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Journal of General Virology (1998), 79, 1299–1307. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Nucleotide sequence of the 3«-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus K.-S. Ling,1 H.-Y. Zhu,1 R. F. Drong,2 J. L. Slightom,2 J. R. McFerson3 and D. Gonsalves1 1 Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA Molecular Biology Research, Pharmacia & Upjohn Co., Kalamazoo, MI 49007, USA 3 USDA Plant Genetic Resources Unit, Cornell University, Geneva, NY 14456, USA 2 The RNA genome of grapevine leafroll-associated closterovirus-3 (GLRaV-3) was cloned as a cDNA generated from GLRaV-3-specific dsRNA, and a partial genome sequence of 13 154 nucleotides (nt) including the 3« terminus was determined. The sequenced portion contained 13 open reading frames (ORFs) potentially encoding, in the 5«–3« direction, proteins of " 77 kDa (ORF1a ; helicase, HEL), 61 kDa (ORF1b ; RNA-dependent RNA polymerase, RdRp), 6 kDa (ORF2), 5 kDa (ORF3, small transmembrane protein), 59 kDa (ORF4 ; heat shock protein 70, HSP70), 55 kDa (ORF5), 35 kDa (ORF6 ; coat protein, CP), 53 kDa (ORF7 ; diverged coat protein, CPd), 21 kDa (ORF8), 20 kDa (ORF9), 20 kDa (ORF10), 4 kDa (ORF11), 7 kDa (ORF12), Introduction The plant virus family Closteroviridae was established to include only those viruses with flexuous filamentous particles longer than 1000 nm (Dolja et al., 1994 ; Agranovsky, 1996). Two genera have been proposed. The genus Closterovirus includes those aphid-transmissible viruses with a monopartite genome of up to 20 000 nucleotides (nt), including beet yellows virus (BYV) (Agranovsky et al., 1994), citrus tristeza virus (CTV) (Karasev et al., 1995) and beet yellow stunt virus (BYSV) (Karasev et al., 1996). The other genus, which includes whitefly-transmissible viruses with a bipartite genome, is represented by lettuce infectious yellows virus (LIYV) (Klaassen et al., 1995). Additional genera may be added as new Author for correspondence : Kai-Shu Ling. Fax 1 315 787 2389. e-mail KL12!cornell.edu The nucleotide sequence data reported in this paper will appear in GenBank under accession number AF037268. and an untranslated region of 277 nt. ORF1b is probably expressed via a M1 ribosomal frameshift mechanism, most similar to that of lettuce infectious yellows virus (LIYV). Phylogenetic analysis using various gene sequences (HEL, RdRp, HSP70 and CP) clearly demonstrated that GLRaV-3, a mealybugtransmissible closterovirus, is positioned independently from aphid-transmissible monopartite closteroviruses (beet yellows, citrus tristeza and beet yellows stunt) and whitefly-transmissible bipartite closterovirus (lettuce infectious yellows, LIYV). However, another alleged mealybug-transmissible closterovirus, little cherry virus, was shown to be more closely related to the whitefly-transmissible LIYV than to GLRaV-3. 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 0001-5256 # 1998 SGM BCJJ Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 05:56:59 K.-S. Ling and others 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., 1995, 1996 ; Klaassen et al., 1995 ; Jelkmann et al., 1997). Seven serologically distinct closteroviruses have been associated with grapevine leafroll (Boscia et al., 1995 ; Choueiri et al., 1996), an important and widespread disease of grapevines (Goheen, 1970). These grapevine leafroll-associated viruses (GLRaV), designated GLRaV 1–7, have high molecular mass CPs ranging from 32 to 43 kDa (Hu et al., 1990 b ; Zimmermann et al., 1990). The exception is GLRaV-2 (Boscia et al., 1995 ; Goszczynski et al., 1996), which has a CP of 22 kDa. Observations suggest that leafroll disease does not spread readily within infected vineyards, except when GLRaV-3 is present. Several laboratories have shown that GLRaV-3 can be transmitted by five species of mealybugs : Planococcus ficus (Rosciglione & Gugerli, 1989 ; Engelbrecht & Kasdorf, 1990), Pseudococcus longispinus (Tanne et al., 1989 ; Petersen & Charles, 1997), Pseudococcus viburni (¯ affinis) (Golino et al., 1995), Pseudococcus calceolariae (Petersen & Charles, 1997) and