Complete Nucleotide Sequence and Genome Organization of Pineapple Mealybug Wilt-Associated Virus-1

Arch Virol (2008) 153:707–714 DOI 10.1007/s00705-008-0051-8

ORIGINAL ARTICLE

Complete nucleotide sequence and genome organization of pineapple mealybug wilt-associated virus-1

M. J. Melzer Æ D. M. Sether Æ A. V. Karasev Æ W. Borth Æ J. S. Hu

Received: 15 October 2007 / Accepted: 13 December 2007 / Published online: 19 February 2008 Springer-Verlag 2008

Abstract Pineapple mealybug wilt-associated virus-1 distinct pineapple mealybug wilt-associated viruses (PMWaV-1; family Closteroviridae, genus Ampelovirus) (PMWaVs) in Hawaiian pineapple, PMWaV-1, -2, and -3 belongs to a complex of mealybug-transmissible viruses [33, 34]. The mealybug-transmissible PMWaV-1 has been found in pineapple worldwide. In this study, the complete placed in the genus Ampelovirus of the positive-strand genome of PMWaV-1 was sequenced and found to be RNA virus family Closteroviridae [24, 34, 35]. Aphid- and 13.1 kb in length, making it the smallest in the family. The whiteﬂy-transmissible members of the family belong to the genome encoded seven open reading frames (ORFs) and genera Closterovirus and Crinivirus, respectively [20, 24]. was unusual for an ampelovirus due to the lack of an In addition to mealybug transmissibility, members of the intergenic region between the RdRp and p6 ORFs, an ORF genus Ampelovirus are characterized by a genome length of encoding a relatively small coat protein (CP), and the 16.9–19.5 kb, a 35–37 kDa coat protein (CP), and an open absence of an ORF encoding a coat protein duplicate reading frame (ORF) encoding a duplicate of the CP (CPd), (CPd). Phylogenetic analyses placed PMWaV-1, plum bark generally located immediately downstream of the CP ORF necrosis stem pitting-associated virus and some grapevine [24]. leafroll-associated viruses in a distinct clade within the The genome of PMWaV-2 has been partially sequenced genus Ampelovirus. and found to be that of a typical ampelovirus, and phylogenetic analyses clearly placed PMWaV-2 within the genus [25]. It has been demonstrated that PMWaV-2 has a role in MWP [31], whereas pineapple infected only with Introduction PMWaV-1 can have signiﬁcantly reduced yield but will not develop symptoms of MWP [31, 32]. To better understand Mealybug wilt of pineapple (MWP) is an economically this disease and the diversity of PMWaVs infecting important disease of pineapple that has been found in major pineapple in Hawaii, it is essential that PMWaV-1 be growing regions of the world [11]. Serological and characterized at the molecular level. In this study, we molecular data have revealed the presence of at least three report the complete nucleotide sequence and genome organization of PMWaV-1 and its phylogenetic relationship with other members of the family Closteroviridae. GenBank accession # AF414119

