Arch Virol DOI 10.1007/s00705-015-2489-9
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a novel velarivirus infecting areca palm in China Hongmei Yu1 • Shuishui Qi1 • Zhaoxia Chang1 • Qiqi Rong1 • Ibukun A. Akinyemi1 Qingfa Wu1
•
Received: 7 February 2015 / Accepted: 6 June 2015 Ó Springer-Verlag Wien 2015
Abstract The complete genome of a novel virus, provisionally named areca palm velarivirus 1 (APV1), was identified in areca palm exhibiting leaf yellowing symptoms in Hainan province, China. The genome of APV1 consists of 16,080 nucleotides and possesses 11 open reading frames (ORFs), sharing 56.4 % nucleotide sequence identity with little cherry virus 1 (NC_001836.1). The genome organization of APV1 is highly similar to that of members of the genus Velarivirus (family Closteroviridae). Phylogenetic analysis placed APV1 together with members of the genus Velarivirus.
Areca palm (Areca catechu L.) is one of the most important commercial crops in Hainan province, China. Yellow leaf disease (YLD) is a serious disease of areca palm in Hainan [5]. Initial symptoms of the disease are the yellowing of leaves in the inner whorl, gradually spreading to the outer whorl of the crown. Stems of the affected palms become spongy and friable, and the conducting strands get destroyed. This disease seriously affects the cultivation, consumption and medicinal usage of areca palm, but there is no clear information about the cause. Viruses of the family Closteroviridae have a positivesense, single-stranded RNA genome and are grouped into four genera: Ampelovirus, Closterovirus, Crinivirus and
Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2489-9) contains supplementary material, which is available to authorized users. & Qingfa Wu [email protected] 1
School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui, China
Velarivirus [8, 9]. The genus Velarivirus includes the viruses grapevine leafroll-associated virus 7 (GLRaV-7), little cherry virus 1 (LChV-1) and cordyline virus 1 (CoV1) [8]. In this study, the nucleotide sequence of the complete genome of a novel velarivirus obtained from the leaves of a diseased areca palm was determined. Areca palm leaves displaying yellowing symptoms were collected from Hainan province, China, in 2012. A small RNA library was prepared from the leaves and sequenced using an Illumina HiSeq-2000 (BGI-ShenZhen, China), yielding 46,613,994 clean reads with sizes between 17 and 30 nucleotides (nt). The small RNAs were assembled into 11,246 contigs with a k-mer of 17 using Velvet [10, 17]. These contigs were compared with the non-redundant nucleotide and protein database of GenBank by BLASTn and BLASTx, respectively. In addition to 2,167 contigs showing C90 % identity and C90 % coverage with the sequences in GenBank by BLASTn, 21 contigs with lengths between 88 and 414 nt were identified by BLASTx (e-value cutoff: 10-3) to have distant similarities with members of the family Closteroviridae, including eight contigs showing highest similarity with LChV-1 (NC_001836.1), five contigs showing highest similarity with GLRaV-7 (NC_016436.1), three contigs showing highest similarity with cordyline virus 4 (JQ599284.2), three other contigs showing highest similarity with cordyline virus 3 (JQ599283.2), one contig showing highest similarity with CoV1 (HM588723.1), and one contig showing highest similarity with cordyline virus 2 (JQ599282.2). In order to further characterize these 21 contigs, the genome sequence of LChV-1 was used as reference to determine the relative positions and orientation of these 21 contigs. Then, RT-PCR and Sanger sequencing were performed to join these contigs together and validate ambiguous nucleotides. The 5’ and 3’ ends of the viral
