5440 Nucleic Acids Research, Vol. 19, No. 19
cDNA sequence of the capsid protein gene and 3' untranslated region of a fanleaf isolate of grapevine fanleaf virus Flora Sanchez, Cathy Chay1, Maria Jos6 Borja, Adib Rowhani1, Javier Romero', George Bruening1 and Fernando Ponz* Departamento de Proteccion Vegetal, CIT-INIA, Apartado 8111, 28080 Madrid, Spain and 1Department of Plant Pathology, University of California, Davis, CA 95616, USA Submitted July 8, 1991 Grapevine Fanleaf Virus (GFLV) is a member of the nepovirus causing infectious degeneration in grapevines (Vitis spp.). Three main types of isolates have been described (fanleaf, yellow mosaic, and vein banding), inducing differential symptomatology in the grapevine host (1). The virus has been reported as one of the most widespread in grapevines, and as the nepovirus of greatest economic importance (2). A fanleaf isolate of GFLV was obtained from a Vitis rupestris cv. St. George from the grapevine collection maintained at Davis. It was further propagated in and purified from Chenopodium quinoa according to Jimenez (3). Genomic RNAs were isolated from purified particles of the fanleaf isolate of the virus and cDNA was synthesized and cloned in vector pCGN 1703 (Calgene, Inc.) following the cloning methodology of Alexander (4). Recombinant clones were identified as corresponding to RNA 1 or 2 according to length and restriction map. Both strands of the cDNA insert of a recombinant clone have been completely sequenced. This cDNA is 2304 bp long, excluding the poly(A) tail, and corresponds approximately to the 3' most two thirds of the viral RNA2. The sequencing method was chain termination using the Sequenase Kit (U.S. Biochemicals). The sequence contains a single long open reading frame and 211 nucleotides of untranslated region, preceding a poly(A) tail of length greater than 75 As. The comparison of the sequence of the coat protein of this isolate with that of isolate F 13 (5) is particularly interesting considering the differences in induced symptoms. F13 is able to encapsulate a satellite RNA, possibly responsible for the severity of symptoms and lack of recovery of GFLV-infected Chenopodium quinoa plants (6). The fanleaf isolate from which we have synthesized cDNA, shows no evidence of an encapsidated satellite, and induces an infection from which C. quinoa is able to recover. The analysis reveals 90% identity with the sequence of GFLV isolate F13 at the nucleotide level and shows only a single unmatched residue in the untranslated region. group,
*
EMBL
accession no.
X60775
By comparison with the deduced amino acid sequence of GFLV isolate F13, the amino terminus of the capsid protein can be assigned to the Gly in position -504 from the carboxy terminus, corresponding to an Arg/Gly cleavage site already proposed for this nepovirus (5). Thus the capsid protein would be 504 aa long and would have a calculated MW of 55,918. At the amino acid level, there is 96% homology between the deduced capsid proteins from the two isolates (14 conservative changes out of 22 in 504 residues). No changes have been found in the 192 amino acids of the deduced polypeptide sequence preceding the capsid
protein. The high degree of homology found between the capsid proteins is in good correlation with the lack of immunological heterogeneity reported for this virus (1). It remains to be seen if some of the detected changes between isolates are related to their ability to encapsidate satellite RNA.
ACKNOWLEDGEMENTS The work in Spain has been supported by Grant AGRO 890078 from the Spanish Granting Agency CICYT. C. Chay and M.J. Borja were holders of predoctoral fellowships from the McKnight Foundation and INIA, respectively.
REFERENCES 1. Hewitt,W.B., Martelli,G., Dias,H.F. and Taylor,R.H. (1970) Descriptions of plant viruses No. 28. Commonwealth Mycological Institute/Association of Applied Biology. 2. Murant,A.F. (1981) In Kurstak,E. (ed.), Handbook of Plant Virus Infections and Comparative Diagnosis. Elsevier/North Holland Biomedical Press. Amsterdam, pp. 197-238. 3. Jimenez,F. (1984) Ph.D. thesis. University of California at Davis, 78pp. 4. Alexander,D.C. (1987) Methods in Enzymology 154, 41-64. 5. Serghini,M.A., Fuchs,M., Pinck,M., Reinbolt,J., Walter,B. and Pinck,L. (1990) J. Gen. Virol. 71, 1433- 1441. 6. Pinck,L., Fuchs,M., Pinck,M., Ravelonandro,M. and Walter,B. (1988) J. Gen. Virol. 69, 233-239.