& M. J. Melzer D. M. Sether W. Borth J. S. Hu ( ) Materials and methods Department of Plant and Environmental Protection Sciences, University of Hawaii, 3190 Maile Way, St. John 310, Honolulu, HI 96822, USA Pineapple, Ananas comosus (L.) Merr. cv. Smooth e-mail: [email protected] Cayenne and a proprietary hybrid, were assayed for the presence of PMWaV-1 and absence of PMWaV-2 and A. V. Karasev Department of Plant, Soil, and Entomological Sciences, PMWaV-3 using RT-PCR and/or tissue blot immunoassays University of Idaho, Moscow, ID 83844, USA (TBIA) [18, 33, 34]. Double-stranded (ds)RNAs were 123 708 Arch Virol (2008) 153:707–714 isolated from the leaf bases of plants testing positive for positive strand of viral dsRNA near the suspected 50 ter- PMWaV-1 [17]. The dsRNAs were resolved by agarose gel minus was tailed with dCTP using terminal electrophoresis, and an approximately 13-kilobase (kb) deoxynucleotidyl transferase (Promega) and used as tem- fragment, assumed to be the replicative form of PMWaV-1, plate in PCR primed by oligo d(G)15 and a virus-specific was excised and used as a template for molecular cloning. primer. All PCR products were cloned into the TA vectors A *5.9-kb dsRNA was also present in some plants pCR2.1 (Invitrogen) or pGEM-T Easy (Promega). Vectors infected with PMWaV-1 and was assumed to be either a were sequenced using either a Sequenase 2.0 kit (USB) or deletion mutant RNA of PMWaV-1 or genetic material of by automated sequencing at the University of Hawaii’s another virus (Fig. 1). Greenwood Molecular Biology Facility or Center for The initial PMWaV-1-specific clone was previously Genomics, Proteomics, and Bioinformatics Research obtained by RT-PCR using degenerate primers [25]. From Initiative. this clone, a step-by-step walking procedure [25] was used To confirm the length and organization of the PMWaV-1 to obtain clones in the 30 and 50 directions. Briefly, a primer genome, dsRNAs isolated from two pineapple plants designed from an existing clone was used to generate infected with PMWaV-1 were probed with a digoxigenin- cDNA extending into unknown sequence. This cDNA was labeled (Roche) DNA sequence spanning the putative then used as a template in PCR which paired the primer 30-terminal ORF and 30 untranslated region (UTR). The used to make the cDNA with an 18-to-22-mer primer prehybridization and hybridization was performed in chosen at random from our collection. Between 20 and 48 PerfectHybTM Plus hybridization buffer (Sigma) at 68C for PCR reactions, each with a different random primer, were 20 min and overnight, respectively. Subsequent washes, performed at each walking step. Amplification products antibody labeling, and CSPD chemiluminescent detection were cloned and sequenced to determine if they repre- were performed following the digoxigenin manufacturer’s sented sequences adjacent to the previous clone, and if so, instructions. the process was repeated. The sequence of the 30 terminus Nucleotide sequences were compared with the non- was obtained via two independent dsRNA polyadenylation redundant amino acid sequence database at the National procedures as previously described [25]. To obtain the Center for Biotechnology Information (NCBI) website sequence of the 50 terminus, cDNA complementary to the using the BLASTX algorithm [4, 5]. The amino acid sequence identity between closteroviruses was determined using the GAP function of SeqWeb v1.2 (GCG Inc.). Amino acid sequences of the helicase (HEL) domain, M 1 2 M 1 2 RNA-dependent RNA polymerase (RdRp), heat shock 70 genomic homolog (Hsp70h), and the coat protein (CP) were used for 10.0 13.1 8.0 phylogenetic analyses. These sequences were aligned using 6.0 5.0 p6/HSP70h ClustalX v1.8 [38], and phylogenetic trees were generated 4.0 4.9 using neighbor-joining (NJ), parsimony, and Bayesian 3.5 p61 methods. PAUP* v4.0b [36] was used to create and boot- 3.0 3.2 2.5 strap NJ and parsimony trees. Tree branches with less than 2.0 60% support following 1,000 bootstrapping replications CP were collapsed. MrBayes v3.1.1 [19, 28] was used to create 1.5 1.6 trees using Bayesian analyses. The fixed-rate amino acid mixed model that allows model switching during the 1.0 p24 analysis was used with 400,000–500,000 generations and 0.75 0.8 sampling every 100 generations. Sequences used in these 0.5 analyses were beet yellow stunt virus (BYSV, accession # U51931); beet yellows virus (BYV, NC_001598); citrus tristeza virus (CTV, U16304); cucurbit yellowing stunt disorder virus (CYSDV, NC_004809/NC_004810); grape- Fig. 1 Analysis of dsRNAs isolated from two pineapple plants (lanes vine leafroll-associated virus-1 (GLRaV-1, AF195822); 1 and 2) infected with PMWaV-1. Left, agarose gel electrophoresis of dsRNAs isolated from *100 g of pineapple tissue. Numbers at the GLRaV-2 (AF039204); GLRaV-3 (AF037268); GLRaV-4 left indicate the size in kilobases (kb) of the digoxigenin-labeled 1-kb (AF039553); GLRaV-5 (AF233934); GLRaV-6 DNA ladder (Mirus) (lane M). Right, autoradiograph of the gel blot (AJ496796); GLRaV-9 (AY072797); little cherry virus-1 0 following hybridization with a p24/3 UTR probe. Arrowheads at the (LChV-1, NC_001836); LChV-2 (AF531505); lettuce right indicate the predicted size (in kb) and location of the PMWaV-1 genomic and subgenomic dsRNAs. The *5.9-kb fragment present in infectious yellows virus (LIYV, NC_003617/NC_003618); lane 1 may be a deletion mutant of PMWaV-1 mint vein banding associated virus (MVBaV, AY548173); 123 Arch Virol (2008) 153:707–714 709 plum bark necrosis stem pitting-associated virus (PBNSPaV, Table 1 Percentage amino acid identity of various gene products EF546442); olive leaf yellowing-associated virus (OLYaV, between PMWaV-1 and selected members of the family Clostero- AJ440010); PMWaV-2 (AF283103); sweet potato chlorotic viridae. Sequence accession numbers and viral abbreviations are in the ‘‘Materials and Methods’’ stunt virus (SPCSV, NC_004123/AJ428555). Genus Virus HELa RdRp Hsp70h CP