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genome were obtained by RACE-PCR and Sanger sequencing. Mapping of small RNAs to the viral genome was performed with Bowtie [11]. ORFs were predicted using ORF Finder. Conserved domains/motifs were analyzed using SMART [12]. Multiple sequence alignment was performed with Multalin [4]. A neighbor-joining phylogenetic tree was generated using MEGA 5 [16]. The complete genome of APV1 consists of 16,080 nt, with a G?C content of 35 %. A total of 4,276,300 (9.2 %) small RNAs were perfectly matched to the APV1 genome, corresponding to 5,622-fold coverage. We also found that 274 assembled contigs showed C95 % identity to the viral genome, covering 95.4 % of the genome. The APV1
genome possesses 11 open reading frames (ORFs) with a 144-nt-long 5’ UTR and a 37-nt-long 3’ UTR (Fig. 1A). The first two ORFs (ORF1a and ORF1b) are involved in replication of the virus [6]. ORF1a (nt 145-6,294) encodes a putative 234-kDa polyprotein that shares 90 % coverage and 30 % identity with the homologous protein of LChV1 (AGB06245.1) and 75 % coverage and 34 % identity with that of GRLaV7 (AEQ59443.1). Conserved domain analysis revealed that the putative ORF1a-encoded protein includes three enzymatic motifis: papain-like protease (LPRO, aa residues 1-393), methyltransferase (MTR, aa residues 455-840) and helicase (HEL, aa residues 1758-2016) (Fig. 1A) [1, 7]. The catalytic Cys and His
Fig. 1 (A) Schematic representation of the genome organization of areca palm virus 1 (APV1). L-PRO, papain-like protease; MTR, RNA methyltransferase; HEL, RNA helicase; RdRp, RNA-dependent RNA polymerase; p4, 4-kDa protein; Hsp70h, heat shock protein 70 homologue; p21, 21-kDa protein; p60, 60-kDa protein; CP, coat protein; CPm, minor coat protein; p26, 26-kDa protein; p18, 18-kDa protein; p19, 19-kDa protein. (B) Phylogenetic tree based on the
deduced amino acid sequence of ORF1a of APV1 and those of members of the family Closteroviridae constructed with MEGA 5 using the neighbour-joining method. Accession numbers and virus names are given directly in the phylogenetic tree. Values at the nodes are the bootstrap values from 1000 replicates, and the bars represent the evolutionary distance
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residues of L-PRO were identified at amino acid positions 325 and 374, respectively, whereas the cleavage site was predicted to be between G393 and A394 [1, 7]. Two 82-aa repeats (aa residues 1-83 and 311-393) sharing 66 % identity were identified in the L-PRO domain of APV1, and these had not been identified in other members of the family Closteroviridae. The termination stop codon for ORF1a is UGA, which is same for all the members of the genus Velarivirus [8]. The putative RNA-dependent RNA polymerase (RdRp) of APV1 is encoded by ORF1b (nt 6,293-7,810), which probably produces a fusion protein of 2554 aa (293 kDa) by a ?1 ribosomal frameshift. The 59-kDa RdRp domain shares 59 % and 57 % identity with the homologous proteins of GRLaV7 (BAM29350.1) and LChV1 (CAA71293.1), respectively. ORFs 2-9 are similar to the correspondingly positioned ORFs in members of the genus Velarivirus. ORF2 (nt 7,812-7,922) putatively encodes a hydrophobic protein of 36 aa (4 kDa; p4), which is similar to the ORF2 of GRLaV7 but shares no significant sequence similarities to GenBank entries. P4 is widely considered to be a transmembrane protein, and the putative APV1 p4 contains a transmembrane domain (aa residues 2-24) [8]. Previous studies indicated that p4 may be a type III membrane protein with the N-terminus located within the lumen, and it is believed to play an important role in cell-to-cell movement [14]. ORF3 (nt 7,978-9,618) encodes a 546-aa (61-kDa) protein that is a HSP70 homologue (HSP70h) and shares 45 % identity with the GRLaV7 Hsp70h (BAM29351.1) and 42 % identity