Nucleotide Sequence Analyses of Genomic Rnas of Peanut Stunt Virus Mi, the Type Strain Representative of a Novel PSV Subgroup from China

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Arch Virol (2005) 150: 1203–1211 DOI 10.1007/s00705-005-0492-2

Nucleotide sequence analyses of genomic RNAs of Peanut stunt virus Mi, the type strain representative of a novel PSV subgroup from China

Brief Report

L. Y. Yan1,2,Z.Y.Xu1, R. Goldbach2, C. Kunrong1, and M. Prins2 1Ministry Key Laboratory of Genetic Improvement for Oil Crops, Oil Crop Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, P.R. China 2Laboratory of Virology, Wageningen University, Wageningen, The Netherlands

Received December 10, 2004; accepted January 4, 2005 Published online March 3, 2005 c Springer-Verlag 2005

Summary. The complete nucleotide sequence of Peanut stunt virus strain Mi (PSV-Mi) from China was determined and compared to other viruses of the genus Cucumovirus. The tripartite genome of PSV-Mi encoded ﬁve open reading frames (ORFs) typical of cucumoviruses. Distance analyses of four ORFs indicated that PSV-Mi differed sufﬁciently in nucleotide sequence from other PSV strains of subgroups I and II to warrant establishment of a third subgroup of PSV. ∗ Peanut stunt virus (PSV) occurs worldwide, infecting primarily legume plants including important crops such as peanut (Arachis hypogaea L.), bean (Phase- olus vulgaris L.), soybean (Glycine max (L.) Merr.), pea (Pisum sativum L.), alfalfa (Medicago sativa L.), yellow lupine (Lupinus luteus L.) and various clovers [12, 21]. PSV is an economically important threat to peanut production in China. Severe epidemics of virus diseases caused by PSV along with Peanut stripe virus (PStV) and Cucumber mosaic virus (CMV) have substantially reduced peanut yields in the major peanut growing areas in northern China since the 1970 s [22]. In addition, disease surveys revealed that black locust trees (Robinia pseudoacacia L.), a popular tree in northern China, are widely infected by PSV

The nucleotide sequence data of RNA1, 2 and 3 of PSV-Mi have been deposited in GenBank under accession numbers AY429431, AY429430 and AY775057. 1204 L. Y.Yan et al. and could act as a primary source of the virus for peanut and other susceptible plants [23, 26]. Peanut stunt virus (PSV) is a species within the genus Cucumovirus in the family Bromoviridae [3]. Other members of the genus are its type member CMV and Tomato aspermy virus (TAV). PSV like other cucumoviruses has a tripartite genome of positive-strand RNAs, designated RNA1, RNA2 and RNA3 in order of decreasing size. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are required for replication. A small overlapping gene (2b) encoded by RNA2 is present in all cucumoviruses sequenced to date and is expressed from subgenomic RNA4A [2]. RNA3 is also dicistronic and encodes the movement protein (MP) and coat protein (CP); the latter is expressed from subgenomic RNA4. RNA4 is packaged into a single virus particle together with RNA3 [14]. Occasionally, PSV and CMV also package a fifth RNA, designated satellite RNA (satRNA), along with their genomic and subgenomic RNAs [16]. PSV was first described in the United States in 1966 (PSV-E). Since then a western strain (PSV-W) was reported in 1969 as a new serotype of PSV different from PSV-E. Isolates and strains from other regions of the world, namely PSV-J, PSV-V, PSV-H, PSV-B, PSV-T, PSV-Tp, and PSV-C, have been reported and differentiated based on host symptomology and serology [12, 21]. The complete nucleotide sequence of the RNAs of PSV-J was first reported by Karasawa et al. in 1992 [7, 8]. Hu and colleagues classified PSV strains into two distinct subgroups based on the RNA sequence of RNA3: subgroup I containing PSV-ER and related strains and subgroup II containing PSV-W [6]. The PSV-Mi, or “mild strain” was identified and characterized in China in 1985. PSV-Micaused common mosaic symptoms in infected peanut plants without obvious stunting. PSV-Mi differed from PSV-E in symptomology induced in other plants as well as serologically [24]. Subsequently six PSV isolates from China, including PSV-Mi, -S, -F and -P from different plants and locations were compared with PSV-E and W by symptomology and serology, revealing that all six Chinese PSV isolates were identical to each other but distinct from PSV-E and W strains. When the nucleotide sequence of the PSV-Mi and S strain CP genes were determined and compared with those of the PSV-E and W CP genes, the nt identity PSV-Mi and S was 99%, but only about 75% between PSV-Mi and PSV-ER, a subgroup I virus. Similar nt identity between PSV-Mi and PSV-W of subgroup II was observed, indicating that PSV-Mi may form a new subgroup of PSV from China [25]. In this report, the complete nucleotide sequence of the three genomic RNAs of the PSV-Mi strain was determined and compared with those of PSV strains in subgroups I and II, CMV and TAV. The results indicated the existence of more genetic diversity among PSV strains than previously reported and that PSV-Mi represented a novel PSV subgroup III. The PSV-Mi strain was propagated on Nicotiana benthamiana and virus was purified according to Francki [4]. Briefly, infected leaves were ground in 2× extraction buffer, (0.5 M citrate buffer, pH 6.5, containing 0.01 M Na2EDTA and 0.1% β-ME), chloroform was added to 10%, and virus was precipitated with 10% polyethylene glycol and purified by differential centrifugation. The genomic Nucleotide sequence of PSV-Mi from China 1205

