COAT PROTEIN GENE SEQUENCE OF AN AUSTRIAN ISOLATE OF GRAPEVINE FANLEAF VIRUS

Sabine Brandt*1, Martin Ibl*2 and Gottfried Himmler*1

*1 Institut für Angewandte Mikrobiologie
   Universität für Bodenkultur
   Nußdorfer Lände 11
   A- 1190 Wien 

*2 CODON Genetic Systems, 
   Weiden am See, Austria

SUMMARY

The nucleotide sequence of the coat protein cistron of an Austrian isolate of grapevine fanleaf virus (GFLV-FC) from Vitis vinifera cv. French Colombard was determined. It shows small differences at the RNA level as well as at the protein level compared to the sequences of already published grapevine fanleaf virus strains. The existing differences may be a result of the natural variations among virus populations or a consequence of selection in a special host plant. As the virus RNA sequence reported here was directly isolated from its natural ligneous host by an immunocapture PCR technique, passage of the virus through a herbaceous host could be avoided and a possible bias introduced by a different host environment was excluded. The sequence similarity of known GFLV coat protein cistrons to the sequence described is as high as among the other strains.

METHODS

Since immunocapture RT-PCR (IC/PCR) proved to be a reliable method for GFLV detection (Brandt et al., 1993), it was used to amplify the coat protein gene from an Austrian GFLV isolate for sequencing purposes. Following sap extraction from naturally GFLV-infected grapevine shoots, immunocapture of the virus, denaturation of the proteins and reverse transcription of the thus released RNA, the synthesized cDNA was amplified by PCR, using oligonucleotide primers selected from homologous RNA2 regions of the GFLV strains "Fanleaf" (Sanchez et al., 1991) and "F13" (Serghini et al., 1990), which spanned the gene. After PCR, the amplified fragments were cloned blunt ended into Sma I-digested and dephosphorylated plasmid vector pBluescript II (Stratagene Inc., La Jolla, USA). Electrocompetent E. coli strain MC 1061 was used for transformation. Purified plasmid-DNA of two individual clones was sequenced according to the Taq-Cycle sequencing protocol for the automated sequencer 373A (Applied Biosystems Inc., Foster City, USA) using Dye-deoxynucleotides and the universal primers "M13-21" and "M13 reverse" respectively.

DISCUSSION

The complete procedure, starting from the sampling of the shoots to the sequencing, was repeated to avoid any bias introduced by nonstatistical sampling. In this way, both strands of four individual cDNA-clones from two independent IC/PCR experiments were sequenced. There was no difference in the sequences of the various clones. The resulting sequence, named GFLV-FC, is presented to show only the coat protein coding region. This region is compared with respective sequences of already published isolates (Fig. 1).

GFLV-F13        GGATTAGCTGGTAGAGGAGTAATCTACATTCCCAAGGATTGCCAGGCAA  2096  
GFLV-FC         GGATTAGCTGGTAGAGGAGTGATTTATATCCCTAAGGATTGCCAGGCAA
GFLV-Fanleaf    GGATTGGCTGGTAGAGGAGTGGTTTATATCCCTAAGGATTGCCAGGCAA
                     o               o o  o  o  o                               	      	     
GFLV-F13        ATAGATACTTGGGCACCCTGAATATACGTGATATGATCTCAGACTTCAA  2145
GFLV-FC         ATAGATATTTGGGCACCCTGAATATACGTGACATGATCTCAGATTTTAA
GFLV-Fanleaf    ACAGATACTTGGGCACCCTGAACATACGTGATATGATTTTAGATTTTAA
                 o     o              o        o     o o   o  o               	  	    	     
GFLV-F13        GGGTGTCCAGTATGAAAAGTGGATAACTGCAGGATTAGTCATGCCTACT  2194
GFLV-FC         GGGTGTTCAGTATGAGAAGTGGATAACCGCGGGATTAGTCATGCCTATT
GFLV-Fanleaf    GGGTGTTCAGTATGAAAAGTGGATAACTGCAGGATTGGTCATGCCTATT
                      o        o           o  o     o          o
	    
