Arch Virol (2010) 155:1713–1715 DOI 10.1007/s00705-010-0767-0
ANNOTATED SEQUENCE RECORD
Molecular characterization of pea enation mosaic virus and bean leafroll virus from the Pacific Northwest, USA B. Vemulapati • K. L. Druffel • S. D. Eigenbrode A. Karasev • H. R. Pappu
•
Received: 12 February 2010 / Accepted: 20 July 2010 / Published online: 10 August 2010 Ó Springer-Verlag 2010
Abstract The family Luteoviridae consists of eight viruses assigned to three different genera, Luteovirus, Polerovirus and Enamovirus. The complete genomic sequences of pea enation mosaic virus (genus Enamovirus) and bean leafroll virus (genus Luteovirus) from the Pacific Northwest, USA, were determined. Annotation, sequence comparisons, and phylogenetic analysis of selected genes together with those of known polero- and enamoviruses were conducted.
Introduction The family Luteoviridae consists of eight viruses assigned to three different genera, Luteovirus, Polerovirus and Enamovirus. This family also consists of 12 unclassified and 20 tentative members [1]. Luteovirus virions are nonenveloped, icosahedral particles that are 25–30 nm in diameter and contain a single molecule of positive-sense ssRNA, 5.7–5.9 kb in size [2]. These viruses are transmitted in a persistent manner by aphid vectors and are mostly restricted to the phloem of infected hosts [3].
The sequences described here were deposited in the GenBank database with the following accession numbers: HM439775 for PEMV-ID and HM439776 for BLRV-WA. B. Vemulapati K. L. Druffel H. R. Pappu (&) Department of Plant Pathology, Washington State University, PO Box 646430, Pullman, WA 99164-6430, USA e-mail:
[email protected] S. D. Eigenbrode A. Karasev Department of Plant, Soil, and Entomological Sciences, University of Idaho, Moscow, ID, USA
Pea enation mosaic virus (PEMV) infects several legume crops, including chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris) and pea (Pisum sativum) [4]. PEMV has a divided genome consisting of two positive-sense ssRNAs. The large RNA (5,706 nucleotides; RNA-1) has nucleotide and deduced amino acid sequence similarities with members of subgroup II luteoviruses [5], while the smaller RNA (4,253 nucleotides; RNA-2) codes for a polymerase and has sequence similarities with viruses in the genera Carmovirus and Tombusvirus [6]. RNA-2 can infect the whole plant by itself, but due to the lack of a coat protein, it depends on RNA-1 for its coat protein and vector-transmission functions. RNA-2, in turn, provides functions related to systemic movement and mechanical transmission [7]. So far, only one complete sequence of PEMV (PEMV-1; accession number L04573) is available in the GenBank database. PEMV genomic RNA contains five open reading frames (ORFs). ORF-1 encodes a 34-kDa protein of unknown function. ORF-2 overlaps the ORF-1 in a unique reading frame and encodes an 84-kDa protein that contains protease-like motifs, which might be involved in posttranslational processing of virus translation products. ORF-3 was found to encode the RNA-dependent RNA polymerase (RdRp) and contain a helicase-like motif typical of RdRp. It is thought to be expressed by a frameshift fusion of the ORF-2 and ORF-3 products. ORF-4 codes for a 21-kDa coat protein (CP), which is separated from ORF-3 by an intergenic region spanning 189 bases. ORF-4 is immediately followed by ORF-5, which codes for a 33-kDa protein and is thought to be an aphid transmission subunit of the virus [8]. Bean leafroll virus (BLRV) belongs to the genus Luteovirus (family Luteoviridae). It occurs in Europe, the
123
1714
Middle East, India, and the USA and infects legumes, including French bean, faba bean (Vicia faba) and pea (Pisum sativum). BLRV is phloem-limited and present at very low concentrations, and it is transmitted by aphids in a persistent manner [9]. So far, only one complete sequence of BLRV (accession number AF441393) is available in the GenBank database. The BLRV genome consists of five ORFs typical of other members of the genus Luteovirus [10]. ORF-1 encodes a protein of 42 kDa and is overlapped by ORF-2 by 15 nt. ORF-2 encodes a protein of 62 kDa. ORF-1 and ORF-2 have been predicted to encode a tombusvirus-like RdRp, where ORF-2 is predicted to be expressed as a translational fusion product of ORF-1 through a frameshift, which is promoted by a ‘slippery’ sequence (GGUUUUU) at the junction of ORF-1 and 2. ORF-3 encodes a 22-kDa coat protein and is separated from ORF-2 by an intergenic region. ORF-4 and ORF-5 are predicted to encode proteins of 16 and 59 kDa, respectively [10]. Our recent surveys of pea and alfalfa fields in the states of Washington and Idaho, USA, have shown the prevalence of PEMV and BLRV. To better understand the genetic diversity of each of these two viruses and to develop improved methods for detection, the complete genomic sequences of one PEMV isolate from Idaho and one BLRV isolate from Washington State were determined in this study.
