MOLECULAR BIOLOGY AND EVOLUTION OF CLOSTEROVIRUSES: Sophisticated Build-up of Large RNA Genomes

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,

mentioning

confidence: 72%

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

Wilhelm¹,

Fechtner

Agranovsky³

1997

“…The partial or complete genome sequences of beet yellows virus (BYV) (Agranovsky et al, , 1994, beet yellow stunt virus (BYSV) (Karasev et al, 1996), citrus tristeza virus (CTV) (Pappu et al, 1994 ;Karasev et al, 1995), lettuce infectious yellows virus (LIYV) (Klaassen et al, 1994(Klaassen et al, , 1995, little cherry virus (LChV) (Keim & Jelkmann, 1996 ;Jelkmann et al, 1997) and GLRaV-3 [Ling et al, 1998 (accompanying paper)] revealed several common features of the closteroviruses, including the presence of heat shock protein 70 (HSP70) and a duplicate (CPd) of the coat protein gene (Agranovsky et al, 1996 ;Dolja et al, 1994 ;Boyko et al, 1992). Characterization of the genome organization of GLRaVs would provide molecular information on the serologically distinct closteroviruses that cause similar leafroll symptoms in grapevine.…”

Section: Introductionmentioning

confidence: 99%

Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.

Zhu¹,

Ling²,

Goszczynski³

et al. 1998

The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5h terminus, was cloned and sequenced. The sequence encompasses nine open reading frames (ORFs) which include, in the 5h to 3h direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3h untranslated region. An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papainlike protease, methyltransferase and helicase. ORF1b potentially encoded a putative RNA-dependent RNA polymerase. The expression of ORF1b may be via a M 1 ribosomal frameshift mechanism, similar to other closteroviruses. A unique gene

“…Fax j1 530 752 5674. e-mail bwfalk!ucdavis.edu ORF 5) ; and the duplicated capsid protein (dCP, ORF 6) (Agranovsky, 1996 ;Dolja et al, 1994 ;Klaassen et al, 1995). Many of these genes are unique to closteroviruses (i.e.…”

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confidence: 99%

“…However, the lack of plasmalemma deposits in cells infected with only LIYV RNA 1, in contrast to their presence in cells infected with both LIYV RNAs 1 and 2, is intriguing. LIYV RNA 2 contains seven ORFs, including the five-ORF hallmark closterovirus gene array (Klaassen et al, 1995 ;Dolja et al, 1994). Whether any of the LIYV RNA 2-encoded gene products have roles in plasmalemma deposit formation is not yet known.…”

mentioning

confidence: 99%

Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts.

Medina

Tian

Wierzchos

et al. 1998

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1-and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2 : (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions ; (b) scattered aggregations of virions within the cytoplasm ; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus of the family Closteroviridae. The LIYV genome is bipartite, divided among two ssRNAs of 8118 and 7193 nucleotides (RNAs 1 and 2 respectively), giving a total of 15 311 nucleotides (Klaassen et al., 1995). On LIYV RNA 2 is the ' hallmark closterovirus gene array ' including five genes encoding : a small hydrophobic protein of as yet unknown function (ORF 1) ; an HSP70 homologue (ORF 2) ; a protein of unknown function (p59, ORF 3) ; the capsid protein (CP,