The Nucleotide Sequence of Satellite RNA in Grapevine Fanleaf Virus, Strain F13

Abstract

SUMMARYThe nucleotide sequence of cDNA copies of grapevine fanleaf virus (strain F13) satellite RNA has been determined. The primary structure obtained was 1114 nucleotides in length, excluding the poly(A) tail, and contained only one long open reading frame encoding a 341 residue, highly hydrophilic polypeptide of Mr 37 275. The coding sequence was bordered by a leader of 14 nucleotides and a 3'-terminal noncoding region of 74 nucleotides. No homology has been found with small satellite RNAs associated with other nepoviruses. Two limited homologies of eight nucleotides have been detected between the satellite RNA in grapevine fanleaf virus and those in tomato black ring virus, and a consensus sequence U. G/UGAAAAU/AU/AU/A at the 5' end of nepovirus RNAs is reported. A less extended consensus exists in this region in comovirus and picornavirus RNA.Grapevine fanleaf nepovirus (GFLV) is responsible for one of the most widespread and damaging viral diseases of grapevine. The isometric particles of about 28 nm in diameter are morphologically and serologically indistinguishable. They sediment as three components in sucrose gradients (Quacquarelli et al., 1976). GFLV has a bipartite RNA genome separately encapsidated, and the virion particles of strain F13 also contain a satellite RNA, called RNA3. This RNA is dependent on the presence of the two genomic RNAs for its multiplication, and directs the synthesis in a wheatgerm translation system of a protein (P3) with an apparent Mr of 39K. The satellite RNA has a polyadenylated 3' end, and probably a protein (VPg) linked to the 5' terminus of the molecule, as do both RNAs of the helper virus. No significant nucleotide sequence homology was detected between RNA3 and the genomic RNAs in Northern hybridization experiments (Pinck et al., 1988).We report here the nucleotide sequence of GFLV strain F13 satellite RNA, obtained by the analysis of full-length cloned cDNA copies. The primary structures of this satellite RNA and its encoded protein were compared with those determined for the different satellites of tomato black ring virus (TBRV) isolates (Hemmer et al., 1987) and other satellite RNAs.GFLV strain F 13, first isolated from a grapevine, Vitis vinifera cv. Muscat, near Frontignan in the south of France (Boubals, 1962), was propagated in Chenopodium quinoa. Virus purification was as in Pinck et al. (1988). Nucleic acids were extracted from purified virus by the conventional SDS-phenol method and concentrated by ethanol precipitation before cloning. cDNA copies were synthesized and cloned according to the method of Heidecker & Messing (1983) with slight modifications. Viral RNAs (5 ~tg) were annealed to linearized oligo(dT)-tailed pUC9 plasmid'DNA. The reaction mixture for the first strand synthesis contained 70 mM-Tris-HC1 pH 8.3, 10 mM-MgCI2, 70 mM-KC1, 2 mM-dithiothreitol, 25 gg/ml actinomycin D, 1 I-tg t Present address: Station de Pathologie V6g6tale, I.N.R.A., Domaine de la Grande Ferrade, 33 i 40 Pont de la Maye, France. 0000-8556 © 1989 SGM

Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1

“…The global similarity between these two sequences is close to 80%, with the highest degree of homology within the 130 first nucleotides. The sequence UGAAAAUUU from nt 2 to 10 is part of the 5' consensus sequence already reported for nepovirus RNAs (Fuchs et al, 1989).…”

“…The length of GFLV RNAs was estimated at about 6800 nucleotides (nt) upon gel electrophoresis under denaturing conditions for RNA1 (Pinck et al, 1988) and at 3774 nt for RNA2 after cloning and complete sequencing (Serghini et al, 1990). An additional smaller satellite RNA (Pinck et al, 1988) has also been identified and is composed of 1114 nt (Fuchs et al, 1989). When translated in vitro in a wheatgerm extract each viral RNA induces the synthesis of a polyprotein corresponding to its entire coding capacity, with apparent Mr values of 225K for RNA1 (Pinck et al, 1988), 122K for RNA2 (Serghini et al, t Permanent address: Escola Tecnica Federal de Quimica do Rio de Janeiro, 121 Rua Senador Furtado, 20270 Rio de Janeiro, Brasil.…”

“…0001-0366 © 1991 SGM 1990) and 37K for RNA3 (Fuchs et al, 1989). Studies on the distribution of genetic functions between genome segments of nepoviruses have shown that RNAI is able to replicate independently in protoplasts (Robinson et al, 1980) and thus encodes the information necessary for viral replication.…”

Biologically Active Transcripts of a Large Satellite RNA from Arabis Mosaic Nepovirus and the Importance of 5' End Sequences for its Replication

“…Since M2R was derived from MaR by cleavage at the unique XmnI site (position 8, Fig. I a), thereby breaking the VpG consensus sequence identified by Fuchs et al (1989), MzR lacked these eight nucleotides at the 5' end. M3R contains the authentic satellite RNA sequence.…”

“…Furthermore, the 5'-terminal 11 nucleotides of this satellite RNA (UAUGAAAAAUU) fit the 5' end consensus sequence of nepoviral RNAs noted by Fuchs et al, (1989). ArMV satellite RNA encodes a polypeptide of 360 amino acids with an Mr of 39K (Liu et al, 1990), which is the major translation product in rabbit reticulocyte lysates (Hellen, 1987).…”