Planococcus citri (Cabaleiro & Segura, 1997). In addition, GLRaV-3 has been reported to be transmitted by the scale insect Pulvinaria vitis L. (Belli et al., 1994). We have investigated GLRaV-3 as part of our long-term goal to characterize the closteroviruses that are associated with the leafroll virus disease. The NY1 isolate of GLRaV-3 used in this study was first characterized in our laboratory by Zee et al. (1987). We demonstrated that closterovirus-like particles were closely associated with the disease and BYV-type vesicles were observed in leafroll-infected tissues (Zee et al., 1987 ; Kim et al., 1989). GLRaV-3-specific antiserum and monoclonal antibodies were produced against purified virions (Zee et al., 1987 ; Hu et al., 1990 a). The molecular mass of the GLRaV-3 CP was estimated on Western blot to be 43 kDa (Hu et al., 1990 b). However, further characterization of its biochemical and molecular properties was hampered due to difficulties in obtaining sufficient amounts of purified virus. Recently, we constructed a cDNA Lambda ZAPII library by cloning cDNA that was synthesized from dsRNA isolated from GLRaV-3infected grapevines and the CP gene was identified (Ling et al., 1997). In this paper, we present the nucleotide sequence of the 3« two-thirds of the GLRaV-3 genome together with an analysis of its relationship with other closteroviruses, and discuss the implications for evolution of closteroviruses. Methods + Virus source and dsRNA isolation. The NY1 isolate of GLRaV3 (Zee et al., 1987 ; Hu et al., 1990 b), which is also referred to as isolate GLRaV 109 by Golino (1992), was used throughout this work. Infected dormant canes or mature leaves were collected from a central New York vineyard and kept at ®20 °C until use. DsRNA was isolated from BDAA grapevine leafroll-diseased vines according to the method described by Hu et al. (1990 b). Full-length dsRNA of GLRaV-3, approximately 18 kb in size, was low melting agarose gel-purified and used as template for the generation of cDNA libraries (Ling et al., 1997) or for PCR. + Identification and sequencing of selected clones. Complementary DNA of GLRaV-3 was synthesized from purified dsRNA and a Lambda ZAPII library was prepared as described (Ling et al., 1997). The CP gene of GLRaV-3 was identified by immunoscreening the cDNA library with GLRaV-3-specific polyclonal and monoclonal antibodies. A degenerate primer (5« GGIGGIGGIACITTYGAYGTITCI, I ¯ inosine, Y ¯ T or C) generated from a conserved amino acid sequence from Motif C of the BYV HSP70 gene (Agranovsky et al., 1991 ; Karasev et al., 1994) was used to select HSP70-positive clones. Clones containing the CP gene (Ling et al., 1997) were used as probes to select clones that may contain sequences beyond the CP gene. In addition, end-sequences from randomly selected clones were used to establish the initial sequence contigs. Further sequence extensions were made by the clone walking strategy, which used sequences that flanked the sequenced contig to probe the library for a clone that might contain an insert extending farther in either the 5« or 3« direction. After sequence assembly, additional gaps were linked by PCR with specific primers on dsRNA template. PCRamplified products were purified from a low melting point agarose gel and sequenced directly or subcloned into a pBlue T-vector (Novagen). To determine the 3« end sequence, poly(A) was added to purified GLRaV-3-specific dsRNAs by yeast poly(A) polymerase (USB) and reverse transcribed using (dT) primer [KSL95-7, 5« GGTCTCGAG(T) ] "& and Moloney murine leukaemia virus (MMLV) reverse transcriptase (Pappu et al., 1994 ; Karasev et al., 1995). Using this first strand cDNA as template, a PCR product was amplified with a (dT) primer and a GLRaV3-specific primer (KSL95-16, 5«CGAGGTAAGATGACTAAACT). The PCR product was either sequenced directly or cloned into a pBlue Tvector prior to sequencing. Selected clones were initially sequenced using universal vector primers (T3 and T7) followed by step-by-step primer extension. The clones were sequenced manually with the Sequenase version 2.0 kit (USB) or with the Taq DyeDeoxy terminator