Arch Virol (2005) 150: 2403–2406 DOI 10.1007/s00705-005-0619-5
Complete nucleotide sequence of an Olive latent virus 1 isolate from olive trees Annotated Sequence Record M. R. F´elix1,3 , J. M. S. Cardoso2,3 , C. M. R. Varanda2,3 , S. Oliveira2,3 , and M. I. E. Clara1,3 ´ de Sanidade Animal e Vegetal, Universidade de Evora, ´Evora, Portugal 2 Departamento de Biologia, Universidade de Evora, ´ ´ Evora, Portugal 3 ICAM, Instituto de Ciˆ encias Agr´arias Mediterrˆanicas, ´ ´ Universidade de Evora, Evora, Portugal
1 Departamento
Received June 23, 2005; accepted July 7, 2005 c Springer-Verlag 2005 Published online August 23, 2005
Introduction Olive latent virus 1 (OLV-1) is a necrovirus belonging to the family Tombusviridae. It is a small icosahedral plant virus, which encapsidates a single stranded positivesense RNA. This virus was first isolated from symptomless olive trees in Italy [7] and afterwards in Jordan and Portugal [10, 4]. OLV-1 was also isolated from symptomatic hosts, such as citrus trees in Turkey [11] and tulips in Japan [9]. Up to now, only one complete genome sequence of an OLV-1 citrus isolate has been determined [8]. This report describes the first full genomic sequence of an OLV-1 isolated from olive trees. Provenance of the virus material GM6 isolate was recovered from fruits of a Portuguese olive tree (cv. Galega vulgar), propagated in Nicotiana benthamiana plants and purified as described previously [3]. The viral RNA was extracted from purified virus particles, using an adaptation of RNeasy Plant Mini Kit (Qiagen). cDNA clones were obtained after a specific RT-PCR assay using, initially primers GP1 5 and GP1 3 [2] and then primers designed based on OLV-1 citrus sequence and obtained from GM6 isolate. PCR products were purified and cloned as described by F´elix et al. [3]. A RACE protocol [5] was adapted and used for cloning the 5 and 3 -terminals, following oligo dA-tailing. For sequencing the terminals of the sequence, multiple clones
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were used. The sequence obtained has been deposited in the GenBank database under the accession number DQ083996. Sequence properties Comparison of olive GM6 isolate complete sequence with that of OLV-1 citrus isolate (accession number NC 001721), revealed that the full length sequence of olive GM6 is 3 nt longer than that of the citrus isolate (3702 versus 3699 nt, respectively), and that the overall nucleotide sequence identity between them is 55%. The genetic organization of the two OLV-1 isolates is similar, both containing five open reading frames (ORFs) (Fig. 1). The 5 -terminal non-coding region (NCR) has the same length (60 nt) and an identical A + U content (58.4%) with a slightly different base ratio (36.7% A, 23.3% C, 18.3% G, 21.7% U and 35% A, 20% C, 21.6% G, 23.4% U for both olive GM6 and citrus isolates, respectively). This A + U content is consistent with the 5 -leader sequence of other plant viruses [6]. The intercistronic regions (ICR) of the olive GM6 genome are very small, ORF2 is overlapped by 14 nt of ORF3, 5 nt separate ORF3 from ORF4 and a 24 nt long ICR is located between ORF4 and ORF5. The 3 -terminal NCR is 258 nt long. ORF1 of olive GM6 isolate (nt 61 to 666) encodes a putative 202 amino acid (aa) polypeptide with a molecular weight (MW) of 23004 Da (p23) which has only 50.4% of sequence identity with the p23 of citrus isolate (Table 1). The readthrough of its amber termination codon allows the translation to continue downstream up to nt 2232, giving rise to a predicted 724 aa long protein, with a MW of 82484 Da (p82). This was identified as the virus RNA dependent RNA
Fig. 1. Schematic representation of the total genomic organization of the olive GM6 isolate of OLV-1. The main ORFs are shown as boxes and the predicted protein size is indicated
Table 1. Amino acid sequence identity between predicted proteins encoded by olive GM6 isolate genome and those of the citrus isolate Amino acid sequence identity (%) Olive GM6