Closterovirus BYV 32 32 32 24 Results CTV 33 31 29 16 GLRaV-2 34 33 30 20 The complete nucleotide sequence of PMWaV-1 was found GLRaV-1 30 33 32 25 to be 13,071 nucleotides in length and was deposited in Crinivirus LIYV 32 26 26 21 GenBank (accession AF414119). To ensure that this rep- Ampelovirus GLRaV-3 31 33 35 26 resented the full-length genome of PMWaV-1 and not a GLRaV-5 – – 58b 56 truncated form, dsRNAs isolated from plants infected with LChV-2 34 37 31 22 PMWaV-1 were probed with a labeled PMWaV-1-specific PMWaV-2 33 31 32 26 sequence. No dsRNAs larger than *13 kb with sequence Unassigned GLRaV-9 51 59 60 54 similarity to PMWaV-1 were present, suggesting that the LChV-1 30 27 26 19 full-length genome was attained (Fig. 1). A *5.9-kb MVBaV 36 35 32 15 dsRNA visible in some plants following gel electrophoresis PBNSPaV 37 37 49 32 did hybridize with the PMWaV-1 probe (Fig. 1), and preliminary sequencing results indicate it is a deletion mutant Values calculated using the Needleman and Wunsch algorithm and of PMWaV-1 (data not shown). oldpep scoring matrix found in the GAP function of the GCG web- based software SeqWeb v1.2 The PMWaV-1 genome was found to contain seven a Amino acid sequence from motifs HEL I to HEL VI [15] putative open reading frames (ORFs; Fig. 2). The ORF b Based on partial sequence nomenclature used here follows the convention used for GLRaV-3, the type member of the genus Ampelovirus [23]. The 50 untranslated region of PMWaV-1 was 353 nt in domain, which has been identified in some family members length and A/T-rich (70.2%). ORF1a was 2077 aa in [10, 24], was present in ORF1a. length, encoding a polyprotein with a predicted molecular The sequence GUUUAACA contains the ORF1a stop mass of 233 kDa. A papain-like protease domain [16] was codon (underlined) and is similar to the sequence presumed identified in ORF1a with the catalytic residues Cys432 and to be involved with the +1 ribosomal frameshift in BYV His475 and a predicted cleavage site at Val491/Gly492. (Fig. 3), BYSV, and GLRaV-2 [1, 21, 39]. Although sim- Methyltransferase and helicase (HEL) domains were also ilar in location, this potential frameshift differs from that present in ORF1a [15, 30]. The HEL of PMWaV-1 was proposed for PMWaV-2 and GLRaV-3 [25] (Fig. 3). The found to be 51% identical to the HEL of the currently asparagine codon in the putative frameshift sequence unassigned GLRaV-9, but \40% identical to the HELs of (italicized above) presumably initiates the 59.8-kDa other members of the family Closteroviridae, including ORF1b, which contains all eight motifs of the RNA- those from the genus Ampelovirus (Table 1). No AlkB dependent RNA-polymerases (RdRp) of positive-strand

Fig. 2 Genome organization of 051015 kb PMWaV-1, PMWaV-2 and PBNSPaV. Boxes representing open reading frames are RdRp p6 p61 p24 vertically offset based on PRO MTR HEL HSP70h CP reading frame, and homologous PMWaV-1 genes or domains are similarly patterned. PRO protease domain; MTR methyltransferase PRO MTR HEL CPd p22 RdRp p5 p20 HSP70h p46 CP p6 domain; HEL helicase domain; ? RdRp RNA-dependent RNA PMWaV-2 polymerase; Hsp70h heat shock protein 70 homolog; CP coat p6 HSP70h CPm protein; CPd coat protein PRO MTR HEL duplicate. The scale gives RdRp p61 CP PBNSPaV approximate size in kilobases