with the LChV1 Hsp70h (NP_045004.1). Alignment of Hsp70 homologues encoded by APV1, beet yellows virus (BYV, genus Closterovirus) and other members of the genus Velarivirus showed that the APV1 HSP70h has a conserved N-terminal region and also contains the conserved residues D7 and E172, which may play significant roles in tail assembly and cell-to-cell movement [2]. ORF5 (nt 10,087-11,628) encodes a putative protein of 513 aa (60 kDa; p60) that shares 24 %-31 % identity with the p61 proteins encoded by members of the genus Velarivirus and contains a conserved Viral_Hsp90 domain (pfam03225). The p60 is a hallmark of closteroviruses that functions together with Hsp70h and CPm in tail assembly and cell-to-cell movement [3]. OFR6 (nt 11,641-12,537) and ORF7 (nt 12,540-14,420) encodes the putative major coat protein (CP) of 298 aa (33 kDa) and minor coat protein (CPm) of 626 aa (72 kDa), respectively. The CP shares highest identity of 35 % with LChV1 CP (ACG69819.1), and CPm shares 22 % identity with CoV1 CPm (ADU03661.1). The CP encapsidates most of the helical nucleocapsid, while CPm, together with Hsp70h and p60, encapsidate a small portion of the 5’ end (the ‘‘tail’’), all of which have functions in virion assembly and cell-to-cell movement [3, 15]. Multiple alignment using the
CPs and CPms of APV1, BYV, and other members of the genus Velarivirus showed that both CP and CPm of APV1 have a conserved arginine (R211 of CP, R546 of CPm) and aspartic acid (D252 of CP, D587 of CPm) that may be involved in those functions [2, 13]. In summary, the APV1 quintuple gene block putatively encodes five proteins (p4, HSP70h, p60, CP and CPm), which are all homologous to proteins encoded by other closteroviruses and may be involved in cell-to-cell movement and virion assembly. A putative protein of 178 aa (21 kDa; p21) is encoded by ORF4 (nt 9,560-10,096, within the quintuple gene block) with no obvious similarity with other proteins in GenBank. The position of ORF4 in APV1 and GRLaV7 is similar, and in each case, it partially overlaps ORF3 and ORF5. It is the largest protein between HSP70h and p60 among the counterparts encoded by members of genus Velarivirus [8]. There is no information about p21 counterparts in other closteroviruses, but interestingly, we found two internal repeats in ORF4 (aa residues 6-85 and 99-178) that may provide insights into its functions. ORF8 (nt 14,396-15,043) encodes a putative protein of 215 aa (26 kDa; p26), ORF9 (nt 15,040-15,489) encodes a putative protein of 149 aa (18 kDa; p18), and ORF10 (nt 15,534-16,043) encodes a putative protein of 169 aa (19 kDa; p19) (Fig. 1A). These three proteins show no significant similarity to other viral proteins, and their functions remain to be determined. In particular, p19, encoded by ORF10, seems to be unique to APV1, as no counterpart is present at the analogous genome position of members of the genus Velarivirus. In order to determined the relationship between APV1 and other closteroviruses, the amino acid sequences of ORF1a from several members of the family Closteroviridae were used to construct a phylogenetic tree, which revealed that APV1 is most closely related to LChV-1 and GLRaV-7, both of which are members of genus Velarivirus (Fig. 1B). Thus, the complete genome sequence suggests that APV1 should be considered a member of a novel species in the genus Velarivirus. This finding will serve as the foundation for further research to study the relationship between the new virus and yellow leaf disease of areca palm. Acknowledgments This study was supported by grants from the National Basic Research Program of China (no. 2014CB138405), the Chinese National Natural Science Foundation (no. 31272011), and the Doctoral Fund of the Ministry of Education of China (20123402110013).
