAACCAATCT GTGTCGTCAA AATCATGGTC 51 TTCTTCCGCT GGTAACGTAA ACCGAGGAGC CTCAGACTCG GACAATTCCA 101 CCACACTCGC GTGGTGGTAC GTGATTTTCT CTTTTGTGTT GTAGATTTGA 151 GTCGAGTCGA GTCATGGACA AATCTGAATC AACCAGTGCC GGTCGTAATC 201 GTCGACGTCG TCCGCGTCGT GGTTCCCGCT CCGCCTCCTC CTCCGCGGAT 251 GCTACACTTA GAGTCCTGTC GCAACAGCTT TCGCGACTTA ATAAGACGTT 301 AGCAGCTGGT CGTCCTACTA TTAACCACCC AACCTTTGTG GGTAGTGAAC 351 GTTGTAAACC TGGATACACG TTCACCTCGA TTACCCTGAA GCCACCGAAA 401 ATAGACAAGG GGTCTTATTA TGGCAAAAGG TTGTTACTTC CTGATTCAGT 451 CACTGAGTTC GATAAGAAGC TTGTTTCGCG CATTCAAGTT CGAGTTAATC 501 CTTTGCCGAA ATTTGATTCT ACCGTGTGGG TGACGGTCCG TAAAGTTCCT 551 GCCTCCTCGG ACCTGTCCGT ATCCGCCATC TCTGCTATGT TTGCGGACGG 601 AGCCTCACCG GTACTGGTTT ATCAGTATGC TGCATCTGGC GTTCAAGCCA 651 ATAACAAATT GTTATATGAT CTTTCAGTGA TGCGCGCTGA TATTGGTGAT 701 ATGAGAAAGT ACGCCGTGCT CGTGTATTCA AAAGACGATG CGCTTGAGAC 751 GGACGAACTA GTACTTCATG TCGACATCGA GCACCAACGC ATTCCCACGT 801 CTGGGGTGCT CCCAGTTTGA ACTCGTGTTT TCCAGGATCC TCCCTCCGTT 351 TTCTGTGGCG GGAGCTGAGT TGGTAGTTTA AAAAAAAAAA Fig. 3 Complete luicleotide sequences of the clone pCP25; 1 163 bases = 5' flanking region; 64- 820 bases = coding sequences for the coat protein; 821-880 = .V untranslated region: 881-890= added poly 'A'residues,
Q. M. R. Haq et al. CHV._P CMV _im CHV__0 CMV y
CMV M CHV_Q
CMV p CHV_.FNY CMV 0 CMV. Y CMV._H CMV_Q CMV..P CMV_ FHY CHV 0
CMV Y CMU M CMV_Q CMV P CMV J'NY CMV 0 CMV__y CMV^_M CMV_Q CMV_ P CMV .FNy CMV_ 0
CMV y CHV_ M
MnKRESTSAGR-NRRRRPRRGSRSASSSADATLRVLSQQLSRLNKTLAAG HDKSESTSACR-HREJRRPURCSRSAPSHAUANFRVI.SQQLsnLNKTLAAG MDKSESTSAGR-NRRRRLRRGSRSASSSSDANFRVI..SQQl,aRLNKTLAAG MLJKSESTSAGR-NRRRRrRRGSHSASS.SADANFRVLSQQLSRLNKTLAAG MDKSGSPNASRTSRRRRPRRGSRSASG-AUAGLRALTQQMLRLNKTLAIG RFTINlfPTFVCSERCKF'GVTETSITLKPPKIDKGSYVGKRLLLF'DSVTEF Ui'TINIlPTFVGSEHCRrGYTFTSITLKPPKIlJRGSVVGKRLLLPDSVTEy
-19 49 49 49 49
RPTINlli-TI V G ' i E T F J Y RPTINHPTFVGSERCRPGyTFTSTTI.RPPKIURCSyyGKRLLLPDSVTEy RPTLNiiPTFVGSESCKPGyTFTSITLKPPEIEKGSyFGRRLSLPDSVTDY
99 99 99 99 99 99
DKKLVSRrQVRVnpr.,PKi DSrvwVTVRKVPASSDLSVSAISAHFADGASP DKKtVSRlylRVNPI,PKFDSTVKVTVlU;VI'ASSDLSVAAISAHFADGASP UKKLVSRIQIRV[lPLPKrDSTVWVTVRKVPASSDLSVAAI5AMFADGA.SP DKKLVSRJQIRVHPI PKrDSTVWVTVIiKVSASSDl.SVAAISAMFADGASP DKKl.V.SRVQlifVNPl PKi l);;'l'VWV'rViiKVI,A;;.';i)l,;;UAAlSAHFADCASP LJKKLVSRlQlRIHI'Lirt US'l'VWVl'VRKVPSSSUL^VAAISAHFGUGNSP
149 149 149 14 9 M9 149
VLVyQyAAFGVQANNKI.LyDlSAMHAUlGUMRKYAVLVYSKDDALETDEL VLVyQyAASGVQArJHKI.LyUI.SAMRAinGDtlRKYAVLVYSKDDALKTDEL VL.vyOYAASGVQ'1'NNKl t,rDI.SAMRADJCDt41!KyAILVYSKDDALETDEL
Vl.HVDlEHyRIPTSGVLPV VIHVUlEHQlUfl'SRVLPV VLHVDVEIIQRIPTSGVLPV VLHVDIEilQRIl'TSRVl.PV
199 199 199 199 199 199
2)3 218 21ii 21B 318
CMV. Q
Fig. 4 Multiple sequence alignment of the coal protein amino acids ofdifTercnl CM V slrains: * - sequences perfectly conserved; • ^ well conserved. Virus acronyms are given iu the text.
