Molecular characterization of Citrus tatter leaf virus historically associated with Meyer lemon trees: complete genome sequence and development of biologically active in vitro transcripts.

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3' end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wild-type virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22 degrees C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5' positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction.

Characterization of the 5'- and 3'-terminal subgenomic RNAs produced by a capillovirus: Evidence for a CP subgenomic RNA.

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein (MP), located within ORF1 in a different reading frame. Organization of the CP sequence as part of the replicase ORF is unusual in capilloviruses. In this study, we examined the capillovirus genome expression strategy by characterizing viral RNAs produced by Citrus tatter leaf virus (CTLV), isolate ML, a Capillovirus. CTLV-ML produced a genome-length RNA of approximately 6.5-kb and two 3'-terminal sgRNAs in infected tissue that contain the MP and CP coding sequences (3'-sgRNA1), and the CP coding sequence (3'-sgRNA2), respectively. Both 3'-sgRNAs initiate at a conserved octanucleotide (UUGAAAGA), and are 1826 (3'-sgRNA1) and 869 (3'-sgRNA2) nts with 119 and 15 nt leader sequences, respectively, suggesting that these two 3'-sgRNAs could serve to express the MP and CP. Additionally, accumulation of two 5'-terminal sgRNAs of 5586 (5'-sgRNA1) and 4625 (5'-sgRNA2) nts was observed, and their 3'-termini mapped to 38-44 nts upstream of the transcription start sites of 3'-sgRNAs. The presence of a separate 3'-sgRNA corresponding to the CP coding sequence and its cognate 5'-terminal sgRNA (5'-sgRNA1) suggests that CTLV-ML produces a dedicated sg mRNA for the expression of its CP.