References 1. Agranovsky AA, Koonin EV, Boyko VP, Maiss E, Frotschl R, Lunina NA, Atabekov JG (1994) Beet yellows closterovirus: complete genome structure and identification of a leader papainlike thiol protease. Virology 198:311–324
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H. Yu et al. 2. Alzhanova DV, Napuli AJ, Creamer R, Dolja VV (2001) Cell-tocell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog. EMBO J 20:6997–7007 3. Alzhanova DV, Prokhnevsky AI, Peremyslov VV, Dolja VV (2007) Virion tails of Beet yellows virus: Coordinated assembly by three structural proteins. Virology 359:220–226 4. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890 5. Daquan LMC, Shabin Y (2001) Identification of pathogens of yellow leaf disease of arecanut in Hainan island. Chinese J Trop Crops 22:43–46 6. Dolja VV, Kreuze JF, Valkonen JPT (2006) Comparative and functional genomics of closteroviruses. Virus Res 117:38–51 7. Jelkmann W, Fechtner B, Agranovsky AA (1997) Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus. J Gen Virol 78:2067–2071 8. Jelkmann W, Mikona C, Turturo C, Navarro B, Rott ME, Menzel W, Saldarelli P, Minafra A, Martelli GP (2012) Molecular characterization and taxonomy of grapevine leafroll-associated virus 7. Arch Virol 157:359–362 9. Karasev AV (2000) Genetic diversity and evolution of closteroviruses. Annu Rev Phytopathol 38:293–324 10. Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7
123
11. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Gen Biol 10:R25 12. Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43:D257– D260 13. Napuli AJ, Alzhanova DV, Doneanu CE, Barofsky DF, Koonin EV, Dolja VV (2003) The 64-kilodalton capsid protein homolog of Beet yellows virus is required for assembly of virion tails. J Virol 77:2377–2384 14. Peremyslov VV, Pan YW, Dolja VV (2004) Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum. J Virol 78:3704–3709 15. Satyanarayana T, Gowda S, Ayllon MA, Dawson WO (2004) Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5’ region. Proc Natl Acad Sci USA 101:799–804 16. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 17. Wu Q, Luo Y, Lu R, Lau N, Lai EC, Li WX, Ding SW (2010) Virus discovery by deep sequencing and assembly of virusderived small silencing RNAs. Proc Natl Acad Sci USA 107:1606–1611
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a novel velarivirus infecting areca palm in China Hongmei Yu1 • Shuishui Qi1 • Zhaoxia Chang1 • Qiqi Rong1 • Ibukun A. Akinyemi1 Qingfa Wu1
•
Received: 7 February 2015 / Accepted: 6 June 2015 Ó Springer-Verlag Wien 2015
Abstract The complete genome of a novel virus, provisionally named areca palm velarivirus 1 (APV1), was identified in areca palm exhibiting leaf yellowing symptoms in Hainan province, China. The genome of APV1 consists of 16,080 nucleotides and possesses 11 open reading frames (ORFs), sharing 56.4 % nucleotide sequence identity with little cherry virus 1 (NC_001836.1). The genome organization of APV1 is highly similar to that of members of the genus Velarivirus (family Closteroviridae). Phylogenetic analysis placed APV1 together with members of the genus Velarivirus.