Materials and methods The PEMV-1 isolate, PEMV-ID, was collected from a naturally infected, commercially grown pea crop in Nez Perce County, Idaho, and the BLRV isolate, BLRV-WA, was from a commercial pea field in Whitman County, Washington. Total plant RNA was extracted individually from the leaves of PEMV-ID- and BLRV-WA-infected pea plants using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Firststrand cDNA synthesis from the total RNA was performed using Superscript-II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A PEMV-1-specific primer pair was designed based on the sequence available in GenBank (L04573). The antisense primer, PEMV5706C (50 -TGCGATACAATCCCAGGAA A-30 ), which started from base 5,706 at the 30 end of the viral genome, was used to synthesize cDNA, which was later subjected to PCR using the antisense primer and a sense primer, PEMV3F (50 -TGAAATAATTGTAAGAAAGCT C-30 ) that started at third base towards the 50 end of the viral genome. To amplify the BLRV genome, a primer pair designed based on the sequence available in GenBank (AF441393) was used. The antisense primer, BLRV5950C (50 -CTTTCGCCACCTTAACAACA-30 ), which started from
123
B. Vemulapati et al.
base 5,950 at the 30 end of the BLRV genome, was used to synthesize cDNA from BLRV. The first-strand cDNA was then used in PCR, using BLRV5950C and the sense primer BLRV4F (50 -AAAGTTGACACCTTTACAAGAG-30 ), which started at the fourth base of the 50 end of the viral genome. Each of the resulting PCR products was cloned into pGEM-T (Invitrogen, Carlsbad, CA, USA) and sequenced by the primer-walking method. The identity of each amplicon was verified using BLAST. Sequences obtained from the clones were assembled into contigs to generate a complete genome sequence of PEMV-1 using ALIGNPlus V 2.0. Nucleotide and deduced amino acid sequence alignments and phylogenetic analyses of the respective ORFs of PEMV-ID and BLRV-WA were done using MEGA 4, and phylogenetic trees were constructed using the neighbor-joining method with 1,000 bootstrap replications [11].
Sequence properties The sequenced region of PEMV-ID contained 5,703 nt, encompassing five ORFs that could potentially encode structural and non-structural proteins. ORF-1 consisted of 912 nt that potentially encode a polypeptide of 34 kDa. ORF-2 contained 2,283 nt and encoded a protein of 84 kDa, which overlapped the ORF-1 by 90%. ORF-3 contained 1,800 nt, and it potentially encodes a protein of 67 kDa. ORF-4 contained 570 nt, encoding the coat protein (CP) of 21 kDa. ORF-5 consisted of 912 nt and encoded a protein of 33 kDa. The 50 end of ORF-5 contained a proline-rich region and was contiguous with the CP. The individual ORFs of the present isolate (PEMV-ID) were compared to the ORFs of PEMV-1, the only complete genome sequence available in GenBank (L04573). The percent nucleotide and deduced amino acid sequence identities of ORFs 1–5 were determined. ORF-1 showed 97.2 and 95.3% nucleotide and deduced amino acid identity, respectively, while these values were 97.0 and 96.4% with ORF-2, 97.2 and 98.6% with ORF-3, 97.0 and 98.9% with ORF-4, and 98.5 and 98.6% with ORF-5. The deduced amino acid sequence of ORF-4 shared a maximum identity of 98.9%, while the lowest identity (95.3%) was found in with ORF-1. A phylogenetic tree that was constructed based on a comparison of the deduced CP amino acid sequences of the PEMV-ID isolate with the existing sequences of PEMV-1 available in GenBank indicated that the PEMV-1 isolates ‘UP58’ and ‘Germany’ were the most closely related to the present isolate (Table 1) and were clustered as a group, separate from the remaining isolates (figure not shown). The sequenced region of BLRV-WA contained 5,888 nt encompassing five ORFs that could potentially code for structural and non-structural proteins. ORF-1 consisted of
Pea enation mosaic virus and bean leafroll virus
1715
Table 1 Comparison of percent nucleotide (Nt) and deduced amino acid (AA) sequence identities of the coat protein (CP) gene of Pea enation mosaic virus-1 isolate PEMV-ID with those of known isolates of PEMV-1 Virus
Strain/isolate/origin
Designation used
GenBank accession number
% Nt identity
% AA identity
PEMV-1
WSG
PEMV-WSG
L04573
98.4
99.4
PEMV-1
AT-
PEMV-AT-
Y09100
98.4
99.4
PEMV-1
AT-D
PEMV-AT-D
Y09098
97.3
99.4
PEMV-1
UP58
PEMV-UP58
AY661882
96.8
100
PEMV-1
Germany
PEMV-FRG
Z48507
96.6
100
PEMV-1
SP
PEMV-SP
AF082833
96.8
95.7
PEMV-1
AT?
