COMPLETE SEQUENCE OF THE CITRUS TRISTEZA VIRUS-RNA GENOME

The sequence of the entire genome of citrus tristeza virus (CTV), Flor ida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 op en reading frames (ORFs) potentially coding for at least 17 protein pr oducts. The 5'-proximal ORF la starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains cha racteristic of (from 5' to 3') two papain-like proteases (P-PRO), a me thyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows clos terovirus (BYV) and potyviruses allowed the prediction of catalytic cy steine and histidine residues as well as two cleavage sites, namely Va l-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein a t these sites would release two N-terminal leader proteins of 54 and 5 5 kDa, respectively, and a 240-kDa C-terminal fragment containing MT a nd HEL domains. The apparent duplication of the leader domain distingu ishes CTV from BYV and accounts for most of the size increase in the O RF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region s uggests that this +1 frameshift probably occurs at a rare arginine cod on CGG and that elements of the RNA secondary structure are unlikely t o be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleava ge of the two N-terminal leaders would yield a 292-kDa protein contain ing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like su pergroup of positive-strand RNA viruses. Two gene blocks or modules ca n be easily identified in the CTV genome. The first includes the repli cative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7 , conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-te rminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), an d three proteins with unknown functions; this gene array is poorly con served among closteroviruses. The genomic double-stranded CTV RNA had an extra G at the 3' terminus of the minus strand and an extra U at th e 3' terminus of the plus strand. (C) 1995 Academic Press, Inc.