Areca palm (Areca catechu L.) is one of the most important commercial crops in Hainan province, China. Yellow leaf disease (YLD) is a serious disease of areca palm in Hainan [5]. Initial symptoms of the disease are the yellowing of leaves in the inner whorl, gradually spreading to the outer whorl of the crown. Stems of the affected palms become spongy and friable, and the conducting strands get destroyed. This disease seriously affects the cultivation, consumption and medicinal usage of areca palm, but there is no clear information about the cause. Viruses of the family Closteroviridae have a positivesense, single-stranded RNA genome and are grouped into four genera: Ampelovirus, Closterovirus, Crinivirus and
Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2489-9) contains supplementary material, which is available to authorized users. & Qingfa Wu [email protected] 1
School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui, China
Velarivirus [8, 9]. The genus Velarivirus includes the viruses grapevine leafroll-associated virus 7 (GLRaV-7), little cherry virus 1 (LChV-1) and cordyline virus 1 (CoV1) [8]. In this study, the nucleotide sequence of the complete genome of a novel velarivirus obtained from the leaves of a diseased areca palm was determined. Areca palm leaves displaying yellowing symptoms were collected from Hainan province, China, in 2012. A small RNA library was prepared from the leaves and sequenced using an Illumina HiSeq-2000 (BGI-ShenZhen, China), yielding 46,613,994 clean reads with sizes between 17 and 30 nucleotides (nt). The small RNAs were assembled into 11,246 contigs with a k-mer of 17 using Velvet [10, 17]. These contigs were compared with the non-redundant nucleotide and protein database of GenBank by BLASTn and BLASTx, respectively. In addition to 2,167 contigs showing C90 % identity and C90 % coverage with the sequences in GenBank by BLASTn, 21 contigs with lengths between 88 and 414 nt were identified by BLASTx (e-value cutoff: 10-3) to have distant similarities with members of the family Closteroviridae, including eight contigs showing highest similarity with LChV-1 (NC_001836.1), five contigs showing highest similarity with GLRaV-7 (NC_016436.1), three contigs showing highest similarity with cordyline virus 4 (JQ599284.2), three other contigs showing highest similarity with cordyline virus 3 (JQ599283.2), one contig showing highest similarity with CoV1 (HM588723.1), and one contig showing highest similarity with cordyline virus 2 (JQ599282.2). In order to further characterize these 21 contigs, the genome sequence of LChV-1 was used as reference to determine the relative positions and orientation of these 21 contigs. Then, RT-PCR and Sanger sequencing were performed to join these contigs together and validate ambiguous nucleotides. The 5’ and 3’ ends of the viral
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H. Yu et al.
genome were obtained by RACE-PCR and Sanger sequencing. Mapping of small RNAs to the viral genome was performed with Bowtie [11]. ORFs were predicted using ORF Finder. Conserved domains/motifs were analyzed using SMART [12]. Multiple sequence alignment was performed with Multalin [4]. A neighbor-joining phylogenetic tree was generated using MEGA 5 [16]. The complete genome of APV1 consists of 16,080 nt, with a G?C content of 35 %. A total of 4,276,300 (9.2 %) small RNAs were perfectly matched to the APV1 genome, corresponding to 5,622-fold coverage. We also found that 274 assembled contigs showed C95 % identity to the viral genome, covering 95.4 % of the genome. The APV1
genome possesses 11 open reading frames (ORFs) with a 144-nt-long 5’ UTR and a 37-nt-long 3’ UTR (Fig. 1A). The first two ORFs (ORF1a and ORF1b) are involved in replication of the virus [6]. ORF1a (nt 145-6,294) encodes a putative 234-kDa polyprotein that shares 90 % coverage and 30 % identity with the homologous protein of LChV1 (AGB06245.1) and 75 % coverage and 34 % identity with that of GRLaV7 (AEQ59443.1). Conserved domain analysis revealed that the putative ORF1a-encoded protein includes three enzymatic motifis: papain-like protease (LPRO, aa residues 1-393), methyltransferase (MTR, aa residues 455-840) and helicase (HEL, aa residues 1758-2016) (Fig. 1A) [1, 7]. The catalytic Cys and His
