Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus.

Abstract

The 5h-terminal genomic region (8597 nt) of little cherry virus (LChV), a mealybug-borne closterovirus, was cloned from double-stranded RNA, and its sequence determined to complete the 16 934 nt sequence of the monopartite LChV RNA genome. In the 5h to 3h direction, the sequence encompasses ORF 1a, encoding the conserved replicative domains of methyltransferase and helicase, and ORF 1b, encoding RNA polymerase. ORFs 1a and 1b partially overlap (in 0/M 1 configuration), and the LChV replicase is probably expressed by ribosomal frameshifting as a fusion product with a molecular mass of 318 kDa. The N-terminal part of the ORF 1a product contains a papain-like cysteine proteinase (PCP) domain with a predicted cleavage site between Gly-619 and Ser-620. The PCP and the upstream protein domains can be aligned with the equivalent parts of the leader proteins encoded by the whitefly-transmitted lettuce infectious yellows and sweet potato sunken vein closteroviruses. Phylogenetic reconstruction based on the aligned RNA polymerase sequences clearly suggests that the aphid-transmissible and whiteflytransmissible closteroviruses represent two distinct evolutionary lineages, with the mealybug-transmissible LChV being the most remote member of the ' whitefly ' lineage.Little cherry disease (LCD) is distributed worldwide in ornamental and sweet cherry and has a great impact on the fruit quality of infected trees. In infected phloem cells,

Nucleotide sequence of the 3'-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus.

“…2 a). However, GLRaV-3 was not placed on the same branch as LChV, the other mealybugtransmissible closterovirus (Jelkmann et al, 1997). Rather, LChV was consistently shown to have a closer relationship with the whitefly-transmissible LIYV, using either of the phylogenetic analysis programs (MegAlign and PHYLIP).…”

“…As first suggested for the expression of ORF1b in BYV (Agranovsky et al, 1994) and later observed in CTV , LIYV (Klaassen et al, 1995), BYSV (Karasev et al, 1996) and LChV (Jelkmann et al, 1997), a j1 ribosomal frameshift mechanism may also be used for the expression of an ORF1a\1b fusion protein in GLRaV-3. However, the socalled ' slippery ' GGGUUU sequence and the stem-loop structure that were proposed to be involved in the BYV frameshift were absent from the GLRaV-3 ORF1a\1b overlap.…”

“…Based on the presence of certain common structures in genome organization, Dolja et al (1994) first proposed the division of a closterovirus genome into four modules : the accessory module of proteinase and vector-specificity factor, the core module of three essential elements for virus replication, methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp), the molecular chaperone module of cellular heat shock protein 70 (HSP70) homologue and a distantly related heat shock protein 90 (HSP90) homologue, and the structural module of coat protein (CP) and its duplicate (CPd). This common genome structure has been observed in all monopartite aphidtransmissible, bipartite whitefly-transmissible, and monopartite mealybug-transmissible closteroviruses (Agranovsky et al, 1994 ;Karasev et al, 1995Karasev et al, , 1996Klaassen et al, 1995 ;Jelkmann et al, 1997).…”

“…genome sequence information becomes available ; for example, the genome sequence of the alleged mealybug-transmissible little cherry closterovirus (LChV) has revealed a typical monopartite genome structure with a relatively large coat protein (46 kDa) (Keim-Konrad & Jelkmann, 1996 ;Jelkmann et al, 1997). Other aphid-or whitefly-transmissible closteroviruses generally have a smaller coat protein, ranging from 22 to 26 kDa (Dolja et al, 1994 ;Agranovsky et al, 1996).…”

“…Four closterovirus genomes have been completely sequenced (Agranovsky et al, 1994 ;Karasev et al, 1995 ;Klaassen et al, 1995 ;Jelkmann et al, 1997). Based on the presence of certain common structures in genome organization, Dolja et al (1994) first proposed the division of a closterovirus genome into four modules : the accessory module of proteinase and vector-specificity factor, the core module of three essential elements for virus replication, methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp), the molecular chaperone module of cellular heat shock protein 70 (HSP70) homologue and a distantly related heat shock protein 90 (HSP90) homologue, and the structural module of coat protein (CP) and its duplicate (CPd).…”