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Journal of General Virology (2000), 81, 273–278. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2 : ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses P. J. Shiel and P. H. Berger Plant Pathology Division/Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339, USA The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus. Apple mosaic virus (ApMV) is in subgroup III of the genus Ilarvirus, which includes Prunus necrotic ringspot virus (PNRSV) and rose mosaic viruses (Rybicki, 1995). The genus Ilarvirus comprises a large group of plant viruses with primarily woody hosts and is divided into subgroups based upon their serological characteristics. This genus, along with the genera Alfamovirus, Cucumovirus, Bromovirus and Oleavirus, constitute the family Bromoviridae. Genera are distinguished by the biological properties of the viruses. The Bromoviridae have positive-sense single-stranded RNA genomes divided into three components designated RNA 1, 2 and 3, and contain an RNA 4 which is a subgenomic messenger for the coat protein Author for correspondence : Philip Berger. Fax j1 208 885 7760. e-mail pberger!uidaho.edu The nucleotide sequence data reported in this article have been deposited with EMBL/GenBank under accession numbers AF174584 and AF174585. 0001-6433 # 2000 SGM of the virus (Rybicki, 1995). Ilarviruses and alfalfa mosaic virus (AMV) (the sole member of the genus Alfamovirus) share the biological trait of genome activation, where mixtures of RNAs 1, 2 and 3 are non-infectious unless either subgenomic RNA 4 or coat protein is added. This function can be fulfilled by RNA 4 or coat protein of heterologous as well as homologous viruses of these genera, but not from other genera (Gonsalves & Fulton, 1977). Biological and chemical RNA protection assays show that both the AMV and ilarvirus 3h non-translated region (NTR) specifically bind to coat protein (AnselMcKinney & Gehrke, 1998). Additional experiments with peptides from the coat protein of tobacco streak ilarvirus (TSV) and the TSV 3h NTR showed that specific binding occurs between these two viruses despite only limited sequence homology (Swanson et al., 1998). AMV is separated from the ilarviruses primarily by its mode of transmission : it is transmitted noncirculatively by at least 14 species of aphids (Jaspers & Bos, 1980). TSV is transmitted in a pollen-mediated manner by thrips (Kaiser et al., 1982). Experimental evidence suggests that thrips are also involved in the transmission of PNRSV (Greber et al., 1992). The mechanism of transmission of many of the ilarviruses, including ApMV, is still unknown. We previously reported the complete nucleotide sequences of ApMV RNA 3 and RNA 4. RNA 3 is bicistronic and encodes the putative movement protein (Shiel et al., 1995). The movement protein is directly translated from RNA 3, whereas the coat protein is translated from the subgenomic mRNA, RNA 4 (Alrefai et al., 1994). This is in agreement with the genome organization of other members of the Bromoviridae, where considerable sequence information is available. If the remainder of the ApMV genome shares the genomic organization of the Bromoviridae, then RNAs 1 and 2 should encode the virus replicases (Rybicki, 1995). Recently, a second open reading frame (ORF) on RNA 2 was identified on all of the ilarviruses for which sequence data are available (Xin et al., 1998). This 2b ORF is expressed in a manner similar to the 2b ORF present in cucumoviruses, even though it encodes a larger peptide and has no sequence similarity to the 2b ORF of cucumoviruses. It is interesting that there is no corresponding 2b ORF in AMV. CHD P. J. Shiel and P. H. Berger While complete nucleotide sequences of AMV and several bromoviruses and cucumoviruses have been available for some time (Brunt et al., 1996), it was only recently that any complete ilarvirus sequences were published (Scott & Ge, 1995 ; Ge et al., 1997 ; Scott et al., 1998). This is probably due in part to the difficulty in handling and manipulating ilarviruses, relative to many other viruses. In addition, most of the known ilarviruses primarily infect woody perennial hosts, where often they are present only in low titre. In general, they are difficult to isolate from their woody hosts and are poorly transmitted to such hosts by mechanical means. ApMV is a pathogen with a diverse natural host-range consisting primarily of woody plants. Besides apple, ApMV naturally infects rose, hazelnut, filbert, horse chestnut, raspberry, birch and hops (Rybicki, 1995). However, due to the labile nature of the virus, isolation has been difficult, and the host-range of the virus has usually been determined using grafts of infected plants (Sweet & Barbara, 1979). Our objective was to characterize the entire genome of ApMV to facilitate detection and further study of this pathogen of woody plants. ApMV was isolated from infected symptomatic apple trees (Malus domestica L.) var. ‘ Golden Delicious ’. The virus was propagated in and purified from inoculated cotyledons of cucumber (Cucumis sativus L. var. Lemon) as previously described (Alrefai et al., 1994). The virus was further purified by 10–40 % sucrose density-gradient centrifugation (SDGC), which separated the virus into an upper band and two closely spaced lower bands. The lower bands (which contain RNA 1 and 2) were separated from the upper band (which contains RNA 3) and concentrated for RNA purification. RNA was extracted from the bottom component of the SDGC-purified virions by adding virus to dissociation buffer containing 1 % SDS and then extracting RNA with phenol heated to 60 mC. Purified ApMV RNA was polyadenylated by using poly(A) polymerase (Gibco\BRL) and used as a template to synthesize the first-strand cDNA with oligo d(T) and Moloney murine leukaemia reverse transcriptase or Superscript II (Gibco\BRL). The first-strand reaction products were used for synthesis of second-strand cDNA, according to the manufacturer’s protocols (Promega). Double-stranded cDNA was blunt-end ligated into the EcoRV site of pBluescript, and these reactions were used to transform E. coli strain DH10b. Plasmids containing sizeable inserts were analysed by restriction enzyme mapping and were further purified for doublestranded DNA sequencing. Each plasmid was treated with 10 mg\ml RNase A (Sigma) at 37 mC for 30 min, extracted with phenol–chloroform and precipitated with ethanol. The DNA was sequenced in both directions by dideoxychain termination method using the Sequitherm Long-Read Cycle Sequencing Kit (Epicentre Technologies) according to the manufacturer’s protocols and analysed using a Li-Cor 4000L automated DNA sequencing system and IRD-41 labelled primers. The 5h-ends of RNA 1 and 2 were cloned by 5h-rapid amplification of cDNA ends (5h-RACE) (Gibco\BRL CHE version 2.0) according to the manufacturer’s protocols and sequenced as above. Nucleic acid and amino acid sequence data were analysed with the University of Wisconsin Genetics Computer Group sequence analysis software package (GCG version 8) at the Center for Visualization, Analysis, and Design of Molecular Sequences at Washington State University. Additional analyses were performed with DNA Strider (version 1.3, CEA, France). The RNA secondary structure of the 5h-noncoding regions was analysed with the RNAFOLD program (RNA Secondary Structure Predictor, version 2.0 ; Scientific and Educational Software). Phylogenetic analysis was done with the program PAUP (Phylogenetic Analysis Using Parsimony, version 4.0). cDNA clones that contained ApMV RNA 1 and 2 were initially identified on the basis of sequences that were homologous to the 3h-NTR of RNA 3 but not to the rest of RNA 3, because the 3h-NTRs of the three RNAs of ApMV (and all other bromoviruses) are the only region which has sequence homology within the genome of each species. In addition to RNA 3, two other sets of clones were found. Using the BLAST program, one of these sets of clones was found to encode peptides similar to the putative polymerase regions of AMV and Citrus leaf rugose virus (CiLRV), which are encoded by RNA 2 of these viruses. The other set of clones encoded peptides similar to the putative helicase region of these viruses, which is encoded by RNA 1. In addition, both sets of clones showed a lesser degree of similarity to the putative polymerase and helicase regions of other members of the Bromoviridae, as well as several tobamoviruses and tobraviruses. Additional clones were then identified and characterized based on homologies to those already obtained and a library of clones was generated for each set. Sixteen nucleotides of the ApMV RNA 1 5h-end and 499 nucleotides of the ApMV RNA 2 5hend were cloned and sequenced by 5h RACE with two sequence-specific primers for each RNA. ApMV has three genomic RNAs and a subgenomic RNA which contains the coat protein cistron. The largest RNA, RNA 1, is 3476 nucleotides long and encodes a single large polypeptide which is similar to the methyltransferase-like and helicase-like domains present in many plant RNA viruses. This RNA encodes