cycle sequencing kit (ABI) on an ABI373 automatic sequencer at Cornell’s New York State Agricultural Experiment Station. Nucleotide sequences were initially analysed using the Genetics Computer Group (GCG) sequence analysis software package (version 7.2). The sequence was assembled with Newgelstart to initialize the GCG fragment assembly system and to support automated fragment assembly. Later, the SeqMan (DNASTAR) computer-aided sequence analysis package was used for sequence assembly. Both sequence assembling programs revealed the same pattern of sequence arrangement. The amino acid sequences were obtained either from the SWISSPROT database or translated from nucleotide sequences from GenBank. The Pearson & Lipman (1988) method of the program MegAlign in the DNASTAR package was used to depict amino acid sequence similarity of GLRaV-3 with respect to other closteroviruses for various gene regions. The Clustal method of MegAlign was also used to generate a putative phylogenetic relationship of GLRaV-3 HEL, RdRp, HSP70 and CP}CPd with respect to the other closteroviruses. With the Clustal method, a preliminary phylogeny was derived from the distances between pairs of input sequences and application of the UPGMA algorithm (Sneath & Sokal, 1973), which guides the alignment of ancestral sequences. The final phylogeny was produced by applying the neighbour-joining method of Saitou & Nei (1987) to the distance and alignment data. To assess more accurately the relationships among closteroviruses, corresponding HEL and RdRp sequences from tobacco mosaic virus (TMV) were used as an Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 05:56:59 GLRaV-3 nucleotide sequence outgroup member. In the case of HSP70, a plant HSP70 (designated pea70HSP) was used as an outgroup. To further confirm the tentative phylogenetic relationship, additional phylogenetic analysis with PHYLIP (version 3.572) (Felsenstein, 1989) was applied, which used the Dayhoff PAM matrix 001 for calculating distance matrices (Dayhoff, 1979), followed by the neighbour-joining method (Saitou & Nei, 1987) for tree construction. Furthermore, Bootstrap was used to obtain a consensus tree for a better assessment of phylogenetic relationships. Results and Discussion Clone selection and nucleotide sequencing The same cDNA library that was used to identify the CP gene (Ling et al., 1997) was also used for the present study. Clone walking, using probes derived from 5« or 3« ends of mapped clones, was used to isolate 200 clones (insert sizes ranging from 300 bp to 3 kb). From this large set of clones, 54 were selected for end-sequence analysis to identify the smaller subset of clones that contained the longest stretch of the GLRaV-3 genome. This subset of 18 clones was then sequenced on both strands. After assembling clones by their overlapping sequences, analysis of the overall sequence revealed two potential gaps. These gaps were completely sequenced by directly sequencing GLRaV-3 genomic PCR products or the subclones of these PCR products (3«-3 and 3«-4 in Fig. 1). A total of 13 154 nt was sequenced and deposited in GenBank (accession number AF037268). Sequence analysis and GLRaV-3 genome organization The genome sequence of GLRaV-3 revealed in the present study potentially encompassed 13 open reading frames (ORFs), tentatively designated ORFs 1a, 1b, and 2–12 following the conventional closterovirus designation (Agranovsky et al., 1994). Major genetic components, such as HEL (ORF1a), RdRp (ORF1b), HSP70 homologue (ORF4), a putative HSP90 homologue (ORF5), CP (ORF6) and CPd (ORF7) were identified. In general, the genome organization of GLRaV-3 is consistent with that expected for a typical monopartite closterovirus (Dolja et al., 1994). Our analysis of ORF1a shows that it is incomplete, missing an expected 5 kb in the 5«-terminal portion. However, the sequence obtained encodes a peptide of 701 amino acids, which covers approximately one-third of the expected ORF1a. Database searches indicated that the C-terminal portion of this protein shared significant similarity with the Superfamily 1 helicase of positive-strand RNA viruses. Pairwise