Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2

M. J. Melzer1, A. V. Karasev2, D. M. Sether1 and J. S. Hu1

Department of Plant Pathology, University of Hawaii, Honolulu, HI 96822, USA1
Department of Microbiology and Immunology, Thomas Jefferson University, Doylestown, PA 18901, USA2

Author for correspondence: John Hu. Fax +1 808 956 2832. e-mail johnhu{at}hawaii.edu


   Abstract
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3'-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5' to 3', potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3' terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family Closteroviridae the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.


   Introduction
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The virus family Closteroviridae includes positive-stranded RNA plant viruses with long, flexuous virions and elongated genomes of up to 20 kb. Aphids, mealybugs and whiteflies are known to transmit these viruses (Agranovsky, 1996 ; Karasev, 2000 ). In recent years the genomes of beet yellows virus (BYV) (Agranovsky et al., 1994 ), citrus tristeza virus (CTV) (Karasev et al., 1995 ), lettuce infectious yellows virus (LIYV) (Klaassen et al., 1995 ) and little cherry virus (LChV) (Jelkmann et al., 1997 ) have been completely sequenced. The genomes of beet yellow stunt virus (BYSV) (Karasev et al., 1996 ), grapevine leafroll-associated virus-1 (GLRaV-1) (Fazeli & Rezaian, 2000 ), GLRaV-2 (Zhu et al., 1998 ) and GLRaV-3 (Ling et al., 1998 ) have been sequenced except for the 5'-terminal regions. Analysis of these genomes has demonstrated a great deal of diversity in gene content and organization within the family Closteroviridae.

Based on the number of genomic RNAs, the closteroviruses are currently divided into two genera. The genus Closterovirus, with BYV as the type member, includes viruses with monopartite genomes. The genus Crinivirus, with LIYV as the type member, includes viruses with bipartite genomes. Based on phylogenetic analyses, a recent proposal by Karasev (2000) suggests closteroviruses be classified by vector rather than the number of genomic RNAs. With this proposal, the genus Closterovirus would include aphid-transmitted members and the genus Crinivirus would include whitefly-transmitted members regardless of their number of genomic RNAs. Karasev (2000) also proposes the establishment of a third genus, Vinivirus, with GLRaV-3 as the type member, to accommodate mealybug-transmitted members. In order to better understand the evolutionary relationships within the family Closteroviridae and perhaps support this proposal, more members must be characterized at the molecular level.

Mealybug wilt of pineapple (MWP) is a major constraint on the global production of pineapple [Ananas comosus (L.) Merr.] (Carter, 1934 , 1942 ; Rohrbach et al., 1988 ; Wakman et al., 1995 ). Gunasinghe & German (1989) isolated flexuous, rod-shaped virus particles (12x1200 nm) and high molecular mass double-stranded (ds)RNA (8·35x106 Da) from MWP-diseased plants, but not from healthy plants. Based on particle morphology and nucleic acid and protein characteristics, they tentatively placed this virus in the genus Closterovirus. Initially designated the ‘pineapple closterovirus’ or PCV (Hu et al., 1996 , 1997 ), the name has been revised to ‘pineapple mealybug wilt-associated virus’ (PMWaV) in keeping with current nomenclature. Recent work has strongly associated PMWaV with MWP (Sether & Hu, 1998 , 2000 ) and identified both grey and pink pineapple mealybugs (Dysmicoccus neobrevipes and D. brevipes, respectively) as vectors of the virus (Sether et al., 1998 ). Immunosorbent electron microscopy has also provided evidence that at least two serotypes of PMWaV are present in Hawaii (Hu et al., 1996 ).

To further study the importance of PMWaV(s) in MWP, the molecular and biochemical properties of PMWaV(s) must first be characterized. In this paper we report the nearly complete nucleotide sequence of PMWaV-2, expand the current closterovirus sequence database, and determine the evolutionary position of PMWaV-2 in relation to other characterized members.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Virus source and dsRNA isolation.
Champaka 153 ‘Smooth Cayenne’ pineapple plants grown under glasshouse conditions were assayed for the presence of PMWaV using a tissue blot immunoassay (TBIA) described by Hu et al. (1997) . Double-stranded RNA was isolated from leaf tissue of TBIA-positive plants as described by Hu et al. (1993) . The dsRNAs were resolved by agarose gel electrophoresis and the band with the highest molecular mass was excised. The gel fragment was then centrifuged through a GF/C filter (Whatman) and the eluate stored at -20 °C.