123 710 Arch Virol (2008) 153:707–714

A PMWaV-1, however not all hydrophobic residues were Q Q C V N PMWaV-1 cag cag ugc guuu aac conserved (data not shown). BYV ccg cag cgg guuu agc P Q R V S ORF5 of PMWaV-1 encoded the CP, with a predicted S A L F E molecular mass of 28.1 kDa. The CP was found to contain PMWaV-2 ucg gcg uua uuuc gag 117 162 GLRaV-3 gcu ggu ugc uuuc gag the invariant consensus amino acid residues S ,R , and A G C F E D203, which are conserved in the CPs of all rod-shaped and B HEL VI HRLVALSRHTISLQYFCIPNKMNKGIGEDVQSIIKLE..ERVAANFVVQQCVNIYLPC...KDVVVPIP filamentous RNA plant viruses [12]. The PMWaV-1 CP | |||||||| | | : .|: :| : |: : | : | || : | .|. : HILVALSRHTQSCVYATLSSKLADKVGAAIDSVTRKEVSDTVLKTFVASALFELIERCSWQQDLEREV. was 56 and 54% identical to the respective CPs of GLRaV-5 and -9, but\33% identical to the CPs from other members LGLGRPPKAHFEAIQSFLDDALNGVGSLLFLSTEEQFQMTDTITNITDFSMSDNDIRFKPIKFFSNQPR of the family Closteroviridae (Table 1). | | .|| : |||:: . | || : |: |: .| | : || : : ..LARCSNSHFYVVNSFLEEMVPGSVSLDYRFFEDDFEFSDHEFLINSCILRDNSVNKLTYRENYIYSF ORF6 of PMWaV-1 encoded a 23.9-kDa protein (p24) that was 40% identical to similarly located ORFs in the IRSQMVVRRQNTLKANILAYEKRNAGVVKSLWHPDVTDEVM.TVVDDFFGSYVDSRKLSEVLVDPVEPN ||| . . :.|||| |:. :| || | : : | | .|: : :| |. | | | genomes of GLRaV-5 and GLRaV-9. These p24 orthologs IRSNIGMPKRNTLKCNLVTFENRNFNVDRDC.YVGCDDFVADALVEKLVNRFFLGNRLFELQSDVVCAN have been referred to as CPd in GenBank (AAK38611 and IFDLADWLLSRTPMGKKALMMELENPVELGTNLNRFKLMVKA PMWaV-1 (1335-1588) AAQ63813), although they share \20% identity with their ..|: |||| | |||: | .|:|||||. AVAASNWIDSRTPSGYKALLSALGGYFYTPDGMSRYKLMVKS PMWaV-2 (1785-2030) respective CP. Likewise, PMWaV-1 p24 is only 15.5% RdRp I identical to the PMWaV-1 CP, with the regions of limited Fig. 3 Analysis of the putative PMWaV-1 ORF1a/1b frameshift similarity scattered throughout the alignment (Fig. 4). In sequence and location. A, Comparison of the nucleotide and amino contrast, the CP and CPd/CPm of other members of the acid sequence alignments at the proposed frameshift site for BYV and family Closteroviridae have pronounced sequence simi- PMWaV-1, and potential frameshift sites for PMWaV-2 and GLRaV- 3. The putative ‘shifty’ codons are underlined. B, Comparison of the larity, primarily in their C-terminal regions (Fig. 4). The putative frameshift location for PMWaV-1 and PMWaV-2. Amino genome of PBNSPaV possesses a 30-terminal ORF acid sequences are from the HEL VI motif of ORF1a to the RdRp I encoding a 25-kDa protein which has been designated CPm Arrows motif of ORF1b. indicate the putative frameshift site, and the based on the presence of S59,R72,D101 (Fig. 5)[3]. It is box indicates the relative location of the proposed BYV frameshift site unclear if this is an ortholog of the p24s of PMWaV-1, GLRaV-5, and GLRaV-9 as the sequence similarities are marginal (13–20% identical, 42–48% similar). An align- RNA viruses [22]. The PMWaV-1 RdRp was 59% identical ment of these ORFs reveals a complete consensus for two to the GLRaV-9 RdRp, but \40% identical to RdRps of sets of arginine and aspartate residues, but not for any other members of the family Closteroviridae, including serine residues (Fig. 5). those from the genus Ampelovirus (Table 1). The 30 UTR of PMWaV-1 was determined from two ORF2 of PMWaV-1 encoded a 5.8-kDa protein (p6) independent dsRNA polyadenylation experiments. In the which contained a hydrophobic N-terminal region (data not first experiment, two clones were sequenced, indicating shown) and had low sequence similarity with equivalent that it was 132 or 131 nt in length and terminated with the proteins in other viruses of the genus Ampelovirus.No sequence 50-...GGGCCG-30 or 50 -...GGGCC-30. Additional intergenic region was present between the RdRp and p6 in clones were not sequenced from this experiment as it was PMWaV-1 (Fig. 2). assumed the dsRNA template for polyadenylation was ORF3 of PMWaV-1 encoded a 55.5-kDa Hsp70h, which truncated. A different dsRNA was used as template for the had 58–60% amino acid identity with the available second experiment, and all three clones sequenced were in sequences for Hsp70h’s of GLRaV-4, GLRaV-5, GLRaV- agreement with a 132-nt 30 UTR terminating with the 6, GLRaV-9, and 49% identity with the PBNSPaV Hsp70h. sequence 50-…GGGCCG-30. In addition to these two pol- Sequence identities with homologs from viruses of other yadenylation experiments, eight step-by-step walking members of the family Closteroviridae, including those attempts and two 30 RACE experiments were unsuccessful from the genus Ampelovirus, were low (\36%) (Table 1). in obtaining PMWaV-1-specific sequence beyond this ORF4 of PMWaV-1 encoded a 61.2-kDa protein (p61) putative terminus. This genome organization and size were which shared 42, 46, 31, and 20% identity with the supported by probing dsRNAs from PMWaV-1-infected orthologs of GLRaV-5, GLRaV-9, PBNSPaV, and plants with labeled sequences from p24 and the putative 30 PMWaV-2, respectively. Sequence identity with similarly UTR. In addition to the genomic dsRNA, the probe located proteins in the genomes of other members of the hybridized with dsRNAs approximately 5, 3.2, 1.5, and family Closteroviridae was low. The C-terminal CP-like 0.7 kb in size, which correspond closely to the predicted domain that was identified in p61 orthologs of other subgenomic (sg)RNA sizes of 4.9, 3.2, 1.6, and 0.8 nt members of the family Closterovirdae [26] was present in (Fig. 1).