GFLV-F13        TTCAAGATAGTTATTAGGCTACCTGCAAATGCCTTTACTGGATTGACAT  2243
GFLV-FC         TTTAAGATAATTGTCAGGCTACCTGCGAATGCCTTTACTGGATTGACTT
GFLV-Fanleaf    TTTAAGATAGTTGTTAGGCTACCTGCAAATGCCTTTACCGGATTGACGT
                  o      o  o o           o           o        o               	      	     
GFLV-F13        GGGTGATGAGCTTTGATGCTTATAACCGGATAACTAGTAGAATTACTGC  2292
GFLV-FC         GGGTGATGAGTTTTGATGCTTATAATCGGATAACTAGCAGAATCACTGC
GFLV-Fanleaf    GGGTGATGAGTTTTGATGCTTATAACCGGATAACTAGTAGAATCACTGC
                          o              o           o     o                  	  	    	     
GFLV-F13        TAGTGCGGATCCTGTATACACCTTGTCAGTCCCACATTGGCTTATCCAC  2341
GFLV-FC         TAGTGCAGATCCTGTGTATACCCTGTCGGTCCCACATTGGCTTATCCAT
GFLV-Fanleaf    TAGTGCAGATCCTGTGTACACCCTATCAGTTCCACATTGGCTTATCCAC
                      o        o  o   o o  o  o                 o
           
GFLV-F13        CATAAGTTGGGCACGTTTTCATGTGAGATAGACTATGGAGAATTGTGTG  2390
GFLV-FC         CACAAGTTGGGCACGTTTTCATGTGAAGTGGACTATGGAGAGTTGTGTG
GFLV-Fanleaf    CATAAGTTGGGCACGTTTTCATGTGAAATAGACTATGGAGAGTTGTGCG
                  o                       o o                 o               	 	     	     
GFLV-F13        GTCATGCTATGTGGTTTAAATCAACCACATTTGAATCTCCAAGGTTGCA  2439
GFLV-FC         GTCATGCTATGTGGTTCAAGTCCACGACATTTGAATCTCCACGGCTACA
GFLV-Fanleaf    GTCATGCTATGTGGTTCAAGACCACGACATTTGAATCTCCAAGGTTGCA
                                o  oo o  o               o  o o 
	    
GFLV-F13        TTTCACGTGTTTAACGGGCAACAACAAAGAGTTAGCGGCAGACTGGCAA  2488
GFLV-FC         TTTCACATGTCTAACGGGCAACAACAAAGAGTTGGCGGCAGACTGGCAA
GFLV-Fanleaf    TTTCACGTGCCTAACGGGTAACAATAAAGAGTTGGCGGCAGACTGGCAA
                      o  oo       o     o        o             
           
GFLV-F13        GCTGTCGTAGAACTATATGCCGAATTGGAAGAGGCCACTTCTTTCCTTG  2537
GFLV-FC         GCTGTCGTAGAACTATATACCGAATTGGAAGAGGCCACTTCTTTTCTTG
GFLV-Fanleaf    GCTGTTGTCGAACTTTATGCTGAATTGGAAGAGGCCACTTCTTTTCTTG
                     o  o     o   o o                       o  
	    
GFLV-F13        GGAAACCAACTTTGGTTTTTGACCCAGGTGTTTTCAATGGCAAATTTCA  2586
GFLV-FC         GGAAACCAACTTTGGTTTTTGACCCAGGGGTGTTCAATGGTAAATTCCA
GFLV-Fanleaf    GGAAACCAACTTTGGTTTTTGACCCAGGTGTTTTTAATGGTAAATTCCA
                                            o  o  o     o     o                	      	     
GFLV-F13        ATTTCTTACTTGCCCTCCCATATTCTTTGATTTAACGGCCGTCACGGCC  2635
GFLV-FC         ATTTTTAACTTGCCCTCCCATATTCTTTGATTTAACAGCCGTCACGGCT
GFLV-Fanleaf    ATTTCTCACTTGCCCTCCCATATTCTTCGATTTGACGGCCGTCACGGCT
                    o o                    o     o  o           o
           