reacted witli CMV-CR antiscrtini. Antiscrtim raised agains! the \irus isolate had a very low titre. However, in Otichterioiiy doLibie-dilTusion tests and Western biot immunoassay. the viral antiserum responded positive!)- with the homologous antigen. Nucleic acid isolated from purified virus particles was infectious when inoculated on A'. rnstiea. Electrophresis of nucleic acid preparations in formaldehyde denaturing agarose gel revealed four bands (Fig. 2) which were unaffected by DNasc treatment. However. SI nuclease- and RNasc-trcated Lines did not show any band, suggesting that the nucleic acid was singlesiranded RNA. Transformation of XL I-blue cells with cDNA clones resulted in more than 2000 white colonies. A clone containing an approxiniatch 900 base pair (bp) fragment, which hybridized with the coat protein probe was selected for further studies and designated as pCP25. The complete nucleotide sequence of the coat protein gene was established after sequencing both the strands of
the full length clone and subclones. The sequence data revealed that the clone pCP25 had an insert of 890 bp containing ihe complete eoat protein gene (Fig. 3). It contained an open reading frame (ORF) of 657 base pairs besides 163 bp upstream of the start eodon (ATG) and 60 bp from the V untranslated region Iblhnvcd by the added poly A tail (iO residues). A protein or218 amino acid residues with a calculated molecular weight of 24041 Da was predicted from the nticlcotide sequences of the coat protein gene. The isolate showed a nueleotide sequence identity of S9 91",, with CMV subgroup I Slrains. namely Fny-CMV. M-CMV. O-CMV and Y-CMV. With Q-CMV. a subgroup II strain, the sequence similarity was signiticantly lower, thai is to say 6S"n studies for separating isolates of CMV into subgroups I and II. The availability of the sequences of the coat protein gene of the isolate (CMV-P) allowed a reliable establishment of relationship with other strains reported from dilierent paits of the world. The isolate (CMV-P) .showed a nucleoiide sequence identity o\' 89-91% with the CMV subgroup ! strains, namely Fny-CMV (Owens a al.. 1990).. M-CMV (Owens er al.. 1990). OCMV (Hayakawa cl al.. 1989) and Y-CMV
(Nitta cr al.. 1988): with CMV subgroup II. namely Q-CMV (Davics & Symons. 1988) it is significantly low. i.e. 7()"o. The deduced amino acid sequences of coat protein of the isolate revealed a high percentage—93-95% of identity with the subgroup I strains. In contrast, identity with subgroup II strain v\as as low as 79'V,,. These results coincide with the lindings of Mayakawa er al. (1989). who ciassiticd the CMV strains. O. D and Y in subgroup I on the basis of high sequence similarity. Similarly. 25 strains of CMV have been placed into two subgroups based on comparison ol' nueieotide sequence from their coat protein genes (Gonda & Symons 1978. Pia77ola er al.. 1979: Owens & Paiukaitis 19S8: Hayakawa er al.. 19X9; and Paiukaitis ei al.. 1992).
ACKNOWLEDGEMENT We thank Dr P. V. Sane, Director. NBRI for his keen interest and providing facility to work. The senior author is thankful to Department of Bioieehnokigy. India for financial support. REFERENCES Da\ics C. Symons RH. I4S8. Further implicaiioti for the evolutionary relationships between tripartite plant viruses based on cucumber mostiic virus RNA3. Viroiogy 165. 216 24. Francki RIB. Hiibili N. 1972. Stabilization of capsid struclurc and enhancement of immunogenicity of cucumber mosaic virus (Q strain) by form aldehyde. Virology'm. 304-15. Francki RIB. Mossop DW, Hatta T. 1474. CMI AAB De.siriprioii nt Phtni I'l'nm'.v No. 213. Gondit TJ. S\mons RH. 1978. The useofhvbridization
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analysis with compiemcnlary DNA lo determine the RNA sequence honiology between strains of plant viruses. Its application to several strains at' cucumoviruses. l'irt>hgyS8. 361. Gubler U. HotTman BJ. t983- A simple and very ctticient method for generatingcDNA libraries. Gene 25.263 9. Hayakawa T. Mizykami M, Nakajima M, Suzuki M. 1989. Complete nucleotide sequence of RNA3 from cucumber mosaic virus (CMV| strain O: comparative study of nucleotide sequences among CMV stnuns O.Q.D and Y. Journal of General I 'irology 70, 499-504. Lot H. Marrou J. Quiot JB, Esvan C. 1972. Contribulion a Tctudcdu virus de la mosaiquedu Concombre (CMV), II. Methods de purifiualion rapide du virus. Annals oj Phytopathology 4. 25 8. Maizel JV. 1971, Polyacrylamide ge) eleetrophoresis of viral proteins. Methods in Virology. Vol. V. Maramorosch K. Koprowski H. eds. New York. London: Academic Press. 179 246. Nilta N. Masuta C. Kuwata S. Takanami \'. 1988. Comparative studies on the nticleotide sequence of cucumber mosaic virus RNA3 between Y strain and Q slritin. Annals of Phytopadiological Socieiy of Japan 54. 516 22. Ouens .1. Shintaku M. .Aeshleniiin P, Tahar SB. Paliikaitis P. 1990, Nucieotide sequence and c\olutioiiary relationships of cucumber mosaic virus (CMv') strains: CMV RNA3. Journal of General r/r