{blacksquare} First-strand cDNA synthesis and cloning strategy.
Viral dsRNA was denatured by heat or 20 mM methylmercury hydroxide, and first-strand cDNAs were synthesized at 37 or 48 °C using Superscript II reverse transcriptase (Gibco BRL/Life Technologies) following the manufacturer’s instructions. Several PCR-based strategies were then used to clone the PMWaV genome from the cDNA templates. First, degenerate primers targeting the conserved ‘A’ and ‘E’ domains of the closterovirus hsp70 gene were used to obtain the initial PMWaV-specific clones (Karasev et al., 1994 ). A step-by-step walking procedure was then used to clone those regions upstream and downstream of the hsp70 domains (Karasev et al., 1994 , 1995 ). Briefly, a PMWaV-2-specific oligonucleotide was used to prime a cDNA which extended past the known sequence. Approximately 20 PCR reactions were then simultaneously performed on this cDNA which combined the PMWaV-2-specific primer with a different random primer. The amplification protocol involved one cycle of 94 °C for 5 min; 45 cycles of 94 °C for 1 min, 33 °C for 1 min; 72 °C for 2·5 min; and a final 72 °C extension for 7 min. Reactions were analysed by agarose gel electrophoresis, and selected amplification products were cloned and sequenced. These served as the basis for the next step of walking along the genome. To clone the 3' terminus, heat-denatured PMWaV-2 dsRNA was polyadenylated with yeast poly(A) polymerase (USB) following the manufacturer’s instructions. Oligo(dT)15 was then used to prime first-strand synthesis, and the cDNA was amplified with a PMWaV-2-specific primer and oligo(dT)15. All PCR products were cloned into either the TA vectors pCR2.1 (Invitrogen), pGEM-T Easy (Promega) or pBluescript KS (Stratagene) modified into a TA vector (Marchuk et al., 1991 ).

{blacksquare} Nucleotide sequencing and analysis.
Both strands of plasmid DNA were sequenced using T3, T7, SP6 or PMWaV-2-specific primers with the Sequenase 2.0 kit (USB) following the manufacturer’s instructions, or by Taq DyeDeoxy terminator cycle sequencing (PE Applied Biosystems). Automated sequencing was done on sequencers at the Guelph Molecular Supercenter (Model ABI377), University of Guelph, Guelph, Canada, at the NCSU DNA Sequencing Facility (ABI377), North Carolina State University, Raleigh, NC, USA or at the Biotechnology/Molecular Biology Instrumentation and Training Facility (ABI373/ABI377), University of Hawaii, Honolulu, HI, USA.

Sequence data were analysed using the web-based GCG package SeqWeb 1.1 (Genetics Computer Group, University of Wisconsin, Madison, WI, USA). BLASTX, based on the Basic Local Alignment Search Tool algorithm (Altschul et al., 1990 , 1994 ), was used to translate nucleotide sequences to amino acid sequences and compare these putative translation products with the non-redundant amino acid sequence database at the National Center for Biotechnology Information website (NCBI). The amino acid sequences of other closteroviruses and phylogenetic outgroups were obtained through the Entrez program at the NCBI.


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Isolation and cloning of PMWaV-2
From the approximately 3 kg of PMWaV-infected pineapple tissue used for dsRNA isolation, only 400–500 ng of viral dsRNA was recovered. This low recovery rate, perhaps related to the low virus titre in infected tissues, is also common to GLRaV-2 (Zhu et al., 1998 ) and GLRaV-3 (Ling et al., 1998 ). The dsRNA band with the highest molecular mass was estimated to be 16 kb and was gel-purified for cloning. Degenerate primers targeting conserved motifs within hsp70 amplified a weak but discrete band of the expected size of about 1 kb. Following cloning and sequencing, it was discovered that the 5'-terminal half of two different but related closterovirus hsp70 genes had amplified. The two viruses were designated PMWaV-1 and PMWaV-2. Twelve clones were generated for PMWaV-2 using the degenerate primer and step-by-step walking strategies (Fig. 1). These overlapping clones encompassed the 3'-terminal 14861 nt of the PMWaV-2 genome which was deposited in GenBank (accession no. AF283103).