comparison of the conserved region (291 amino acids) shows a 29±3 % similarity to BYSV, 28±9 % to BYV, 28±6 % to LIYV, 27±5 % to CTV and 26±1 % to LChV (Table 1). Six motifs conserved in helicases of positive-strand RNA virus Superfamily 1 (Hodgman, 1988 ; Koonin & Dolja, 1993) were also observed (data not shown). Phylogenetic analysis suggests that GLRaV3, which is transmitted by mealybugs (e.g. Cabaleiro & Sequra, Fig. 1. Schematic presentation of the GLRaV-3 genome, and positions of the selected cDNA clones that were used to determine the nucleotide sequence. Clones 3«-3 and 3«-4 were obtained from PCR amplification to bridge two gaps and other clones were selected from the cDNA library. Table 1. Percentage amino acid sequence similarity between GLRaV-3 and other closteroviruses for different genes in the virus genomes Amino acid sequence similarity was calculated from a pairwise comparison of sequences using the Pearson and Lipman method of the DNASTAR sequence analysis package. BYV CTV LIYV BYSV LChV HEL RdRp HSP70 p55 CP 28±9 27±5 28±6 29±3 26±1 39±2 37±9 33±1 36±0 32±2 23±5 23±8 22±0 24±0 22±7 11±2 11±4 11±8 12±2 11±4 12±3 12±6 15±8 11±6 13±2 1997) is located in an independent lineage from the aphidtransmissible (BYV, CTV and BYSV) and whitefly-transmissible (LIYV) closteroviruses (Fig. 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). ORF1b overlaps the last 113 nt of ORF1a and terminates at the UAA codon at positions 3711–3713. This ORF potentially encodes a protein of 533 amino acid residues, including the first methionine, with a calculated molecular mass of 60 678 Da. Database searches revealed that this protein has a significant similarity to the Supergroup 3 RdRp of the positive-strand RNA viruses. Pairwise comparison of GLRaV-3 with BYV, CTV, LIYV, BYSV and LChV over a 313 amino acid sequence fragment revealed striking amino acid sequence similarity among eight conserved motifs (data not shown). The best alignment was with BYV, with 39±2 % similarity, followed by CTV (37±9 %), BYSV (36±0 %), LIYV (33±1 %), and LChV (32±2 %) (Table 1). Using RdRp sequences (313 amino acids), a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 05:56:59 BDAB K.-S. Ling and others (a) (b) (c) (d) Fig. 2. Phylogenetic relationship of closteroviruses as determined from homologue genes. These relationship were determined using amino acid sequences expected to contain HEL (a), RdRp (b), HSP70 (c) and CP/CPd (d). In the case of HEL and RdRp, corresponding tobacco mosaic virus (TMV) sequences (J02415) were used as a non-closterovirus outgroup. In the case of HSP70, the pea HSP70 (L03299) was used as a non-closterovirus outgroup. In the case of CP, sweet potato sunken vein virus (SPSVV) CP (X80995) was also included. To facilitate the alignment, only 250 amino acid residues in the C-terminal portion of CP (LChV and GLRaV-3) and CPd (LIYV, LChV and GLRaV-3) were used. The length of each pair of branches represents the distance between sequence pairs. The scale beneath the tree measures the distance between sequences. Numbers indicate total substitution events. similar pattern of phylogenetic relationships to that of the HEL was observed (Fig. 2 b). As first suggested for the expression of ORF1b in BYV (Agranovsky et al., 1994) and later observed in CTV (Karasev et al., 1995), LIYV (Klaassen et al., 1995), BYSV (Karasev et al., 1996) and LChV (Jelkmann et al., 1997), a 1 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. Direct comparison of the GLRaV-3 ORF1a}1b overlap with other closteroviruses showed a striking amino acid sequence similarity around the putative frameshift point to LIYV BDAC (Klaassen et al., 1995) (data not shown). Additional experiments on in vitro expression of GLRaV-3 genomic RNA are needed in order to determine whether a large fusion protein is actually produced. ORF2 potentially encodes a small peptide with a calculated molecular mass of 5927 Da (p6) followed by a long intergenic region of 1067 nt. There are no counterpart ORFs in the BYV and LChV genomes, but in CTV (Karasev et al., 1995), LIYV (Klaassen et al., 1995) and BYSV (Karasev et al., 1996) larger