Citrus isolate
p23
p82 (RdRp)
p8
p6
p30 (CP)
50.4
97.3
93.2
100
87.7
Genome sequence of OLV-1 from olive
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polymerase (RdRp) following a BLAST search [1]. The GDD motif, as well as other typical polymerases sequences [13], was identified. This p82 has 97.3% of aa sequence identity with that of the OLV-1 citrus isolate (Table1). ORF3 (nt 2219–2437) and ORF4 (nt 2443–2610) encode two small peptides, with 73 aa and a MW of 8051 (p8) and 56 aa and a MW of 6268 (p6), respectively, which appear to be involved in the cell to cell movement of virus particles [12]. Sequence identity of olive GM6 p8 and p6 predicted products with those of citrus isolate are 93.2% and 100% respectively (Table 1). The olive GM6 p8 protein differs from that of OLV-1 citrus in 5 aa residues. ORF 5 is located in the 3’-terminal, between the nt 2635 and 3444, encodes a protein with 270 aa long and a MW of 29853 (p30) corresponding to the viral coat protein and it shows a sequence identity of 87.7% with that of citrus isolate (Table 1). These sequence data are in accordance with previous results on identification of olive GM6 isolate as an OLV-1 species based on ELISA reactions and CP gene sequence [3]. However, some remarkable differences between the genomic sequences of these two OLV-1 isolates can be observed. Furthermore, the olive GM6 isolate produces only local symptoms after mechanical inoculation onto N. bentamiana plants, in contrast with the systemic symptoms induced by the citrus isolate in the same host [11]. These differences could be related to the amino acid differences found in the coat protein, which is involved in long distance movement and in the p8 cell to cell movement peptide. Acknowledgements This work was supported by funds of Funda¸ca˜ o para a Ciˆencia e Tecnologia (FCT)/SAPIENS research project (POCTI/AGR/36230/) co-financed by EU-FEDER. The authors would like to thank Maria M´ario Azedo for her excellent technical assistance.
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7. Gallitelli D, Savino V (1985) Olive latent virus 1, an isometric virus with a single RNA species isolated from olive in Apulia, Southern Italy. Annu Appl Biol 106: 295–303 8. Grieco F, Savino V, Martelli GP (1996) Nucleotide sequence of the genome of a citrus isolate of Olive latent virus 1. Arch Virol 141: 825–838 9. Kanematsu S, Taga Y, Morikawa T (2001) Isolation of Olive latent virus 1 from Tulip in Toyama Prefecture. J Gen Plant Pathol 67: 333–334 10. Martelli GP, Sabanadzovic S, Savino V, Abu-Zurayk AR, Masannat M (1995) Virus-like diseases and viruses of olive in Jordan. Phytopathol Medit 34: 133–136 11. Martelli GP,Yilmaz MA, Savino V, Baloglu S, Grieco F, G¨uld¨ur ME, Greco N, Lafortezza R (1996) Properties of a citrus isolate of Olive latent virus 1, a new necrovirus. Eur J Plant Pathol 102: 527–536 12. Pantaleo V, Grieco F, Castellano MA, Martelli GP (1999) Synthesis of infectious transcripts of Olive latent virus 1: genes required for RNA replication and virus movement. Arch Virol 144: 1071–1079 13. Poch O, Sauvaget I, Delarue M, Tordo N (1989) Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J 8: 3867–3874 Author’s address: Maria R. F´elix, Departamento de Sanidade Animal e Vegetal, Univer´ ´ sidade de Evora, Apartado 94, 7002-554 Evora, Portugal; e-mail:
[email protected]
Verleger: Springer-Verlag GmbH, Sachsenplatz 4–6, 1201 Wien, Austria. – Herausgeber: Dr. M. H. V. Van Regenmortel, ´ Ecole Sup´erieure de Biotechnologie de Strasbourg (ESBS), Parc d’Innovation, Boulevard S´ebastian Brandt, 67400 Illkirch, France. – Redaktion: Sachsenplatz 4–6, 1201 Wien, Austria. – Satz und Umbruch: Thomson Press (India) Ltd., Chennai, India. – Offsetdruck: Holzhausen Druck & Medien GmbH, Holzhausenplatz 1, 1140 Wien, Austria. – Herstellungsort: Wien, Austria. – Printed in Austria.