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Fig. 1. Schematic representation of the PMWaV-2 genome and select overlapping clones. Boxes represent the ten PMWaV-2 ORFs identified in this study with the corresponding number above. Horizontal bars represent clones used for sequencing, with their respective designations below. The scale above gives approximate size in kb. PRO, papain-like protease domain; MTR, methyltransferase domain; HEL, helicase domain; RdRp, RNA-dependent RNA polymerase; HSP70, cellular heat shock protein 70 homologue; CP, coat protein; CPd, coat protein duplicate.

 
Genome organization and sequence analysis of PMWaV-2
Ten open reading frames (ORFs) were identified within the PMWaV-2 genome in this study (Fig. 1). These ORFs were designated 1a, 1b and 2 through 9 following the convention used for BYV, the type member of the genus Closterovirus.

The incomplete ORF1a potentially encodes a large polyprotein (>204 kDa) containing PRO, MTR and HEL domains. The PRO domain was located in the 5'-region of ORF1a and was identified by its homology with PRO domains of other closteroviruses. The putative catalytic cysteine and histidine residues were at positions 189 and 235, respectively, with the predicted cleavage site occurring between Gly-382 and Gly-383.

The region of ORF1a immediately downstream of the PRO domain was identified as an MTR domain based on homology with other closteroviruses and members of the Sindbis-like group of positive-strand RNA viruses. The amino acid residues spanning the six MTR motifs (Rozanov et al., 1992 ) of PMWaV-2 were 33, 30, 30 and 26% identical to the same regions in the BYV, CTV, LIYV and LChV genomes, respectively. The C-terminal region of ORF1a contained the seven conserved motifs associated with the Superfamily 1 helicase of positive-strand RNA viruses (Gorbalenya & Koonin, 1993 ). This region also shared significant similarity with helicases of other closteroviruses, most notably that of GLRaV-3 (Table 1).


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Table 1. Percentage amino acid identity between PMWaV-2 and select closteroviruses for various gene products

 
ORF1b overlapped the last 53 nt of ORF1a and potentially encoded a 559 amino acid polypeptide with a predicted molecular mass of 64·8 kDa. Database searches revealed that this protein has significant similarity with, and contains the eight conserved motifs common to, the RdRps of positive-strand RNA viruses (Koonin, 1991 ; Koonin & Dolja, 1993 ). Sequence analysis revealed that this putative RdRp of PMWaV-2 is most closely related to that of GLRaV-3, sharing 71·5% similarity at the amino acid level (Table 1).

It is proposed that ORF1bs of BYSV (Karasev et al., 1996 ), BYV (Agranovsky et al., 1994 ), CTV (Karasev et al., 1995 ), LChV (Jelkmann et al., 1997 ), LIYV (Klaassen et al., 1995 ), GLRaV-1 (Fazeli & Rezaian, 2000 ), GLRaV-2 (Zhu et al., 1998 ) and GLRaV-3 (Ling et al., 1998 ) are expressed by a +1 ribosomal frameshift. Different models have been proposed for the frameshift mechanism, but none appear to accommodate all virus members. In PMWaV-2, it appeared that ORF1b could also be expressed by a +1 ribosomal frameshift. Two stem–loop structures, as well as a pseudoknot structure, flanked the ORF1a/1b overlap, although their significance in the frameshift mechanism is unclear, since such structures were common throughout the genome. The sequence UUUC is present in the PMWaV-2 ORF1a/1b overlap as well as the ORF1a/1b overlap for GLRaV-1 (Fazeli & Rezaian, 2000 ) and GLRaV-3 (Ling et al., 1998 ). This sequence encodes phenylalanine in both the 0 and +1 frame, allowing the possibility of a ‘shifty’ tRNAPhe to induce the frameshift.

The start codon of ORF2 was located 624 nt downstream of the RdRp stop codon and potentially initiated a 47 amino acid protein with a predicted molecular mass of 5·3 kDa. Designated p5, the N-terminal region of this putative protein is hydrophobic. Putative proteins of similar size, hydrophobicity and location within the genome are also present in other closteroviruses (see Dolja et al., 1994 ; Klaassen et al., 1995 ) yet share no significant sequence similarity to p5.