RNAs were SDS-phenol extracted from purified virions and precipitated with ethanol [18]. The 5 terminal sequence of PSV-Mi RNAs 1, 2 and 3 were obtained by using a5 Rapid Amplification of cDNAs Ends (RACE) kit (Invitrogen) according to the manufacturer’s protocol with 1 µg of viral RNA template and primers designed on the basis of conserved sequences located at ∼100 nt from the 5 end of RNA1, 2 and 3 of PSV-ER, J and W: PSV-1 5AGAGCCGTGGGAGGCTACC3; PSV-2 5CCACATCTTCGGGAGTGTC3; PSV-3 5TCGGGGCTGAATAATA TCT3. For RT-PCR amplification and sequencing of each of the three genomic segments of PSV-Mi, 5 forward (F) primers were designed based on the sequences obtained from 5 RACE fragments while intermediate (MD) forward and reverse (R) and 3 reverse PCR primers were designed based on conserved sequences of PSV-ER, J and W. The following primers were employed: PSV RNA1-F 5GTTTTATCAAGAGCG TACGG3 (nt 1–20); PSV RNA1 MD-F 5GTCAGCAGCCTGTGCAAGGCG3 (nt 1685–1701); PSV RNA1 MD-R 5GTCGACGA CTGAGGATCTCC3 (nt 1697–1716); PSV RNA 1R 5TGGTCT CCTATGG AGCC3 (nt 3331–3347); PSV RNA2-F 5GTTTTATCAAGAGC GTACGG3 (nt 1–20); PSV RNA2 MD-F 5GAGAACCTGCAATTGTATG AGCATATGG3 (nt 1384–1411); PSV RNA2 MD-R 5GTATCAGCCACA ACC GGCTTAAC (nt 1422–1444); PSV RNA2 R1 5TGGTCTCCTATGG (nt 3050–3037); PSV RNA3 F1 5GTTTTATACCACTGAGATCTG3 (nt 1–22); PSV RNA3 MD-F 5CCCCGGATCCATGGCATCTTC AGGATCTGG3 (nt 1228–1248); PSV RNA3 MD-R 5TAGCGTGAGCGT CCACTTT AGC3 (nt 1290–1310); PSV RNA 3R 5CCCCGGATCCTGGTCTCCTT3 (nt 2210–2219). BamHI sites in primers PSV RNA3 MD-F and R are underlined. PCR amplification of the first strand cDNA reaction (5 µl) with 5 F/MD-R or MD-F/3 R primer pair for each genomic RNA utilized 0.2 U Taq DNA polymerase, 0.5 µl 10 mM dNTPs and the following reaction protocol: 94 ◦C for 3 min followed by 30 cycles of 94 ◦C for 30 s, 50 ◦C for 30 s, 72 ◦C for 2.5 min and ending with 72 ◦C for 7 min. The resulting amplicons were purified and ligated into the pGEM-Teasy vector (Promega). Sequences were generated initially using the universal T7 and Sp6 sequencing primers and then using internal primers designed as sequence was generated. Sequence was obtained bi-directionally from at least two independent cDNA clones. The sequence was analyzed using DNAstar and Clustal X (using default parameters) was used to align nt and deduced amino acid (aa) sequences [20]. MEGA version 2.1 was used for neighbor-joining analysis and genetic distance was estimated by Kimura’s two-parameter distance method [19]. The bootstrap analysis consisted of 1000 replications. The lengths of RNA1, 2 and 3 of PSV-Mi were determined to be 3347, 2966 and 2170 nt, respectively. Like for all cucumoviruses, there are 5 predicted ORFs in the PSV-Mi genome. ORF1a (nt 86–3100 of RNA1) is predicted to encode a 1a protein of 1005 aa’s with a calculated Mr of 111,604. The 1a protein deduced sequence includes an N-proximal putative methyltransferase domain, involved in the 1206 L. Y.Yan et al. capping of genomic and subgenomic RNAs, and a C-proximal putative helicase (superfamily 1) domain [17]. The PSV-Mi ORF2a (nt 82–2620 of RNA2) is predicted to encode a 2a protein of 846 aa’s with a calculated Mr of 94,647. Motifs characteristic of an RNA-dependent RNA polymerase [9] are located in the deduced aa sequence of the 2a protein. ORF2b, 279 nts in length (nt 2438–2716 of RNA2), which partly overlaps