ORFs (33, 32 and 33 kDa respectively) are found in this position. ORF3 potentially encodes a small peptide of 45 amino acids with a calculated molecular mass of 5090 Da (p5). Direct comparison with other closteroviruses suggests that this is a small hydrophobic transmembrane protein (data not shown). ORF4 potentially encodes a protein of 549 amino acids with a calculated molecular mass of 59 113 Da (p59). A search in GenBank revealed significant similarity to the HSP70 family of chaperones with the highest similarity to those from closteroviruses. A multiple amino acid sequence alignment of GLRaV-3 p59 with HSP70 homologues of closteroviruses showed a sequence similarity among eight conserved motifs (A–H) (Fig. 3). Functionally important motifs (A–C) that are characteristic of all proteins containing the ATPase domain of the HSP70 type (Bork et al., 1992) were also preserved in GLRaV-3 p59, which suggests that this HSP70 chaperone-like protein may also possess ATPase activity on its N-terminal domain and protein–protein interaction on its C-terminal domain (Dolja et al., 1994). Inspection of a tentative phylogenetic relationship among closterovirus HSP70s and a cellular HSP70 shows that closterovirus HSP70s are grouped together and separated from the plant HSP70 (Fig. 2 c). Two mealybugtransmissible viruses (LChV and GLRaV-3) were placed between the aphid-transmissible and whitefly-transmissible viruses. Pairwise comparison of p59 with other closterovirus HSP70s showed the highest percentage amino acid similarity to BYSV (24±0 %), followed by CTV (23±8 %), BYV (23±5 %), LChV (22±7 %) and LIYV (22±0 %) (Table 1). ORF5 potentially encodes a protein of 483 amino acids with a calculated molecular mass of 54 852 Da (p55). The deduced protein did not have significant sequence similarity to proteins observed in GenBank except to the similarly sized and positioned ORF of other closteroviruses. Direct comparison with counterparts from closteroviruses revealed very limited sequence similarity, with 12±2 % to BYSV, 11±8 % to LIYV, 11±4 % to CTV, 11±4 % to LChV and 11±2 % to BYV, respectively (Table 1). Moreover, two conserved regions of HSP90 homologue previously delineated in BYV and CTV were not identified in p55 of GLRaV-3 (data not shown). ORF6 was previously identified as the CP gene (Ling et al., 1997). Besides four conserved amino acids (N, R, G and D) identified in all closterovirus CPs, there was very limited sequence similarity (11±6–15±8 %) with LIYV, LChV, CTV, BYV and BYSV (Table 1). ORF7 potentially encodes a protein Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 05:56:59 GLRaV-3 nucleotide sequence Fig. 3. Amino acid sequence alignment of the HSP70 homologue of GLRaV-3 with respect to the homologues from BYV, CTV, LIYV, LChV and BYSV. The eight conserved motifs (A–H) of cellular HSP70 are overlined. Consensus amino acids are shown above the alignment in cases where a majority of the residues match (4 out of 6). of molecular mass 53 104 Da (p53, CPd) and was identified as a CPd gene based on the presence of four conserved closterovirus coat protein residues (N, R, G and D) in the Cterminal portion of this protein. Analysis of the phylogenetic relationship of closteroviruses using CPs and their diverged copies again placed GLRaV-3 in an independent lineage from aphid-transmissible and whitefly-transmissible closteroviruses. In addition, sequences of GLRaV-3 CP and its diverged copy Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 05:56:59 BDAD K.-S. Ling and others were more closely related to one another than their counterparts were in the other mealybug transmissible closterovirus (LChV) (Fig. 2 d). The remaining five ORFs downstream of CPd (ORFs 8–12) potentially encode proteins of 185, 177, 179, 36 and 60 amino acids with calculated molecular mass values of 21 248 (ORF8, p21), 19 588 (ORF9, p19.6), 19 652 (ORF10, p19.7), 3 933 (ORF11, p4) and 6768 Da (ORF12, p7), respectively. Searches of current databases using the BLAST program did not reveal statistically significant homologies for these proteins. Although these genes were organized in a similar fashion to