Little cherry virus-2: sequence and genomic organization of an unusual member of the Closteroviridae.

The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.

Complete nucleotide sequence of cherry necrotic rusty mottle virus.

A virus closely related to cherry green ring mottle virus (CGRMV) was isolated from a tree displaying typical symptoms of cherry necrotic rusty mottle disease. We have named this virus cherry necrotic rusty mottle virus (CNRMV) and report here its complete genomic sequence as determined from overlapping cloned cDNAs. CNRMV has a genome of 8,432 nucleotides excluding the 3' poly(A) sequence and codes for 7 significant open reading frames (ORFs). Five of these ORFs are conserved among all fovea-, allexi-, potex- and carlaviruses and code for a methyltransferase/helicase/polymerase polypeptide, the triple gene block movement proteins and the coat protein. Two further ORFs, ORFs 2a and 5a, are nested completely within ORFs 2 and 5, respectively. The putative translation products from these ORFs display sequence similarity with putative translation products from two similarly nested ORFs present in the CGRMV genome. The function of these two ORFs is unknown, nor are they conserved among other related viruses.

Detection and Partial Characterization of a Second Closterovirus Associated with Little Cherry Disease, Little cherry virus-2.

ABSTRACT Little cherry disease (LChD) is a complex and serious viral disease of cherry. Although originally described almost 70 years ago, there has been little progress in identifying the causal agent of the disease due to the difficulty in obtaining purified virus from infected trees. This problem was partially overcome in 1997 when the compete sequence of a closterovirus associated with LChD, Little cherry virus (LChV), was published. This virus could be associated with some, but not all, trees with LChD, indicating that another virus was also involved. We report here the partial characterization of a second closterovirus associated with LChD, Little cherry virus-2 (LChV-2), and in order to differentiate the two LChD-associated viruses, we refer to LChV as Little cherry virus-1 (LChV-1). LChV-2 is a new closterovirus with molecular similarities to Grapevine leafroll-associated virus-1 (GLRaV-1) and GLRaV-3 but only distantly related to LChV-1. Based on limited sequence comparisons, LChV-2 is the same virus previously identified in association with LChD in Canada. In reverse transcription-polymerase chain reaction detection assays using specific oligonucleotide primers to either LChV-1 or LChV-2, 27 of 28 isolates of LChD tested positive to one or both of these viruses originating from Europe and North America. These results would further confirm the association of LChV-2 with LChD. One isolate, however, tested negative to both LChV-1 and LChV-2, indicating that while this report brings us a step closer to understanding LChD, further work is required to confirm the causal agents of LChD.

Nucleotide sequence of tomato ringspot virus RNA1.

The nucleotide sequence of tomato ringspot nepovirus (TomRSV) RNA1 has been determined. TomRSV RNA1 is 8214 nucleotides in length, excluding the 3' poly(A) tail, and contains a single long open reading frame (ORF) of 6591 nucleotides beginning at the first AUG codon at nucleotide position 78. This ORF accounts for 80% of the RNA1 sequence and would give rise to a polyprotein with a predicted molecular mass of 244 kDa. Amino acid sequence comparisons between portions of the TomRSV RNA1-encoded polyprotein and proteins encoded by several members of the picornavirus superfamily have provided information concerning the genomic organization and putative functions of TomRSV-encoded proteins. The putative TomRSV protease retains a conserved histidine residue present in the proteases encoded by members of the como-, poty- and poliovirus groups which is thought to be involved in dipeptide cleavage site recognition. Interestingly, this histidine residue is replaced by a leucine in the proteases of other sequenced nepoviruses. This suggests that the TomRSV protease shares dipeptide cleavage site specificity with that of como-, poty- and picornaviruses rather than the other nepoviruses.

Comparison of the 5' and 3' termini of tomato ringspot virus RNA1 and RNA2: evidence for RNA recombination.

The sequences of the 5' terminal 1140 and 3' terminal 1546 nt of tomato ringspot virus (TomRSV) RNA1 have been determined. These sequences share a high degree of nucleotide sequence similarity with the previously determined TomRSV RNA2 sequence. Eighty-eight percent of the 5' terminal 907 nt of TomRSV RNA1 and RNA2 contain identical nucleotide residues; the first 459 nt are identical at all positions, whereas the next 447 nt are identical at only 75.8% of the nucleotide positions. The region of similarity includes not only the 5' nontranslated leader but also sequence probably encoding polyproteins. The 3' terminal 1533 nt of TomRSV RNA1 and RNA2 are identical and are noncoding. The sequences common to RNA1 and RNA2 account for almost 35% of the total genomic sequence. 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.

Nucleotide sequence of tomato ringspot virus RNA-2.

The sequence of tomato ringspot virus (TomRSV) RNA-2 has been determined. It is 7273 nucleotides in length excluding the 3' poly(A) tail and contains a single long open reading frame (ORF) of 5646 nucleotides in the positive sense beginning at position 78 and terminating at position 5723. A second in-frame AUG at position 441 is in a more favourable context for initiation of translation and may act as a site for initiation of translation. The TomRSV RNA-2 3' noncoding region is 1550 nucleotides in length. The coat protein is located in the C-terminal region of the large polypeptide and shows significant but limited amino acid sequence similarity to the putative coat proteins of the nepoviruses tomato black ring (TBRV), Hungarian grapevine chrome mosaic (GCMV) and grapevine fanleaf (GFLV). Comparisons of the coding and non-coding regions of TomRSV RNA-2 and the RNA components of TBRV, GCMV, GFLV and the comovirus cowpea mosaic virus revealed significant similarity for over 300 amino acids between the coding region immediately to the N-terminal side of the putative coat proteins of TomRSV and GFLV; very little similarity could be detected among the non-coding regions of TomRSV and any of these viruses.