Fig. 1 (A) Schematic representation of the genome organization of areca palm virus 1 (APV1). L-PRO, papain-like protease; MTR, RNA methyltransferase; HEL, RNA helicase; RdRp, RNA-dependent RNA polymerase; p4, 4-kDa protein; Hsp70h, heat shock protein 70 homologue; p21, 21-kDa protein; p60, 60-kDa protein; CP, coat protein; CPm, minor coat protein; p26, 26-kDa protein; p18, 18-kDa protein; p19, 19-kDa protein. (B) Phylogenetic tree based on the
deduced amino acid sequence of ORF1a of APV1 and those of members of the family Closteroviridae constructed with MEGA 5 using the neighbour-joining method. Accession numbers and virus names are given directly in the phylogenetic tree. Values at the nodes are the bootstrap values from 1000 replicates, and the bars represent the evolutionary distance
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Novel velarivirus infecting areca palm in China
residues of L-PRO were identified at amino acid positions 325 and 374, respectively, whereas the cleavage site was predicted to be between G393 and A394 [1, 7]. Two 82-aa repeats (aa residues 1-83 and 311-393) sharing 66 % identity were identified in the L-PRO domain of APV1, and these had not been identified in other members of the family Closteroviridae. The termination stop codon for ORF1a is UGA, which is same for all the members of the genus Velarivirus [8]. The putative RNA-dependent RNA polymerase (RdRp) of APV1 is encoded by ORF1b (nt 6,293-7,810), which probably produces a fusion protein of 2554 aa (293 kDa) by a ?1 ribosomal frameshift. The 59-kDa RdRp domain shares 59 % and 57 % identity with the homologous proteins of GRLaV7 (BAM29350.1) and LChV1 (CAA71293.1), respectively. ORFs 2-9 are similar to the correspondingly positioned ORFs in members of the genus Velarivirus. ORF2 (nt 7,812-7,922) putatively encodes a hydrophobic protein of 36 aa (4 kDa; p4), which is similar to the ORF2 of GRLaV7 but shares no significant sequence similarities to GenBank entries. P4 is widely considered to be a transmembrane protein, and the putative APV1 p4 contains a transmembrane domain (aa residues 2-24) [8]. Previous studies indicated that p4 may be a type III membrane protein with the N-terminus located within the lumen, and it is believed to play an important role in cell-to-cell movement [14]. ORF3 (nt 7,978-9,618) encodes a 546-aa (61-kDa) protein that is a HSP70 homologue (HSP70h) and shares 45 % identity with the GRLaV7 Hsp70h (BAM29351.1) and 42 % identity with the LChV1 Hsp70h (NP_045004.1). Alignment of Hsp70 homologues encoded by APV1, beet yellows virus (BYV, genus Closterovirus) and other members of the genus Velarivirus showed that the APV1 HSP70h has a conserved N-terminal region and also contains the conserved residues D7 and E172, which may play significant roles in tail assembly and cell-to-cell movement [2]. ORF5 (nt 10,087-11,628) encodes a putative protein of 513 aa (60 kDa; p60) that shares 24 %-31 % identity with the p61 proteins encoded by members of the genus Velarivirus and contains a conserved Viral_Hsp90 domain (pfam03225). The p60 is a hallmark of closteroviruses that functions together with Hsp70h and CPm in tail assembly and cell-to-cell movement [3]. OFR6 (nt 11,641-12,537) and ORF7 (nt 12,540-14,420) encodes the putative major coat protein (CP) of 298 aa (33 kDa) and minor coat protein (CPm) of 626 aa (72 kDa), respectively. The CP shares highest identity of 35 % with LChV1 CP (ACG69819.1), and CPm shares 22 % identity with CoV1 CPm (ADU03661.1). The CP encapsidates most of the helical nucleocapsid, while CPm, together with Hsp70h and p60, encapsidate a small portion of the 5’ end (the ‘‘tail’’), all of which have functions in virion assembly and cell-to-cell movement [3, 15]. Multiple alignment using the