a predicted open reading frame of 1046 amino acids (aa) with a relative molecular mass (Mr) of 118 416. These are comparable to the protein products of the RNA 1 of AMV, CiLRV and elm mottle virus (EMoV). These sequences have homology to molecules that have been demonstrated to act as methyltransferases and helicases. The nucleotide sequence of ApMV RNA 2 was also determined. This sequence is 2979 nucleotides in length and contains a single large ORF. The predicted amino acid sequence of this ORF shows similarity to the corresponding protein product of AMV and other ilarvirus sequences that are currently available. It encodes a predicted peptide product that is 875 aa in length with an Mr of 99 866. This is comparable to (a) (b) (c) CHF ApMV RNA 1 and 2 sequences Fig. 1. Phylogenetic trees inferred by parsimony analysis of (a) the putative methyltransferase/helicase protein, (b) the polymerase protein and (c) the movement protein of members of the Bromoviridae. Horizontal branch lengths are proportional to evolutionary distance. Analysis was done under exhaustive search conditions. The number on each branch is the result of bootstrap analysis. TMV is used as the outgroup in trees (a) and (b) ; tree (c) is unrooted. Serogroups denoted with an asterisk (*) contain definable zinc-finger domains. See text and Table 1 for definitions of virus acronyms. P. J. Shiel and P. H. Berger Table 1. GenBank accession numbers of sequences used in this study (a) Methyltransferase/helicase motifs Accession no. Accession no. Virus Helicase Polymerase Virus Helicase Polymerase AMV BMV CiLRV CMV EMoV L00163 X58456 U23715 D16403 U57047 K02702 X58457 U17726 D10209 U34050 PDV RBDV SpLV TMV TSV U57648 S51557 U93192 X02144 U80934 * S51557 U93193 X02144 U75538 * Sequence not published. (b) Putative movement protein, coat protein, and 3h-NTR of RNA 3 Virus acronyms not otherwise defined in the text : AV, Asparagus virus ; CVV, Citrus variegation virus ; HdMV, Hydrangea mosaic virus ; LRMV, lilac ring mottle virus. Virus AMV-4M AMV-S ApMV-A ApMV-G AV-2 CiLRV CVV EMoV-1 EMoV-2 HdMV LRMV PDV-Sc PDV-Sk PNRSV-CH9 PNRSV-CH30 Accession no. K02703 X00819 U15608 S78319 X86352 U17390 U17389 U57048 U85399 U35145 U17391 L28145 U31310 AF034992 AF034994 Virus PNRSV-CH38 PNRSV-CH39 PNRSV-CH57 PNRSV-CH61 PNRSV-CH71 PNRSV-G PNRSV-Mi PNRSV-P5 PNRSV-Pr PNRSV-Sc PNRSV-Sp1 PNRSV-Sp2 PNRSV-SW6 SpLV TSV the protein products of the RNA 2 of AMV, CiLRV and EMoV. The RNA 2 ORFs of ApMV, like AMV, CiLRV, EMoV, raspberry bushy dwarf virus (RBDV), spinach latent virus (SpLV) and TSV, share sequence homology, especially in the region of the consensus signature motif DX [FYWLCA]X – DXn[STM]GX TX [NE]XnGDD $ $ $ !" found in most virus-encoded RNA-dependent RNA polymerases (Buck, 1997). In addition, all of these proteins appear to have a predominance of acidic residues near the aminoterminal end, and a similar predominance of basic residues near the carboxyl terminus. Unlike all of the ilarviruses that have been sequenced to date, ApMV does not contain a second ORF (ORF 2b), and is similar to AMV in that respect. When GenBank was searched with the BLAST and TFASTA programs for sequences similar to ApMV RNA 1 and CHG Accession no. AF034991 AF034990 AF034993 AF034989 AF034995 S78312 AF013285 L38823 AF013286 U57046 U03857 Y07568 AF013287 U93194 X00435 2, results indicated strong similarities between these sequences and proteins of AMV, brome mosaic virus (BMV), CiLRV, cucumber mosaic virus (CMV), EMoV, prune dwarf virus (PDV), RBDV, SpLV, tobacco mosaic virus (TMV) and TSV. ApMV appeared to be more closely related to AMV and PDV than to other viruses. Phylogenetic inferences were obtained using both parsimony and the distance-based neighbourjoining methods. Since dendrograms generated by either method were topologically indistinguishable, only results using parsimony are presented here (Fig. 1). These dendrograms show that both sequences are more closely related to AMV than to the ilarviruses from other subgroups. The putative movement protein on RNA 3 shows the highest degree of homology to the other ilarviruses that infect rosaceous hosts, particularly PNRSV (Fig. 1). Next in similarity is the movement protein of AMV, which is clearly between the ApMV RNA 1 and 2 sequences Fig. 2. Alignment and secondary structural features of the 5h NTR of ApMV RNA 1 and RNA 2. Initiation codons for the large ORFs of both RNAs are boxed. Bases involved in secondary structure are underlined. subgroup III ilarviruses and other ilarvirus subgroups. This is in contrast to the