| Summary: | Complementary DNA (cDNA) to tomato ringspot (TomRSV) nepovirus RNA1 and RNA2 was synthesized and cloned. Overlapping cDNA clones corresponding to over 99% of the TomRSV genome were obtained and used to determine the nucleotide sequence of RNA1and RNA2. The 5' termini of RNA1 and RNA2, which were not present in any cDNA clone analyzed, were determined by using viral RNA as a template in dideoxynucleotide sequencing reactions. TomRSV RNA1 is 8,214 nucleotides (nt) in length, excluding the 3'poly(A) tail, and contains a single long open reading frame (ORF) which accounts for 80%of the nucleotide sequence and has the capacity to encode a polyprotein of 244 kDa. Comparisons between the deduced TomRSV RNA1-encoded polyprotein sequence with those encoded by the RNA1 components of the nepoviruses tomato black ring (TBRV),grapevine chrome mosaic (GCMV) and grapevine fan leaf (GFLV), the B RNA component of the plant comovirus cowpea mosaic virus (CPMV), and the genomic RNAs from the plant potyvirus tobacco etch (TEV) and the animal poliovirus, identified a similarly ordered set of conserved amino acid sequence domains in common with these other viruses. The putative functions for the conserved amino acid sequence domains are, in order from the N-terminus, a protease co-factor, NTP-binding domain, VPg, protease, and an RNA-dependent RNA polymerase. Possible cleavage sites have been identified between each of the conserved domains which would release each of the functional domains from the large polyprotein. TomRSV RNA2 is 7273 nt in length excluding the 3' poly(A) tail and contains a single long ORF, accounting for 78% of the nucleotide sequence, with the capacity to encode a polyprotein of 207 kDa. The TomRSV coat protein gene was localized to the 3' end of the ORF by comparison of the TomRSV coat protein amino acid composition to the amino acid composition of all regions of the deduced RNA2 encoded polyprotein sequence and by comparisons with the amino acid sequences of the TBRV, GCMV and GFLV coat proteins which are encoded by their respective RNA2 components. Sequences potentially involved in viral cell-to-cell movement were also localized on the TomRSV RNA2polyprotein sequence, N-terminal of the putative coat protein sequence that may also include a set of three tandem repeats of 38 to 53 amino acids. Extensive nucleotide sequence similarity accounting for almost 35% of the total TomRSV genomic RNA was observed between the 3' termini and between the 5' termini of TomRSV RNA1 and RNA2.Eighty-eight percent of the 5' terminal 907 nt of TomRSV RNA1 and RNA2 are identical and include both coding and noncoding sequences while the 3' terminal 1533 nt of RNA1and RNA2 are identical and noncoding. It is possible that the similar sequences at both ends of TomRSV RNA1 and RNA2 are a result of recombination between these two genomic RNA components during viral replication. Examination of potential translation initiation sites on TomRSV RNA identified two in-frame AUG triplets at position 78 and441. Expression from both sites were assayed in vitro and in protoplasts which identifiedAUG78 as the site of translation initiation. Full-length cDNA clones corresponding toTomRSV RNA1 and RNA2 were constructed and fused to the bacteriophage T7 RNA promoter. RNA transcripts synthesized from constructs in vitro using 17 RNA polymerase were tested for infectivity by inoculation onto Chenopodiunt amaranticolor and assaying for viral symptoms. Transcripts were found to be noninfectious.
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