ORF2a on RNA2, encodes a 2b protein with a calculated Mr of 10,748. PSV-Mi RNA3 encodes ORF3a and ORF3b separated by an internal non-coding region (IR) of 248 nt. ORF3a (nt 117–980 of RNA3) encompasses 864 nt and encodes the movement protein with a calculated Mr of 30,942. The deduced aa sequence of this protein contains the conserved motif MSVPQVLCAITRTVTAEAEGSIRIYLADLGDNE typical of the 30 K movement protein superfamily [14]; within this 33 aa conserved region, PSV strains exhibit eight variable residues compared to only one variable residue among CMV strains [6]. PSV-Mi ORF3b (nt 1229–1882 of RNA3) encodes the viral coat protein with a calculated Mr of 23,643. The 5 untranslated regions (UTR’s) of RNA1 and 2 of PSV-Mi like any particular isolate in genus Cucumovirus are highly conserved. The 5UTR of PSV RNA3, like those of all other cucumoviruses, contains the conserved UG tract (5GUGUUGUUGUUGGU3) that is required for efficient accumulation of RNA3 [1]. Like other members of the genus Cucumovirus, the 3UTRs of RNA1, 2 and 3 of PSV-Mi are highly conserved and contain a 40 nt sequence (5GATCGGGTTGTCCATCCAGCTAACGGCTAAAATGGTCAGT3) conserved in all Cucumovirus RNAs, which is also important for RNA accumulation [10]. Among the UTR’s, the 5UTRs of RNA1 and 2 of all PSV strains are most conserved as the 5UTR of PSV-Mi RNA1 and 2 were 83.5 ∼ 96.1% identical to those of the PSV-ER and W strains whereas the 5UTR of RNA3 had only 78.4 ∼ 79.8% identity with the 5UTR of RNA3 to PSV-ER and W (PSV-ER and -W share over 90% identity in this region). In contrast to the 5UTR, the 3UTR of RNA1, 2 and 3 among the PSV strains showed more diversity. PSV-Mi RNA1, 2 and 3 had 74 ∼ 82.6% identity with the 3UTRs of these RNAs of PSV-ER and -W. Sequence identities among the RNAs, ORFs and predicted proteins of PSV-Mi strain with PSV-ER and -W (representative of PSV subgroups I and II), CMV-Fny and TrK (representative of CMV subgroups I and II), and TAV-KC are shown in Table 1. PSV-Mi RNA1, 2 and 3 were 75.2 ∼ 78.8% identical to those of PSV-ER and 74.0 ∼ 79.6% to those of PSV-W. Identities of PSV-Mi RNA1 and 2 to those of CMV and TAV were similar, below 70% for RNA1 and below 60% for RNA2. The RNA3 sequence of PSV-Mi was more closely related to TAV than to CMV. The GenBank accession number of RNA1, 2, 3 of the cucumoviruses strains used in these alignments were: PSV-J (D11126, D11127, D00668), PSV-ER (U15728, U15729, U15730), PSV-W (U33145, U33146, U31366), CMV-Fny (D00356, D00355, D10538), CMV-Y (D12537, D12538, D12499), CMV-Q (X02733, X00985, M21464), CMV-TrK7 (AJ007933, AJ007934, L15336) and TAV-KC (AJ320273, AJ320274, AJ237849). Nucleotide sequence of PSV-Mi from China 1207 Sequence indentities of RNAs, ORFs and encoded proteins of PSV-Mi strain with other PSV strains, CMV and TAV Complete ORF1a%nt %nt Complete %aa %nt ORF2a %nt %aa ORF2b %nt %aa %nt Complete ORF3a %nt %aa %nt %aa ORF3b Table 1. PSV-ER(I)PSV-W(II) 78.8CMV-Fny 79.6CMV-Trk 67.2 78.3 67.1 79.5 87.7 68.0 87.9 75.2 68.0 74.2 74.0 74.2 58.0 74.7 58.4 73.6 78.0 59.0 77.0 67.4 60.2 57.4 37.9 74.2 59.3 51.4 51.2 77.7 50.9 46.2 76.7 48.1 57.5 78.3 58.8 78.9 72.4 64.8 69.5 75.7 66.5 61.2 75.2 72.1 62.6 52.8 67.6 53.6 49.1 49.5 TAV-KC 64.5 64.9 69.7 58.5 58.8 59.6 49.8 42.4 68.1 75.4 72.0 68.3 71.4 Viruses RNA1 RNA2 RNA3 1208 L. Y.Yan et al.: Nucleotide sequence of PSV-Mi from China