GFLV-F13        CTTAGGAGTGCTGGGCTGACATTGGGGCAAGTCCCAATGGTTGGCACCA  2684
GFLV-FC         CTCAAGAGTGCTGGGCTGACATTGGGACAAGTCCCAATGGTTGGCACCA
GFLV-Fanleaf    CTTAAGAGTGCTGGGCTGACATTGGGACAAGTCCCAATGGTTGGCACTA
                  o o                     o                    o
           
GFLV-F13        CTAAGGTTTATAATCTAAACAGCACTCTTGTGAGTTGTGTTTTGGGTAT  2733
GFLV-FC         CCAAGGTTTATAATTTAAACAGTACTCTTGTGAGTTGTGTTTTGGGTAT
GFLV-Fanleaf    CTAGGGTTTATAATCTAAATAGCACTCTCGTAAGCTGTGTTTTGGGTAT
                 o o          o    o  o     o  o  o       
          
GFLV-F13        GGGAGGTACTGTTAGAGGGAGGGTGCACATTTGTGCGCCAATCTTCTAC  2782
GFLV-FC         GGGGGGTACTATTAGAGGAAAAGTACACATTTGTGCACCAATCTTCTAT
GFLV-Fanleaf    GGGAGGTACTATTAGAGGAAAGGTGCACATTTGTGCTCCAATCTTCTAT
                   o      o       o oo  o           o           o

GFLV-F13        AGTATTGTTTTATGGGTCGTTAGTGAGTGGAACGGGACCACTATGGACT  2831 
GFLV-FC         AGTATTGTTTTGTGGGTTGTTAGCGAGTGGAACGGGACCACTATGGATT
GFLV-Fanleaf    AGTATTGTTTTGTGGGTCGTTAGTGAGTGGAACGGGACCACTATGGATT
                           o     o     o                       o
           
GFLV-F13        GGAATGAACTTTTTAAGTATCCCGGGGTGTATGTGGAAGAGGACGGAAG  2880
GFLV-FC         GGAATGAACTTTTTAAGTATCCCGGGGTGTATGTGGAAGAGGATGGAAG
GFLV-Fanleaf    GGAATGAACTTTTTAAGTATCCCGGGGTGTATGTGGAAGAGGACGGAAG
                                                           o                 
	     
 GFLV-F13        TTTTGAAGTAAAGATTCGCTCTCCATATCACCGAACTCCTGCCAGATTG  2929
GFLV-FC         TTTTGAAGTTAAAATTCGCTCTCCATATCACCGAACGCCTGCTAGATTG
GFLV-Fanleaf    TTCTGAAGTCAAAATTCGCTCTCCATATCACCGAACGCCTGCTAGATTG
                  o      o  o                       o            
           
GFLV-F13        CTTGCTGGTCAAAGTCAGAGAGACATGAGCTCTCTTAATTTTTATGCAA  2978
GFLV-FC         CTTGCTGGTCAAAGTCAGAGAGACATGAGCTCTCTAAATTTTTATGCAA
GFLV-Fanleaf    CTTGCTGGTCAAAGTCAGAGGGACATGAGCTCTCTAAATTTTTACGCAA
                                 o              o        o
           
GFLV-F13        TAGCAGGACCTATCGCTCCTTCGGGTGAGACTGCGCAACTTCCTATTGT  3027
GFLV-FC         TAGCAGGACCCATCGCTCCATCGGGTGAGACCGCACGACTTCCTATCGT
GFLV-Fanleaf    TAGCAGGGCCTATCGCTCCATCGGGTGAGACTGCGCGACTTCCTATCGT
                       o  o        o           o  o o         o
           
GFLV-F13        TGTGCAGATAGATGAAATTGTGCGCCCAGATCTCTCTTTACCAAGTTTT  3076
GFLV-FC         TGTACAGATTGAGGAAATTGTGCGTCCAGACTTCTCTCTACCGAGTTTT
GFLV-Fanleaf    TGTGCAGATTGAAGAAATTGTGCGCCCGGACTTATCTCTACCAAGCTTT
                   o     o  o           o  o  oo o   o    o  o
           