CPs and CPms of APV1, BYV, and other members of the genus Velarivirus showed that both CP and CPm of APV1 have a conserved arginine (R211 of CP, R546 of CPm) and aspartic acid (D252 of CP, D587 of CPm) that may be involved in those functions [2, 13]. In summary, the APV1 quintuple gene block putatively encodes five proteins (p4, HSP70h, p60, CP and CPm), which are all homologous to proteins encoded by other closteroviruses and may be involved in cell-to-cell movement and virion assembly. A putative protein of 178 aa (21 kDa; p21) is encoded by ORF4 (nt 9,560-10,096, within the quintuple gene block) with no obvious similarity with other proteins in GenBank. The position of ORF4 in APV1 and GRLaV7 is similar, and in each case, it partially overlaps ORF3 and ORF5. It is the largest protein between HSP70h and p60 among the counterparts encoded by members of genus Velarivirus [8]. There is no information about p21 counterparts in other closteroviruses, but interestingly, we found two internal repeats in ORF4 (aa residues 6-85 and 99-178) that may provide insights into its functions. ORF8 (nt 14,396-15,043) encodes a putative protein of 215 aa (26 kDa; p26), ORF9 (nt 15,040-15,489) encodes a putative protein of 149 aa (18 kDa; p18), and ORF10 (nt 15,534-16,043) encodes a putative protein of 169 aa (19 kDa; p19) (Fig. 1A). These three proteins show no significant similarity to other viral proteins, and their functions remain to be determined. In particular, p19, encoded by ORF10, seems to be unique to APV1, as no counterpart is present at the analogous genome position of members of the genus Velarivirus. In order to determined the relationship between APV1 and other closteroviruses, the amino acid sequences of ORF1a from several members of the family Closteroviridae were used to construct a phylogenetic tree, which revealed that APV1 is most closely related to LChV-1 and GLRaV-7, both of which are members of genus Velarivirus (Fig. 1B). Thus, the complete genome sequence suggests that APV1 should be considered a member of a novel species in the genus Velarivirus. This finding will serve as the foundation for further research to study the relationship between the new virus and yellow leaf disease of areca palm. Acknowledgments This study was supported by grants from the National Basic Research Program of China (no. 2014CB138405), the Chinese National Natural Science Foundation (no. 31272011), and the Doctoral Fund of the Ministry of Education of China (20123402110013).
References 1. Agranovsky AA, Koonin EV, Boyko VP, Maiss E, Frotschl R, Lunina NA, Atabekov JG (1994) Beet yellows closterovirus: complete genome structure and identification of a leader papainlike thiol protease. Virology 198:311–324
123
H. Yu et al. 2. Alzhanova DV, Napuli AJ, Creamer R, Dolja VV (2001) Cell-tocell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog. EMBO J 20:6997–7007 3. Alzhanova DV, Prokhnevsky AI, Peremyslov VV, Dolja VV (2007) Virion tails of Beet yellows virus: Coordinated assembly by three structural proteins. Virology 359:220–226 4. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890 5. Daquan LMC, Shabin Y (2001) Identification of pathogens of yellow leaf disease of arecanut in Hainan island. Chinese J Trop Crops 22:43–46 6. Dolja VV, Kreuze JF, Valkonen JPT (2006) Comparative and functional genomics of closteroviruses. Virus Res 117:38–51 7. Jelkmann W, Fechtner B, Agranovsky AA (1997) Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus. J Gen Virol 78:2067–2071 8. Jelkmann W, Mikona C, Turturo C, Navarro B, Rott ME, Menzel W, Saldarelli P, Minafra A, Martelli GP (2012) Molecular characterization and taxonomy of grapevine leafroll-associated virus 7. Arch Virol 157:359–362 9. Karasev AV (2000) Genetic diversity and evolution of closteroviruses. Annu Rev Phytopathol 38:293–324 10. Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7
123
11. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Gen Biol 10:R25 12. Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43:D257– D260 13. Napuli AJ, Alzhanova DV, Doneanu CE, Barofsky DF, Koonin EV, Dolja VV (2003) The 64-kilodalton capsid protein homolog of Beet yellows virus is required for assembly of virion tails. J Virol 77:2377–2384 14. Peremyslov VV, Pan YW, Dolja VV (2004) Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum. J Virol 78:3704–3709 15. Satyanarayana T, Gowda S, Ayllon MA, Dawson WO (2004) Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5’ region. Proc Natl Acad Sci USA 101:799–804 16. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 17. Wu Q, Luo Y, Lu R, Lau N, Lai EC, Li WX, Ding SW (2010) Virus discovery by deep sequencing and assembly of virusderived small silencing RNAs. Proc Natl Acad Sci USA 107:1606–1611