The start codon of ORF3 was located 13 nt downstream of the p5 stop codon and potentially initiated a 541 amino acid protein with a predicted molecular mass of 59·1 kDa.

Database searches revealed that the putative protein is homologous to members of the HSP70 family of molecular chaperones, and closely related to those found in other closteroviruses. The putative PMWaV-2 HSP70 protein was most homologous to the HSP70 of GLRaV-3 (Table 1). Like the HSP70s of other closteroviruses, the PMWaV-2 HSP70 contained the eight conserved motifs (A–H) associated with cellular HSP70 genes (Ting & Lee, 1988 ). Despite their relatedness to cellular HSP70s, phylogenetic analysis revealed that closterovirus HSP70s are distinct from their counterparts in plants (Fig. 2b; Ling et al., 1998 ).



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Fig. 2. Dendrograms depicting phylogenetic relationships of closteroviruses as determined from homologous genes. These relationships were determined from putative RdRp (a), HSP70 (b) and CP (c) amino acid sequences of the respective viral genomes. For RdRp analysis, the corresponding sequence from Arabidopsis thaliana (ARAB) (accession no. CAA09894) was used as an outgroup. For HSP70 analysis, the corresponding sequence from Oryza sativa (RICE) (CAA47948) was used as an outgroup. For CP analysis, the corresponding sequence from tobacco mosaic virus (TMV) (P03576) was used as an outgroup. The dendrograms were generated by the PileUp analysis in the GCG web-based software SeqWeb 1.1.

 
The ORF4 start codon was located 238 nt downstream of the HSP70 homologue stop codon and potentially initiated a 403 amino acid protein with a predicted molecular mass of 46·4 kDa. Designated p46, the function of this putative protein is unknown and a search of databases did not identify significantly similar proteins except its counterpart (p55) in GRLaV-3 (Table 1).

The start codon of ORF5 was located 75 nt downstream of the p46 stop codon and potentially encoded a 302 amino acid protein with a predicted molecular mass of 33·8 kDa. ORF5 was identified as the CP gene of PMWaV-2 based on the high homology to GRLaV-3 CP, and the moderate homology to CPs of other closteroviruses (Table 1). Alignment of the CP amino acid sequences of BYV, CTV, LIYV, GLRaV-2, GLRaV-3 and PMWaV-2 revealed the invariant consensus of amino acid residues S, R and D (data not shown) (Dolja et al., 1991 ). A large discrepancy has been identified between the PMWaV-2 CP predicted molecular mass of 33·8 kDa, and the 23·8 kDa observation given by Gunasinghe & German (1989) using SDS–PAGE. Based on the sequence information provided in this study, we believe that the 23·8 kDa protein identified previously was non-viral in origin. This hypothesis is further supported by the fact that the CP of GLRaV-3, which shares high sequence homology with the PMWaV-2 CP, is very similar in size (35 kDa).

The ORF6 start codon was located 33 bp downstream of the CP stop codon. This ORF potentially encoded a 491 amino acid protein with a predicted molecular mass of 55·8 kDa. It was identified as the CPd based on the high homology with GRLaV-3 CPd and moderate homologies with the CPd of other closteroviruses (Table 1). The S, R and D residues conserved in the CP were also conserved in the CPd (data not shown).

The start codon for ORF7 was located 8 nt upstream of the CPd stop codon and potentially initiated a 172 amino acid protein with a predicted molecular mass of 19·7 kDa. Designated p20, this putative protein is homologous to p21 in GLRaV-3, with a 22% identity and 40% similarity between the amino acid sequences. The function of p20 in PMWaV-2 and p21 in GLRaV-3 is unknown; they share no significant sequence homology with other proteins in the databases (this study and Ling et al., 1998 ).

The start codon (AUG) for ORF8 shares the UG residues of the stop codon (UGA) for ORF7. ORF8 potentially encoded a 194 amino acid protein with a predicted molecular mass of 22·3 kDa. Designated p22, this protein shares no significant homology with any other closterovirus sequence, however, it is similar (20% identity; 30% similarity) with the N-terminal region of a viral capsid-associated protein of nuclear polyhedrosis viruses (Lu & Carstens, 1992 ).