GFLV-F13        GAAGATGACTATTTCGTATGGGTGGATTTTTCTGAATTCACTCTTGATA  3125
GFLV-FC         GAAGATGATTATTTTGTGTGGGTGGATTTTTCTGAATTTACCCTTGATA
GFLV-Fanleaf    GAAGATGATTATTTTGTATGGGTGGATTTTTCTGAGTTCACCCTTGACA
                        o     o  o                 o  o  o     o
           
GFLV-F13        AAGAAGAAATTGAGATTGGTTCTCGTTTCTTCGATTTCACTTCGAATAC  3174
GFLV-FC         AAGAAGAAATCGAAATTGGATCTCGTTTCTTTGATTACACTTCAAATAC
GFLV-Fanleaf    AAGAAGAAATTGAGATTGGATCTCGTTTTTTCGATTATACTTCAAGCAC
                          o  o     o        o  o    oo     o oo 
           
GFLV-F13        TTGTAGGGTATCTATGGGTGAAAATCCGTTTGCTGCAATGATTGCCTGC  3223
GFLV-FC         TTGTAGGGTGTCCATGGGGGAAAACCCATTTGCTGCAATGATTGCCTGT
GFLV-Fanleaf    TTGTAGAGTGTCTATGGGAGAAAATCCGTTTGCTGCAATGATTGCCTGT
                      o  o  o     o     o                       o
           
GFLV-F13        CATGGATTGCATAGTGGTGTATTAGACCTCAAACTCCAATGGAGTCTGA  3272
GFLV-FC         CATGGATTGCATAGCGGTGTTTTAGACCTCAAATTCCAATGGAGTTTAA
GFLV-Fanleaf    CATGGATTACATAGTGGTGTTCTGGATCTCAAATTTCAGTGGAGTTTGA
                        o     o     oo o  o      o o  o      o o
           
GFLV-F13        ACACCGAATTCGGCAAGAGCAGCGGGAGCGTTACCATCACGAAGCTGGT  3321
GFLV-FC         ACACCGAATTTGGTAAGAGCAGCGGGAGCGTTATTATCACGAAACTAGT
GFLV-Fanleaf    ACACCGAATTTGGCAAGAGCAGCGGGAGCGTTACTATCACAAAACTGGT
                          o  o                   oo     o  o  o
           
GFLV-F13        GGGTGATAAGGCCATGGGTCTGGACGGACCTTCTCACGTTTTTGCCATA  3370 
GFLV-FC         GGGTGATAAAGCCACGGGTTTGGATGGGCCTTCACAGGTCTTTGCCATA
GFLV-Fanleaf    GGGTGATAAAGCCACGGGTCTGGACGGGCCTTCACAGGTTTTTGCCATA
                         o    o    o    o  o     o  o  o     
           
GFLV-F13        CAAAAACTAGAGGGAACTACAGAGTTGTTGGTTGGGAATTTTGCAGGAG  3419
GFLV-FC         CAGAAACTAGAGGGAGCTACGGAATTGTTGATTGGGAATTTTGCAGGAG
GFLV-Fanleaf    CAAAAACTAGAGGGAGCTACAGAATTGTTGATTGGGAATTTTGCAGGAG
                  o            o    o  o      o               
           
GFLV-F13        CAAACCCAAACACTCGTTTTTCCCTTTATAGTCGCTGGATGGCAATTAA  3468
GFLV-FC         CAAACCCAAATACTCATATCTCCCTCTATAGTCGATGGATGGCAATTAA
GFLV-Fanleaf    CAAACCCAAATACTCATATCTCCCTCTATAGTCGGTGGATGGCAATTAA
                          o    o o o     o        o        
           
GFLV-F13        ATTGGATCAAGCAAAGAGTATTAAAGTACTCCGCGTTTTGTGCAAGCCC   3509
GFLV-FC         GCTAGACCAAGCAAAGAGCATTAAAGTGCTCCGTGTTTTGTGCAAGCCG
GFLV-Fanleaf    ACTGGATCAAGCAAAGAGTATTAGAGTACTCCGTGTTTTGTGTAAGCCT
                oo o  o           o    o   o     o        o     o
	    