123 Arch Virol (2008) 153:707–714 711

1 50 GLRaV-5 ~~~~MEVTLC LSKVKEKLPV TCDEIFYLRN AGVYAT...... VMPPER GLRaV-9 ~~~~MEVTLY LSKAKEDLPV ACDETFYLRN ARVYAT...... VMPPER PMWaV-1 ~~~~MERIIL VSKDKNLLNA NEEEIFYKVK YSLFMR...... AKEGNE PBNSPaV MKILKMSSVV LRVVRNNTFD PRDVTRRAAD LSLFVKHRDG FVYYINPANT Consensus ~~~~------

51 100 GLRaV-5 LSFFYDK... LSVG..NNND RNKYRELFQF LDNLGNVVAT NNGNFIKFLD GLRaV-9 LSFFYDK... LSVG..NNSD RNKYRELFQF LDNLGNVVAT NNGNFIKFLD PMWaV-1 CSFFYDK... LNVF..SDSS RRRYAELFSF LDSLGNVVAT NNETFLNFLD PBNSPaV PLFVFTRSCG GFVGSTENQE RMECEVAAQM CYGLGEVWLT NSPYVISNID Consensus --F------V------R------LG-V--T N------D

101 150 GLRaV-5 VDSVMEYFRR RYPDGGNLSS INKTPSSDYC CLTVPALTTF LNSVCGCIEG GLRaV-9 VDSVMEYFRR RYPDGSSLSN MNKTPSSDYC CLTVPALTTF LNSVCGCLEG PMWaV-1 GLSVDDYMLR RYGGRGKREL SEGEKRAPYL TIPVPALETL WGVIEESGKK PBNSPaV VVYPAEKFLG IHQTQFQVGS IGWNVVNDLS TLGIDDITVV LGGYVLDGMP Consensus ------

151 200 GLRaV-5 LTTVLAVGFT RKSDT..DEL WSFKNTFVVF ENSGEISTFY GDSMDTSNSA GLRaV-9 LTTVLAVGFT RKDDT..DEL WSFKDTFVVF ENSGVISTFY GDSMDTSNSA PMWaV-1 LHKVHCVGFV RNRGSSCDEL YDFDRSFAVF DYGEGFEVVY GGSVDSSNVA PBNSPaV TLDDNDTNGV RLSSIAVVKY KNNEQWTYKL CVKEEVNIKF PPVRDCFRML Consensus ------R------D-----

201 227 GLRaV-5 LLEVLDSFGS TSGKTDFEKV VSLLSI~ GLRaV-9 LLEVLDSFGS ASGKTDFEKV VNLLSI~ PMWaV-1 LKGKLKILKE AEEKTGLEKV VGILSEI PBNSPaV VASKTNKSPI HRFRQSFNFG SA~~~~~ Consensus ------

Fig. 5 Amino acid alignment of the PMWaV-1 p24, its orthologs in GLRaV-5 and GLRaV-9, and the 25-kDa 30-terminal ORF of PBNSPaV. The highlighted S, R, and D of the PBNSPaV ORF are proposed by Alrwahnih et al. [3] to be the hallmark residues of rod- shaped and ﬁlamentous RNA virus coat proteins. The residues in white type on a black background may also be candidates. The alignment was created using the ‘Pretty’ function and structgappep scoring matrix [27] of the GCG web-based software SeqWeb 1.1

Fig. 4 Graphic representation of the low sequence similarity between the CP (x-axis) and p24 (y-axis) of PWMaV-1 (top). In contrast, their evolution (Fig. 6). PMWaV-2, GLRaV-1, and pronounced sequence similarity exists between the CP and CPd of GLRaV-3 represent one group, and PMWaV-1, GLRaV-4, PMWaV-2 (bottom left) and BYV (bottom right), particularly in the GLRaV-5, GLRaV-6, GLRaV-9, and PBNSPaV represent C-terminal regions of the proteins (upper right corner of graphs). Numbers on the axes correspond to amino acid residue positions for the other (Fig. 6). These two groups within the genus the proteins. Graphs were generated using the ‘Compare’ function of Ampelovirus were also strongly supported by NJ and par- the GCG web-based software SeqWeb v1.2 simony analyses using the Hsp70h and CP, although not all viruses were represented in these analyses due to lack of One distance-based method (NJ) and two character- available sequence information (data not shown). based methods (parsimony and Bayesian) were used to resolve the phylogenies of the HEL domain, RdRp, Hsp70h, and CP of PMWaV-1 and other members, tenta- Discussion tive members, and unassigned members of the family Closteroviridae. All trees generated in this study supported Molecular characterization of the PMWaV-1 genome has the genera Closterovirus and Crinivirus by creating revealed several key features that distinguish it from a monophyletic groups consisting of members and tentative prototypical ampelovirus. Based on partial sequence data members of the respective genus. Most analyses also sup- available in GenBank, the ampelovirus GLRaV-5, tentative ported the genus Ampelovirus, by assigning members and ampeloviruses GLRaV-4 and GLRaV-6, and the currently tentative members to a monophyletic group. However, undesignated GLRaV-9 may also share some or all of these polytomies generated by NJ and parsimony analyses using features. The recently sequenced genome of PBNSPaV the HEL domain and the RdRp depicted the genus as also appears to share some, but not all of these features [3]. polyphyletic, with two distinct groups (data not shown). First, the long intergenic region between the RdRp and Except for the CP analysis, Bayesian resolved all of the p6 ORFs characteristic of the genus Ampelovirus [20]is polytomies created by the other methods and placed all absent in PMWaV-1. In the atypical genome of the ampeloviruses and tentative ampeloviruses in a monophy- mealybug-transmissible LChV-2/USA6b ampelovirus, the letic group composed of two groups that diverged early in ORFs of a putative 14-kDa read-through protein and 123 712 Arch Virol (2008) 153:707–714