CTV in terms of position and size, direct pairwise comparisons did not reveal obvious sequence relationships. The 3« untranslated region (UTR) consisted of 277 nt, for which sequence database searches found no significant match except to the p20 sequence identified in another isolate of GLRaV-3 (Habili et al., 1995). Prediction of extensive secondary structure in the 3« UTR suggested that this region of the GLRaV-3 RNA may play a role in viral RNA replication. The sequence information provided by Habili et al. (1995) showed that they have cloned the region near the 3« terminus of the GLRaV-3 genome. However, we extended this 3« end sequence by 16 nt which was in agreement with the size predicted by their primer extension experiment (Habili et al., 1995). As observed in CTV (Karasev et al., 1995) and BYSV (Karasev et al., 1996), a G nucleotide of nonviral origin was added to the dsRNA. We also observed what may be an additional G in the dsRNA of GLRaV-3. In addition to our earlier sequencing of the CP gene and its flanking regions (Ling et al., 1997), we have now sequenced about two-thirds of the GLRaV-3 genome. These reports are the first direct evidence showing that the high molecular mass dsRNA (C 18 kb) isolated from infected vines is actually derived from the genome of GLRaV-3. Analysis of the 13 ORFs identified in the present study indicates that the genome organization of GLRaV-3 bears significant similarity to other typical closteroviruses. Another cDNA library was obtained from dsRNA of an Italian isolate of GLRaV-3 and selected clones reacted specifically to dsRNA isolated from GLRaV-3-infected vines on a Northern blot (Saldarelli et al., 1994). However, sequence information was not reported. Habili et al. (1995) sequenced a small (1012 nt) part of dsRNA isolated from GLRaV-3-infected vines from Australia and identified a putative ORF (p20). However, a pairwise comparison between that sequence and our GLRaV-3 genome suggested that their p20 sequence was probably derived from a defective interference (DI) sequence. When we analysed the p20 sequence by breaking it down into two parts, the 5« part of 662 nt in p20 mapped to the Nterminal portion of ORF5 (position 6829–7491) with a similarity of 97±9 % at the nucleotide level and 97±2 % at the amino acid level. The 3« part (350 nt) of p20 corresponded to the major region of the 3« UTR (position 12787–13140) with 92±8 % nucleotide similarity. Mawassi et al. (1995 a, b) showed BDAE that DI RNAs are rather commonly associated with CTV infection. Thus, it is not surprising that DI RNAs are present in GLRaV-3-infected plants. Several serologically distinct closteroviruses have been shown to induce a similar type of leafroll symptom on grapevines ; moreover, mixed infections of different viruses in a plant are common (Boscia et al., 1995 ; Choueiri et al., 1996). Thus, the cDNA library developed in the present study may have contained clones from different viral genomes. In fact, when we used a degenerate primer to HSP70 as a probe to screen the cDNA library, we identified some clones with sequences that were not related to that of GLRaV-3. Thus, it became even more important that the CP gene of GLRaV-3 was first identified (Ling et al., 1997). We are confident that the sequence information presented here is of the GLRaV-3 genome because we used the clone-walking strategy with the CP gene as the anchor point. Except for two gaps that were bridged over with PCR (3«-3 and 3«-4), all other sequences were confirmed by at least two overlapping clones. However, some sequence heterogeneity was still observed in the GLRaV3-specific clones. Final sequence information from these ambiguous positions (106 nt or 0±8 % of the total nucleotide sequence) was determined by majority rule with at least one additional overlapping clone. Information regarding the genome of GLRaV-3 gives a better understanding of closteroviruses that are associated with grapevine leafroll disease and adds to the fundamental knowledge of closteroviruses. Based on a tentative division of the closterovirus genome (Dolja et al., 1994), Agranovsky (1996) suggested that the