Identical to the overlap between ORF7 and ORF8, the sequence AUGA initiates ORF9 and terminates ORF8. ORF9 potentially encoded a 50 amino acid protein with a predicted molecular mass of 5·6 kDa. Designated p6, this putative protein shares no significant homology with any other sequence in the databases; however, it is similar in size and genome location to p7 in GLRaV-3 (Fig. 3).



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Fig. 3. Comparison of the genome organization of PMWaV-2 and select characterized closteroviruses. Boxes represent open reading frames (ORFs), and homologous genes or domains are similarly patterned. Open boxes represent ORFs whose function is unknown or are not well conserved among members. Dotted lines represent unknown sequence. The above scale gives size in kb. Protein designations as in Fig. 1.

 
The 3'-untranslated region (UTR) was composed of 132 nt which did not share any significant similarity with other viral nucleotide sequences in the databases. The 3'-terminal nucleotide residues of PMWaV-2 were CC which are also conserved in the BYSV, BYV and CTV genomes. Consistent with other closteroviruses, the 3'-UTR of PMWaV-2 was predicted to contain extensive secondary structure, including stem–loop and hairpin formations. The conserved sequence reported by Karasev et al. (1996) in the 3'-UTRs of BYSV, BYV and CTV, however, was not present in the 3'-UTR of PMWaV-2.

PMWaV-2, a mealybug-transmissible virus, has a genome organization typical of a monopartite closterovirus. PMWaV-2 contains the components required for replication and amplification of viral RNA (Peremyslov et al., 1998 ), virion assembly (Agranovsky et al., 1995 ) and cell-to-cell movement (Peremyslov et al., 1999 ; Alzhanova et al., 2000 ) in BYV. Whether PMWaV-2 has components which enhance RNA accumulation is unknown. In BYV, the 3'-terminal protein p21 has been associated with enhanced RNA accumulation (Peremyslov et al., 1998 ); however, no homologue to p21 is present in PMWaV-2. If BYV p21 is responsible for protecting RNA from degradation, perhaps either p20 or p22 in PMWaV-2 has similar protective qualities but a different mode of action.

Phylogenetic analyses using the RdRp (Fig. 2a), HSP70 (Fig. 2b) and CP (Fig. 2c) genes of various closteroviruses reveal that PMWaV-2 is firmly embedded within the closterovirus clade. PMWaV-2 is distant from aphid-transmissible viruses such as BYV, BYSV and CTV as well as the whitefly-transmissible LIYV. Instead, PMWaV-2 is consistently located on the same branch as GLRaV-1 and GLRaV-3, two non-aphid-transmitted members (Fig. 2). This consistent grouping supports the establishment of the third closterovirus genus, Vinivirus, of which PMWaV-2 would be a member. As well, the phylogenetic analyses performed here support the current proposal for closterovirus classification based on vector type (Karasev, 2000 ). Sequence comparison and phylogenetic analyses suggest PMWaV-2 is most closely related to GLRaV-3 and GLRaV-1 (Table 1; Fig. 2). This close relationship to GLRaV-1 is especially interesting considering that GLRaV-1 is unique among characterized closteroviruses in its possession of two distinct CPds (Fazeli & Rezaian, 2000 ). At least seven distinct closteroviruses have been identified in diseased grapevine (Boscia et al., 1995 ; Choueiri et al., 1996 ). GLRaV-1, -2 and -3 are well characterized at the molecular level, and show significant differences in their genome organization. This study has confirmed the earlier reports of more than one PMWaV by Hu et al. (1996) , and it will be interesting to see whether distinct PMWaVs are as varied as are the GLRaVs. Also, the possibility of identifying more or less severe strains of PMWaV may greatly help future efforts to control MWP.


   Acknowledgments
 
The authors thank Drs W. Borth and W. Dawson for their suggestions in preparation of this manuscript. This work was supported, in part, by a grant from the State of Hawaii Governor’s Agricultural Coordinating Committee (Contract No. 87-12), the Specific Cooperative Grant agreement 59-5320-5-693 between the USDA and the University of Hawaii, and by the endowment in honour of J.R. and Addie S. Graves. This is Journal Series 4513 of the Hawaii Institute of Tropical Agriculture and Human Resources.


   Footnotes
 
The GenBank accession number of the sequence reported in this paper is AF283103.


   References
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Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 6 July 2000; accepted 11 October 2000.