A b Fig. 6 Unrooted phylograms depicting relationships within the PMWaV-1 family Closteroviridae using the RdRp (A), CP (B), and Hsp70h LChV-2 GLRaV-1 (C). The phylogram using the HEL domain was essentially the same as the RdRp. Trees were generated by Bayesian analyses using the PMWaV-2 GLRaV-9 BLOSUM ﬁxed-rate amino acid model. Numbers on branches are posterior probabilities and used as an indicator of branch support. 1.00 0.95 Branches with posterior probabilities\0.70 were collapsed. Sequence 1.00 GLRaV-3 0.91 accession numbers and viral abbreviations are in the Materials and PBNSPaV 1.00 1.00 Methods; (p) refers to a partial sequence. The ovals in D encompass 0.92 the current, tentative, and proposed members of the three Clostero- 1.00 GLRaV-2 viridae genera; those outside the ovals as well as GLRaV-9 and MVBaV 1.00 BYV PBNSPaV are currently unassigned members of the family 1.00 BYSV the CPd have been found in this location; however, a small 1.00 (0.3 kb) intergenic region persists [29]. This intergenic SPCSV CTV region is also absent in PBNSPaV [3]. Second, with pre-

0.1 LIYV LChV-1 dicted molecular masses of 28.1–29.5 kDa, the CPs of PMWaV-1, GLRaV-5, and GLRaV-9 would represent, by far, the smallest in the genus, whose members typically B GLRaV-1 MVBaV have molecular masses of 35–39 kDa [24]. Indeed, these GLRaV-9 GLRaV-5 PMWaV-2 CPs are more similar in size to those found in members of PMWaV-1 1.00 the genera Crinivirus and perhaps Closterovirus [20, 24]. 1.00 GLRaV-3 1.00 PBNSPaV 1.00 The CP of PBNSPaV is predicted to be 36 kDa, well within

1.00 the typical range of ampeloviruses [3]. The third distin- 0.70 LChV-2 guishing characteristic of the PMWaV-1 genome is the 1.00 absence of a CPd. Other members of the family Clostero- LChV-1 1.00 viridae possess at least one CPd that presumably arose 1.00 through duplication of the CP in an ancestral closterovirus 0.98 CTV 0.81 [9, 13]. While composed mostly of CP, BYV virions also SPCSV possess a short heterogeneous ‘tail’ whose essential com- LIYV GLRaV-2 ponents include CPd, Hsp70h and p64 [2, 7, 8, 26]. A 0.1 BYSV BYV similar virion architecture has also been shown for CTV and LIYV [14, 37], suggesting this is a common charac- C teristic of the family. Each of the tail components is Ampelovirus essential for cell-to-cell movement of BYV virions [6, 7], PMWaV-2 GLRaV-3 making the absence of a CPd in the PMWaV-1 genome PBNSPaV conspicuous. Alzanhova et al. [8] have suggested that prior PMWaV-1 GLRaV-1 to the acquisition of CPd, this tail could have been formed

1.00 GLRaV-4 entirely with HSP70h and p61 orthologs. Further studies 1.00 1.00 GLRaV-9 0.96 GLRaV-6 are needed to determine if PMWaV-1 virions possess a tail, LChV-2 1.00 1.00 1.00 GLRaV-5(p) and if so, its composition. 1.00

1.00 Orthologs of p24 in GLRaV-5 and GLRaV-9 have been MVBaV identiﬁed as CPd in GenBank. The p24 of PMWaV-1 does 1.00 BYV not appear to be a paralog of the PMWaV-1 CP, and 1.00

0.99 1.00 1.00 therefore cannot be accurately described as CPd. An ORF BYSV 0.99 LChV-1 in PBNSPaV of similar size and location, but with limited 1.00 sequence similarity to p24, has been designated CPm based GLRaV-2 1.00 on the presence of specific S, R, and D residues common to LIYV the CPs of all rod-shaped and filamentous RNA viruses [3, CTV 12]. Using a structural alignment matrix, which specifically Closterovirus SPCSV CYSDV OLYaV scores substitutions based on a-carbon position [27], we 0.1 Crinivirus found the R and D residues specified by Alrwahnih et al. [3] were also conserved in PMWaV-1 p24 and its ortho-