GFLV-F13        CGTCCAGGCTTTAGCTTTTATGGTAGAACCAGTTTCCCAGTCTAG  3554  
GFLV-FC         CGTCCAGGCTTCAGTTTTTATGGAAGAACTAGTTTCCCAGTCTAG
GFLV-Fanleaf    CGTCCAGGTTTCAGTTTTTATGGAAGAACCAGTTTTCCAGTCTAG
                        o  o  o         o     o     o
                o = differences between the three sequences

Fig 1. Alignment of the coat protein cistron cDNA sequences of three 
       GFLV isolates. GFLV-FC (this paper), GFLV-Fanleaf and GFLV-F13 
       (Position numbering according to Serghini et al.)

A comparison of the translation products of the coat protein genes is shown in Fig. 2. It can be seen that most of the nucleotide differences at RNA level represent silent mutations. At protein level, most of the amino acid differences are changes within the same amino acid group (Fig. 2).

GFLV-F13        GLAGRGVIYIPKDCQANRYLGTLNIRDMISDFKGVQYEKWITAGLVM  727
GFLV-FC         GLAGRGVIYIPKDCQANRYLGTLNIRDMISDFKGVQYEKWITAGLVM
GFLV-Fanleaf    GLAGRGVVYIPKDCQANRYLGTLNIRDMILDFKGVQYEKWITAGLVM
                       o                     o                 
                          
GFLV-F13        PTFKIVIRLPANAFTGLTWVMSFDAYNRITSRITASADPVYTLSVPH  774	
GFLV-FC         PIFKIIVRLPANAFTGLTWVMSFDAYNRITSRITASADPVYTLSVPH
GFLV-Fanleaf    PIFKIVVRLPANAFTGLTWVMSFDAYNRITSRITASADPVYTLSVPH
                 o   oo                                        

GFLV-F13        WLIHHKLGTFSCEIDYGELCGHAMWFKSTTFESPRLHFTCLTGNNKE  821
GFLV-FC         WLIHHKLGTFSCEVDYGELCGHAMWFKSTTFESPRLHFTCLTGNNKE
GFLV-Fanleaf    WLIHHKLGTFSCEIDYGELCGHAMWFKTTTFESPRLHFTCLTGNNKE
                             o             o                   

GFLV-F13        LAADWQAVVELYAELEEATSFLGKPTLVFDPGVFNGKFQFLTCPPIF  868
GFLV-FC         LAADWQAVVELYTELEEATSFLGKPTLVFDPGVFNGKFQFLTCPPIF
GFLV-Fanleaf    LAADWQAVVELYAELEEATSFLGKPTLVFDPGVFNGKFQFLTCPPIF
                            o                                  

GFLV-F13        FDLTAVTALRSAGLTLGQVPMVGTTKVYNLNSTLVSCVLGMGGTVRG  915
GFLV-FC         FDLTAVTALKSAGLTLGQVPMVGTTKVYNLNSTLVSCVLGMGGTIRG
GFLV-Fanleaf    FDLTAVTALKSAGLTLGQVPMVGTTRVYNLNSTLVSCVLGMGGTIRG
                         o               o                  o  

GFLV-F13        RVHICAPIFYSIVLWVVSEWNGTTMDWNELFKYPGVYVEEDGSFEVK  962
GFLV-FC         KVHICAPIFYSIVLWVVSEWNGTTMDWNELFKYPGVYVEEDGSFEVK
GFLV-Fanleaf    KVHICAPIFYSIVLWVVSEWNGTTMDWNELFKYPGVYVEEDGSSEVK
                o                                          o   

GFLV-F13        IRSPYHRTPARLLAGQSQRDMSSLNFYAIAGPIAPSGETAQLPIVVQ  1009
GFLV-FC         IRSPYHRTPARLLAGQSQRDMSSLNFYAIAGPIAPSGETARLPIVVQ
GFLV-Fanleaf    IRSPYHRTPARLLAGQSQRDMSSLNFYAIAGPIAPSGETARLPIVVQ
                                                        o      