To whom correspondence should be addressed
'On leave of absence at Plant Molecular Biology Center. Northern Illinois University, Dekalb, IL 60115. USA
cDNA sequence of the capsid protein gene and 3' untranslated region of a fanleaf isolate of grapevine fanleaf virus Flora Sanchez, Cathy Chay1, Maria Jos6 Borja, Adib Rowhani1, Javier Romero', George Bruening1 and Fernando Ponz* Departamento de Proteccion Vegetal, CIT-INIA, Apartado 8111, 28080 Madrid, Spain and 1Department of Plant Pathology, University of California, Davis, CA 95616, USA Submitted July 8, 1991 Grapevine Fanleaf Virus (GFLV) is a member of the nepovirus causing infectious degeneration in grapevines (Vitis spp.). Three main types of isolates have been described (fanleaf, yellow mosaic, and vein banding), inducing differential symptomatology in the grapevine host (1). The virus has been reported as one of the most widespread in grapevines, and as the nepovirus of greatest economic importance (2). A fanleaf isolate of GFLV was obtained from a Vitis rupestris cv. St. George from the grapevine collection maintained at Davis. It was further propagated in and purified from Chenopodium quinoa according to Jimenez (3). Genomic RNAs were isolated from purified particles of the fanleaf isolate of the virus and cDNA was synthesized and cloned in vector pCGN 1703 (Calgene, Inc.) following the cloning methodology of Alexander (4). Recombinant clones were identified as corresponding to RNA 1 or 2 according to length and restriction map. Both strands of the cDNA insert of a recombinant clone have been completely sequenced. This cDNA is 2304 bp long, excluding the poly(A) tail, and corresponds approximately to the 3' most two thirds of the viral RNA2. The sequencing method was chain termination using the Sequenase Kit (U.S. Biochemicals). The sequence contains a single long open reading frame and 211 nucleotides of untranslated region, preceding a poly(A) tail of length greater than 75 As. The comparison of the sequence of the coat protein of this isolate with that of isolate F 13 (5) is particularly interesting considering the differences in induced symptoms. F13 is able to encapsulate a satellite RNA, possibly responsible for the severity of symptoms and lack of recovery of GFLV-infected Chenopodium quinoa plants (6). The fanleaf isolate from which we have synthesized cDNA, shows no evidence of an encapsidated satellite, and induces an infection from which C. quinoa is able to recover. The analysis reveals 90% identity with the sequence of GFLV isolate F13 at the nucleotide level and shows only a single unmatched residue in the untranslated region. group,
*
EMBL
accession no.
X60775
By comparison with the deduced amino acid sequence of GFLV isolate F13, the amino terminus of the capsid protein can be assigned to the Gly in position -504 from the carboxy terminus, corresponding to an Arg/Gly cleavage site already proposed for this nepovirus (5). Thus the capsid protein would be 504 aa long and would have a calculated MW of 55,918. At the amino acid level, there is 96% homology between the deduced capsid proteins from the two isolates (14 conservative changes out of 22 in 504 residues). No changes have been found in the 192 amino acids of the deduced polypeptide sequence preceding the capsid
protein. The high degree of homology found between the capsid proteins is in good correlation with the lack of immunological heterogeneity reported for this virus (1). It remains to be seen if some of the detected changes between isolates are related to their ability to encapsidate satellite RNA.
ACKNOWLEDGEMENTS The work in Spain has been supported by Grant AGRO 890078 from the Spanish Granting Agency CICYT. C. Chay and M.J. Borja were holders of predoctoral fellowships from the McKnight Foundation and INIA, respectively.
REFERENCES 1. Hewitt,W.B., Martelli,G., Dias,H.F. and Taylor,R.H. (1970) Descriptions of plant viruses No. 28. Commonwealth Mycological Institute/Association of Applied Biology. 2. Murant,A.F. (1981) In Kurstak,E. (ed.), Handbook of Plant Virus Infections and Comparative Diagnosis. Elsevier/North Holland Biomedical Press. Amsterdam, pp. 197-238. 3. Jimenez,F. (1984) Ph.D. thesis. University of California at Davis, 78pp. 4. Alexander,D.C. (1987) Methods in Enzymology 154, 41-64. 5. Serghini,M.A., Fuchs,M., Pinck,M., Reinbolt,J., Walter,B. and Pinck,L. (1990) J. Gen. Virol. 71, 1433- 1441. 6. Pinck,L., Fuchs,M., Pinck,M., Ravelonandro,M. and Walter,B. (1988) J. Gen. Virol. 69, 233-239.
To whom correspondence should be addressed
'On leave of absence at Plant Molecular Biology Center. Northern Illinois University, Dekalb, IL 60115. USA