123 Arch Virol (2008) 153:707–714 713 logs. A second set of R and D residues in the C-terminal References region of the alignment were also conserved in all sequences (Fig. 5). Since only the R and D residues are 1. Agranovsky AA, Koonin EV, Boyko VP, Maiss E, Frotschl R, conserved in the CP-like domain of p61 [26], the PMWaV- Lunina NA, Atabekov JG (1994) Beet yellows closterovirus:complete genome structure and identification of a leader 1 p24 may function as a CPm, although this needs to be papain-like thiol protease. Virology 198:311–324 verified experimentally. 2. Agranovsky AA, Lesemann DE, Maiss E, Hull R, Atabekov JG Members of the family Closteroviridae are known for (1995) ‘‘Rattlesnake’’ structure of a filamentous plant RNA virus their long RNA genomes, the largest of the plant viruses built of two capsid proteins. Proc Natl Acad Sci USA 92:2470– 2473 [13]. However, at a modest 13.1 kb in length, the genome 3. Alrwahnih M, Uyemoto JK, Falk BW, Rowhani A (2007) of PMWaV-1 has considerably less coding capacity than Molecular characterization and detection of plum bark necrosis those of other fully sequenced closteroviruses, which range stem pitting-associated virus. Arch Virol 152:2197–2206 from 14.2 to 19.3 kb [3, 24]. Dolja et al. [13] have recently 4. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 proposed an evolutionary scenario for the family Closte- 5. Altschul SF, Boguski MS, Gish W, Wooton JC (1994) Issues roviridae in which the genome of the common ancestor for in searching molecular sequence databases. Nature Genet the family looks remarkably similar in size and organiza- 6:119–129 tion to that of PMWaV-1. The only differences from 6. Alzhanova DV, Hagiwara Y, Peremyslov VV, Dolja VV (2000) Genetic analysis of the cell-to-cell movement of beet yellows PMWaV-1 in the proposed progenitor are the presence of closterovirus. Virology 268:192–200 an intergenic region between the RdRp and p6 ORFs and 7. Alzhanova DV, Napuli A, Creamer R, Dolja VV (2001) Cell-to- the lack of p24. Could PMWaV-1 represent an extant cell movement and assembly of a plant closterovirus: roles for the version of this ancestral closterovirus that has acquired the capsid proteins and Hsp70 homolog. EMBO J 20:6997–7007 8. Alzhanova DV, Prokhnevsky AI, Peremyslov VV, Dolja VV p24 ORF during its evolution as other closteroviruses have (2007) Virion tails of Beet Yellows Virus: coordinated assembly done with their 30-terminal ORFs? Or is PMWaV-1 a by three structural proteins. Virology 359:220–226 streamlined ampelovirus that has shed unneeded genetic 9. Boyko VP, Karasev AV, Agranovsky AA, Koonin EV, Dolja VV material? (1992) Coat protein duplication in a filamentous RNA virus of plants. Proc Natl Acad Sci USA 89:9156–9160 Phylogenetic analyses suggest an early split in the 10. Bratlie MS, Drabløs F (2005) Bioinformatic mapping of AlkB evolution of the genus Ampelovirus, in agreement with homology domains in viruses. BMC Genomics 6:1. doi: previously published studies [24]. NJ and parsimony 10.1186/1471-2164-6-1 analyses had difficulty resolving basal branching when 11. Carter W (1942) Geographic distribution of mealybug wilt with some other insect pests of pineapple. J Econ Entomol 35:10–15 using the HEL domain and RdRp, resulting in polytomies. 12. Dolja VV, Boyko VP, Agranovsky AA, Koonin EV (1991) These polytomies, which consisted of a closterovirus, Phylogeny of capsid proteins of rod-shaped and filamentous RNA crinivirus, and two ampelovirus clades, could be inter- plant viruses: two families with distinct patterns of sequence and preted as evidence of a second mealybug-transmissible probably structure conservation. Virology 184:79–86 13. Dolja CC, Kreuze JF, Valkonen JPT (2006) Comparative and genus in the family Closteroviridae, although Bayesian functional genomics of closteroviruses. Virus Res 117:38–51 analyses could consistently interpret the genus Ampelovirus 14. Febres VJ, Ashoulin L, Mawassi M, Frank A, Bar-Joseph M, as monophyletic with two major groups. Manjunath KL, Lee RF, Niblett CL (1996) The p27 protein is The inclusion of PMWaV-1 into the genus Ampelovirus present at one end of citrus tristeza virus particles. Phytopathol- ogy 86:1331–1335 requires substantial relaxation of the taxon properties. With 15. Gorbalenya AE, Koonin EV (1993) Helicases: amino acid a 13.1-kb genome, a 28-kDa CP, and lack of a CPd, the sequence comparisons and structure–function relationship. Curr only link PMWaV-1 has with other ampeloviruses is the Opin Struct Biol 3:419–429 vector. If the genomes of PBNSPaV, GLRaV-4, -5, -6, and 16. Gorbalenya AE, Koonin EV, Lai MMC (1991) Putative papain- related thiol proteases of positive-strand RNA viruses. FEBS -9 resemble PMWaV-1 as much as sequence homology and Letters 288:201–205 phylogenetic data indicate, it may be appropriate for this 17. Hu JS, Gonsalves A, Sether D, Ullman DE (1993) Detection of group to form a second mealybug-transmissible genus in pineapple closterovirus, a possible cause of mealybug wilt of the family Closteroviridae. pineapple. Acta Hort 334:411–416 18. Hu JS, Sether DM, Liu XP, Wang M (1997) Use of a tissue blotting immunoassay to examine the distribution of pineapple Acknowledgments The authors thank Dr. A. Rowhani for provid- closterovirus in Hawaii. Plant Dis 81:1150–1154 ing preliminary information on PBNSPaV. This work was funded, in 19. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian part, by a grant from the Hawaii Department of Agriculture (Contract inference of phylogeny. Bioinformatics 17:754–755 No. 49048), a grant through the Tropical/Subtropical Agricultural 20. Karasev AV (2000) Genetic diversity and evolution of clostero- Research (T-STAR) Program (Project No. HAW00985-10145), and a viruses. Annu Rev Phytopathol 38:293–324 grant from the USDA-CSREES Special Research Grants Program- 21. Karasev AV, Nikolaeva OV, Mushegian AR, Lee RF, Dawson Pest Management Alternatives (Project No. HAW00937-G under WO (1996) Organization of the 30terminal half of beet yellow agreement 2003-34381-13659, and by an endowment in honor of stunt virus genome and implications for the evolution of closte- J.R. and Addie S. Graves. roviruses. Virology 221:199–207