ApMV coat protein, which shows similarity only to the other ilarviruses, as attempts to produce a multiple sequence alignment of the ilarviruses with AMV or the other viruses were unsuccessful (data not shown). The 5h-NTR of ApMV RNA 1 is 78 nucleotides long and the 5h-NTR of ApMV RNA 2 is 79 nucleotides long. The RNAs show extensive homology (77 %), with the first 31 nucleotides identical to each other. These nucleotides are involved in base-pairing for predicted stem–loop structures in the 5h-NTR (Fig. 2). In addition, both RNAs have a second predicted stem–loop structure immediately before their respective translation initiation codons. These two stem–loops have similar sequences but have subtle and perhaps significant differences. The stem–loop of RNA 2 includes two of the four bases immediately upstream of the initiation codon, whereas in RNA 1 the stem structure ends six nucleotides away from the initiation codon. The ‘ context ’ nucleotides immediately surrounding the initiation codon have profound effects on the ability of messenger RNAs to translate protein products, and this secondary structure present on the 5h-NTRs of RNA 1 and 2 may affect expression of their proteins (Taylor et al., 1987). Both loop sequences can potentially interact with other nucleotides in the 5h-NTR. The GAAAAG loop sequence in the RNA 2 stem–loop is complementary to CUUUUC and $) %$ could form a pseudoknot structure. There is no detectable similarity between the 5h-NTR of RNA 1 and 2 and the 5h-NTR of RNA 3. In addition, there does not appear to be any consensus internal control region (ICR) on the 5h-NTR of RNA 1 or 2, as was previously observed on RNA 3 of ApMV and some of the other Bromoviridae (Shiel et al., 1995). This also appears to be the case for RNA 1 and 2 of AMV and the known ilarvirus sequences. However, there is an eight base palindrome sequence (GAUUAAUC) that occurs at nucleotide 36 of both RNAs of CiLRV and EMoV. ApMV RNA 1 and 2 also has an eight nucleotide palindromic sequence in an A–U-rich area immediately after the 5h stem–loop at base 22 (AAUUAAUU) (Fig. 2). This is in contrast to the 5h-NTR sequence of RNA 3, which is a 13 nucleotide palindrome, is G–C-rich, and has homology to the consensus ICR 2 sequence (Shiel et al., 1995). Since ApMV is more closely related to AMV than to the other ilarviruses in all of its gene products except for its coat protein, a strictly linear descent of these viruses from a single progenitor seems unlikely. This conclusion is most clearly supported by comparing the viruses that have a known zincfinger domain in their coat protein, and those that either don’t bind zinc or use a different motif for this purpose. TSV and the zinc-finger-containing coat proteins are most distantly related to ApMV, whereas the ilarviruses without a zinc-finger motif are intermediate. Phylogenetic analysis of movement proteins results in groupings according to the natural host-range of the CHH P. J. Shiel and P. H. Berger virus. Furthermore, the methyltransferase\helicase as well as the polymerase domains of ApMV (zinc-finger domain present) appear to be more closely related to AMV and to PDV than to any of the other ilarviruses (Fig. 1). These data further support the idea that functional domains of the virus were obtained from disparate host genes or other co-infecting ilarviruses rather than by linear descent of these viruses from a single progenitor. A polyphyletic origin of the ilarviruses could also have been the result of recombination events. It is possible that AMV originated from ancestors in common with ApMV, PNRSV and PDV, and acquired its coat protein gene from another virus (or perhaps a plant gene), enabling it to be transmitted by aphids, but retained its requirement for coat protein in genome activation. In conclusion, phylogenetic analysis of available sequences of the protein products encoded by ApMV RNA 1 and 2 and the putative movement protein encoded by RNA 3 indicate that ApMV is more closely related to AMV than to the other ilarviruses. These relationships with other related viruses differ from those observed with coat proteins of ApMV, which is more closely related to the ilarviruses than to AMV. It has been noted by other researchers that perhaps AMV should be included as a member of the ilarviruses, rather than as a distinct genus (Sanchez-Navarro & Pallas, 1997 ; Scott et al., 1998). Phylogenetic analysis of the ApMV genome supports this relationship by placing AMV between ApMV and other ilarviruses. 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