PEMV-AT?
Y09099
94.7
98.9
Table 2 Comparison of percent nucleotide (Nt) and deduced amino acid (AA) sequence identities of the coat protein (CP) gene of Bean leafroll virus isolate BLRV-WA with those of known isolates of BLRV Virus
Strain/isolate/origin
Designation used
GenBank accession number
BLRV
USA
BLRV-US1
AF441393
99.8
99.4
BLRV
USA
BLRV-US2
U15978
99.3
99.4
BLRV
Germany
BLRV-FRG
X53865
99.3
100.0
1,107 nt that potentially encode a polypeptide of 42 kDa. ORF-2 contained 1,629 nt and encoded a protein of 62 kDa. ORF-3 contained 591 nt, and it potentially encoded a CP of 22 kDa. ORF-4 contained 429 nt, encoding a protein of 16 kDa, and ORF5 consisted of 1,580 nt, encoding a protein of 59 kDa. The individual ORFs of BLRV-WA were compared to the respective ORFs of the BLRV sequence available in GenBank (AF441393). ORF-1 showed 97.5 and 99.4% nucleotide and deduced amino acid identity, respectively, while these values were 96.4 and 97.7% with ORF-2, 98.6 and 99.4% with ORF-3, 99 and 97.8% with ORF-4, and 96.5 and 98.2% with ORF-5. The deduced amino acid sequences of ORFs 1 and 3 shared a maximum identity of 99.4% with the other ORFs, while the lowest identity (97.7%) was seen with ORF-2. A phylogenetic tree that was constructed based on a comparison of the deduced CP amino acid sequences of the BLRV-WA isolate with the existing sequences of BLRV available in GenBank indicated that the BLRV-WA isolate shared 100% sequence identity with the BLRV ‘Germany’ isolate (Table 2) and clustered as a separate group from the remaining isolates (figure not shown). Acknowledgments This research was supported in part by funding from the USA Dry Peas and Lentil Council and a grant (# 2008511010-4522) from USDA NIFA RAMP. PPNS # 5546, Department of Plant Pathology, College of Agricultural, Human and Natural Resource Sciences, Agricultural Research Center, Project # WNP0 0545, Washington State University, Pullman, WA 99164-6430, USA.
% Nt identity
% AA identity
References 1. Smith HG, Barker H (eds) (1999) The Luteoviridae. CABI Publishing, Wallingford, pp 297–302 2. Hull R (2002) Family Luteoviridae. In: Matthews plant virology. Academic Press, pp 36:107–151 3. Casper R (1998) Luteoviruses. In: Koenig R (ed) The plant viruses: polyhedral virions with monopartitie RNA genomes. Plenum Press, New York, pp 235–258 4. Hagedorn DJ (1984) Compendium of pea diseases. American Phytopathological Society, St. Paul 5. Demler SA, Borkhsenious ON, Rucker DG, de Zoeten GA (1994) Assessment of autonomy of replicative and structural functions encoded by the luteo-phase of pea enation mosaic virus. J Gen Virol 75:997–1007 6. Demler SA, Rucker DG, de Zoeten GA (1993) The chimeric nature of the genome of Pea enation mosaic virus: the independent replication of RNA2. J Gen Virol 74:1–14 7. Demler SA, Rucker-Feeny DG, Skaf JS, de Zoeten GA (1997) Expression and suppression of circulative aphid transmission in Pea enation mosaic virus. J Gen Virol 78:511–523 8. Demler SA, de Zoeten GA (1991) The nucleotide sequence and luteovirus-like nature of RNA 1 of an aphid non-transmissible strain of Pea enation mosaic virus. J Gen Virol 72:1819–1834 9. Ashby JW (1984) Bean leafroll virus. In: Descriptions of plant viruses. CMI/AAB Descr. PI. Viruses No. 286, 4 pp 10. Domier LL, Mc Coppin NK, Larsen RC, D’Arcy CJ (2002) Nucleotide sequence shows that Bean leafroll virus has a Luteovirus-like genome organization. J Gen Virol 83:1791–1798 11. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
123