123 714 Arch Virol (2008) 153:707–714

22. Koonin EV (1991) The phylogeny of RNA-dependent RNA poly- 31. Sether DM, Hu JS (2002) Closterovirus infection and mealybug merases of positive-strand RNA viruses. J Gen Virol 72:2197–2206 exposure are necessary for the development of mealybug wilt of 23. Ling K-S, Zhu H-Y, Drong RF, Slightom JL, McFerson JR, pineapple disease. Phytopathology 92:928–935 Gonsalves D (1998) Nucleotide sequence of the 30-terminal two- 32. Sether DM, Hu JS (2002) Yield impact and spread of pineapple thirds of the grapevine leafroll-associated virus-3 genome reveals mealybug wilt associated virus-2 and mealybug wilt of pineapple a typical monopartite closterovirus. J Gen Virol 79:1299–1307 in Hawaii. Plant Dis 86:867–874 24. Martelli GP, Agranovsky AA, Bar-Joseph M, Boscia D, 33. Sether DM, Karasev AV, Okumura C, Arakawa C, Zee F, Kislan Candresse T, Coutts RHA, Dolja VV, Falk BW, Gonsalves D, M, Busto J, Hu JS (2001) Differentiation, distribution, and Jelkmann W, Karasev AV, Minafra A, Namba S, Vetten HJ, elimination of two different pineapple mealybug wilt-associated Wisler GC, Yoshikawa H (2002) The family Closteroviridae viruses found in pineapple. Plant Dis 85:856–864 revised. Arch Virol 147:2039–2044 34. Sether DM, Melzer MJ, Busto J, Zee F, Hu JS (2005) Diversity 25. Melzer MJ, Karasev AV, Sether DM, Hu JS (2001) Nucleotide and mealybug transmissibility of ampeloviruses in pineapple. sequence, genome organization, and phylogenetic analysis of Plant Dis 89:450–456 pineapple mealybug wilt-associated virus-2. J Gen Virol 82:1–7 35. Sether DM, Ullman DE, Hu JS (1998) Transmission of pineapple 26. Napuli AJ, Alzhanova DV, Doneanu CE, Barofsky DF, Koonin mealybug wilt-associated virus by two species of mealybug EV, Dolja VV (2003) The 64-kilodalton capsid protein homolog (Dysmicoccus spp.). Phytopathology 88:1224–1230 of Beet yellows virus is required for assembly of virion tails. 36. Swofford DL (2000) PAUP*: Phylogenetics using parsimony J Virol 77:2377–2384 (*and other methods). Sinauer Associates, Sunderland, MA 27. Risler JL, Delorme MO, Delacroix H, Henaut A. (1988) Amino 37. Tian T, Rubio L, Yeh HH, Crawford B, Falk BW (1999) Lettuce acid substitutions in structurally related proteins. A pattern rec- infectious yellows virus: in vitro acquisition analysis using par- ognition approach. Determination of a new and efficient scoring tially purified virions and the whitefly Bemisia tabaci. J Gen matrix. J Mol Biol 204:1019–1029 Virol 80:1111–1117 28. Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian 38. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins phylogenetic inference under mixed models. Bioinformatics 19: DG (1997) The ClustalX windows interface: flexible strategies 1572–1574 for multiple sequence alignment aided by quality analysis tools. 29. Rott ME, Jelkmann W (2005) Little cherry virus-2: sequence and Nucl Acids Res 24:4876–4882 genomic organization of an unusual member of the Closterovir- 39. Zhu H-Y, Ling K-S, Goszczynski DE, McFerson JR, Gonsalves idae. Arch Virol 150:107–123 D (1998) Nucleotide sequence and genome organization of 30. Rozanov MN, Koonin EV, Gorbalenya AE (1992) Conservation grapevine leafroll-associated virus-2 are similar to beet yellows of the putative methyltransferase domain: a hallmark of the virus, the closterovirus type member. J Gen Virol 79:1289–1298 ‘Sindbis-like’ supergroup of positive-strand RNA viruses. J Gen Virol 73:2129–2134

123