The nucleotide sequence of the genomic RNA2 (3774 nucleotides) of grapevine fanleaf virus strain F13 was determined from overlapping cDNA clones and its genetic organization was deduced. Two rapid and efficient methods were used for cDNA cloning of the 5' region of RNA2. The complete sequence contained only one long open reading frame of 3555 nucleotides (1184 codons, 131K product). The analysis of the Nterminal sequence of purified coat protein (CP) and identification of its C-terminal residue have allowed the CP cistron to be precisely positioned within the polyprotein. The CP produced by proteolytic cleavage at the Arg/Gly site between residues 680 and 681 contains 504 amino acids (Mr 56019) and has hydrophobic properties. The Arg/Gly cleavage site deduced by N-terminal amino acid sequence analysis is the first for a nepovirus coat protein and for plant viruses expressing their genomic RNAs by polyprotein synthesis. Comparison of GFLV RNA2 with M RNA of cowpea mosaic comovirus and with RNA2 of two closely related nepoviruses, tomato black ring virus and Hungarian grapevine chrome mosaic virus, showed strong similarities among the 3' non-coding regions but less similarity among the 5' end non-coding sequences than reported among other nepovirus RNAs.

SUMMARYThe F 13 isolate of grapevine fanleaf virus (GFLV) differs from other isolates in that it induces severe symptoms on Chenopodium quinoa. We show here that its particles contain three RNA species with sizes, estimated by electrophoresis in agarose gels containing formaldehyde, of 6800 nucleotides (RNA 1), 3900 nucleotides (RNA2) and 1150 nucleotides (RNA3). The three RNA species are polyadenylated and probably have a genome-linked protein at their 5' end. RNA1 and RNA2 are known to be genomic RNAs. Evidence for the satellite nature of RNA3 came from Northern blot analysis with DNA probes. A probe originating from the 3' end region of RNA3 and corresponding to one-third of the molecule did not hybridize with either RNA1 or RNA2. Conversely 3'-terminal cDNA probes of RNA1 and RNA2 did not hybridize significantly to RNA3. Further proof of the satellite nature of RNA3 is that it depends on RNA1 and RNA2 for its multiplication in C. quinoa. RNA3 acts as mRNA in wheatgerm extract and directs the synthesis of a Mr 39000 protein.

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

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