closteroviruses be divided into three modules. For GLRaV-3, the 5« accessory module including the protease and putative vector transmission factor is yet to be identified. The core module, which includes key domains of the RNA replication machinery (MTR-HEL-RdRp), is conserved throughout the alphavirus supergroup and our analysis reveals that the HEL and RdRp domains are conserved in GLRaV-3 (Fig. 4). The MTR domain of GLRaV-3 is yet to be identified. The chaperone-CP module, HSP70-p59-CP-CPd, is conserved in closteroviruses including GLRaV-3 (Fig. 4), which further supports the inclusion of GLRaV-3 as a member of the closterovirus family (Hu et al., 1990 b ; Martelli & Bar-Joseph, 1991 ; Coffin & Coutts, 1993). Sequence comparison and phylogenetic analysis of GLRaV3 with respect to other closteroviruses using several conserved domains clearly demonstrate that this mealybug-transmissible closterovirus is at an intermediate position on the closterovirus evolutionary cascade, with aphid-transmissible (BYV, CTV and BYSV) and whitefly-transmissible (LIYV) closteroviruses at two extremes (Fig. 2). Although the apple mealybug Phenacoccus aceris has been shown to transmit little cherry disease (Raine et al., 1986), several viruses have been recovered from infected plants (Jelkmann, 1995 ; Eastwell & Bernardy, 1996). Thus, additional experiments must be done to show that LChV (Jelkmann et al., 1997) is transmitted by mealybugs. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 05:56:59 GLRaV-3 nucleotide sequence Fig. 4. Comparison of the genome organization of GLRaV-3 with four complete closteroviruses (BYV, CTV, LIYV and LChV) and one partial closterovirus (BYSV). Protein homologues are shown by similar boxed patterns. PRO, papainlike proteinase ; MTR, methyltransferase of type 1 ; HEL, RNA helicase of Superfamily 1 ; RdRp, RNA polymerase of Supergroup 3 ; HSP70, HSP70-related protein ; CP, capsid protein. RBP, RNA-binding protein. Interestingly enough, comparisons of sequences between LChV and GLRaV-3 did not reveal a close relationship in the phylogenetic analysis (Fig. 2) or similarity test (Table 1), although they both possess a large CP (35–46 kDa) and CPd (53–76 kDa). All aphid-transmissible closteroviruses contain smaller (22–26 kDa) CP and CPd while the whiteflytransmitted LIYV has a small CP but a large CPd. Moreover, the genomes of LIYV, LChV and GLRaV-3 have the CPd downstream of CP. Do these characteristics affect vector specificity? Additional sequence information on the 5« portion of GLRaV-3 genome is necessary to get more insight on the genes that may affect vector specificity. Agranovsky (1996) suggested that the putative vector transmission factor may be located in the 5« accessory module, which is yet to be identified in GLRaV-3. Based upon major differences in genome format and organization between BYV, CTV and LIYV, and a phylogenetic analysis, Dolja et al. (1994) proposed the establishment of the new family Closteroviridae, which includes at least two genera, Closterovirus (BYV) and a group of whitefly-transmissible viruses (e.g. LIYV ; Klaassen et al., 1995) with a bipartite genome. Our work on genome organization and phylogenetic analysis of GLRaV-3, along with evidence that it is transmitted by mealybugs (Rosciglione & Gugerli, 1989 ; Tanne et al., 1989 ; Engelbrecht & Kasdorf, 1990 b ; Golino et al., 1995 ; Petersen & Charles, 1997 ; Cabaleiro & Segura, 1997), suggest that a new (third) genus under the family Closteroviridae should also be established. This research was supported in part by USDA}ARS Cooperative Agreement no. 58-2349-9-01 with the USDA Clonal Repository at Geneva, New York and by the Cornell Center for Advanced Technology (CAT) in Biotechnology, which is sponsored by the New York Science and Technology Foundation and Industrial Partners. We thank Dr A. V. Karasev at University of Florida for a critical review of the manuscript and Mr James Van Ee of the BioResource Center’s computing facility at Cornell University for assistance in phylogenetic analysis. References Agranovsky, A. A. (1996). 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