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.

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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.

The nucleotide sequence of the genomic RNA 1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-FI 3) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picornavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-FI3 RNAI. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the snbstrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease eofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.

Abstract. The 3' terminal nucleotide of turnip yellow mosaic virus (TYMV) RNA (23-25 S) may be esterified with valine in the presence of ATP and an enzyme preparation from Escherichia coli. The nucleotide composition near the valine-binding site is different for TYMV RNA and tRNAVaL from cabbage, as shown by comparison of the valine adducts of nucleotides labeled with radioactive valine in T1 RNase digests. Consequently, host tRNAVal is not involved in the observed charging of TYMV RNA with valine. The TYMV RNA appears to have a tRNA-like structure, at or near its 3' end, that is recognized by three different enzymes which specifically catalyze reactions involving tRNA.We have recently shown that RNA extracted from Turnip Yellow Mosaic Virus (TYMV) binds valine when incubated with ATP and cell-free extracts from Escherichia coli that are devoid of nucleic acids. The amino acid is bound by an ester linkage to the 3' terminal nucleotide of the RNA molecule, in a manner analogous to that encountered in aminoacyl-tRNAs.I Purified E. coli valyltRNA synthetase catalyses this reaction, but only after the RNA isolated from TYMV has had an AMP residue linked to its 3' end, from an ATP donor, by tRNA nucleotidyltransferase (EC 2.7.7.25; manuscript in preparation).In our previous paper,' we argued against the possibility that valine is bound to host tRNAVal entrapped within the virus particle. We now present experiments that further confirm our conclusion that valine is indeed bound to TYMV RNA. Our results also exclude the existence of a covalent linkage between host tRNAVal, or part of its structure and the 3' end of TYMV RNA. All of our data suggest that the viral RNA bears a tRNAVal-like structure in the vicinity of its 3' terminus.Methods and Materials. Extraction of RNA from TYMV: TYMV, isolated from infected cabbage (Brassica chinensis) leaves, was kindly supplied to us by Dr.L. Bosch (Leiden), Dr. J. M. Bov6 (Versailles), and Dr. L. Hirth (Strasbourg). RNA was extracted from the virus particles by phenol treatments in the presence of bentonite,3 and stored at -30'C in 0.01 M sodium cacodylate, pH 6.Extraction of tRNA from Chinese cabbage: RNA was extracted from uninfected cabbage leaves by the phenol method, in the presence of 0.01 M Tris (pH 7.4)-0.125 mM EDTA-1% sodium dodecylsulfate, and precipitated by ethanol. The RNA that is soluble in 1.5 M NaCl was isolated, incubated with DNase, and chromato-1345