GFLV-F13        IDEIVRPDLSLPSFEDDYFVWVDFSEFTLDKEEIEIGSRFFDFTSNT  1056
GFLV-FC         IEEIVRPDFSLPSFEDDYFVWVDFSEFTLDKEEIEIGSRFFDYTSNT
GFLV-Fanleaf    IEEIVRPDLSLPSFEDDYFVWVDFSEFTLDKEEIEIGSRFFDYTSST
                 o      o                                 o  o 

GFLV-F13        CRVSMGENPFAAMIACHGLHSGVLDLKLQWSLNTEFGKSSGSVTITK  1103
GFLV-FC         CRVSMGENPFAAMIACHGLHSGVLDLKFQWSLNTEFGKSSGSVIITK
GFLV-Fanleaf    CRVSMGENPFAAMIACHGLHSGVLDLKFQWSLNTEFGKSSGSVTITK
                                           o               o   

GFLV-F13        LVGDKAMGLDGPSHVFAIQKLEGTTELLVGNFAGANPNTRFSLYSRW  1150
GFLV-FC         LVGDKATGLDGPSQVFAIQKLEGATELLIGNFAGANPNTHISLYSRW
GFLV-Fanleaf    LVGDKATGLDGPSQVFAIQKLEGATELLIGNFAGANPNTHISLYSRW
                      o      o         o    o          oo      

GFLV-F13        MAIKLDQAKSIKVLRVLCKPRPGFSFYGRTSFPV  1184
GFLV-FC         MAIKLDQAKSIKVLRVLCKPRPGFSFYGRTSFPV
GFLV-Fanleaf    MAIKLDQAKSIRVLRVLCKPRPGFSFYGRTSFPV
                           o                      

Fig.2. Alignment of the coat protein sequences of the three 
       GFLV isolates GFLV-FC (this paper), GFLV-Fanleaf and GFLV-F13 
       (Position numbering according to Serghini et al.)

o = differences between two of the three sequences

The sequence of the GFLV-FC coat protein cistron was established from virus directly isolated from a naturally infected, ligneous host plant and could therefore be regarded as more definitive than sequences from viruses isolated from an experimental herbaceous host. However, as the percentage of similarity is almost the same between the three strains (Table 1), it is probable that the herbaceous host passage of the virus isolates has a minimal effect on genetic variation.

Similarity between:		RNA	      Protein
GFLV-Fanleaf a)/GFLV-FC b)	91.45% 	      97.62%
GFLV-F13 c)/GFLV-FC		89.97% 	      96.04%
GFLV-F13/GFLV-Fanleaf		90,52 %       95.63%

a) reference Sanchez et al.
b) this paper
c) reference Serghini et al.

Table 1. Percentage of similarity between three GFLV coat proteins and coat protein gene sequences.

ACKNOWLEDGEMENTS

We thank Eva Obermayr and Annelies Klima for technical support. This work was funded by the Förschungsförderungsfonds für die Gewerbliche Wirtschaft and the Federal State of Lower Austria.

REFERENCES

Brandt, S., Himmler, G., and Katinger, H. (1993). Anwendung der Immunocapture Polymerase Chain Reaction (IC/PCR) für den Nachweis von Rebviren aus holzigem Material. Mitteilungen Klosterneuburg 43, 143-147.

Matthews, R. E. F. (1991) Plant Virology. 3rd ed. Academic Press, New York. Sanchez, F., Chay, C., Borja, M. J., Rowhani, A., Romero, J., Bruening, G., and Ponz, F. (1991). cDNA sequence of the capsid protein gene and 3´-untranslated region of a fanleaf isolate of grapevine fanleaf virus. Nucleic Acids Res. 19, 5440.

Serghini, M. A., Fuchs, M., Pinck, M., Reinbolt, J., Walter, B., and Pinck, L. (1990). RNA2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location. J. Gen. Virol. 71, 1433-1441.

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