In addition to the strains used in the identity calculations in Table 1, PSV-J, and CMV-Y and -Q were included in a phylogenetic analysis of nucleotide sequences of the 1a, 2a, 3a and CP genes shown in Fig. 1. Interestingly, in contrast to the 1a and 2a genes which form three distinct clusters containing CMV, TAV, and PSV, respectively, in the 3a and CP genes, PSV strains are more closely related to TAV than to CMV. In each of these trees, PSV-Mi diverges from the previously established PSV subgroups (I containing PSV-J and PSV-ER and II containing PSV-W) and appears to form a distinct new PSV subgroup. As shown in Table 1, the sequence identities of RNA1, 2 and 3 of PSV-Mi were 78.8%, 75.2% and 77.9% in comparison with those of PSV-ER in PSV subgroup I and 79.6%, 74.0% and 77.2% with those of PSV-W in subgroup II. Hajimorad and co-workers proposed that the nt sequence identities between the strains in the same subgroup should be greater than 90%, those between the strains in different subgroups (but within the same species) should be in the range of 70% to 80%, and those between different Cucumovirus species should be less than 70%, but greater than 50% [5]. Within these guidelines, our results demonstrate that the PSV-Mi strain should be placed in a new subgroup within the Peanut Stunt Virus species, which we tentatively named subgroup III. We propose that PSV-Mi serves as the type member of this new subgroup. PSV-E and W in subgroups I and II were first reported in the United States. At present, strains of both of these subgroups have been reported worldwide [11, 21]. PSV widely occurs in the peanut growing areas in Northern China, including the Shandong, Hebei, Henan and Jiangsu provinces [22]. Our research has shown that the PSV isolates or strains from different source plants and regions in China were genetically closely related to each other based on serology and sequence identity of the CP gene [25]. Thus, other PSV strains from China likely belong with PSV-Mi in subgroup III. Genetic diversity is high among PSV strains worldwide. In addition to the Chinese isolate described here, other PSV strains cannot be placed into the present subgroups I and II. Hajimorad reported a PSV isolate (PSV-I) from Iran, that was possibly related to the strains of subgroup II based on Northern blot hybridization. Sequence analyses of parts of RNA1 and 2 of PSV-I showed it to be closer to the W strain (88.8% and 86.7% identities within the regions sequenced) than to the E strain (79.1% and 77.5%), but could not be definitively placed into subgroup II; it was therefore named as ‘untypical old world’ strain. Identities of the PSV-I RNA1 and 2 sequence are 81.3% and 78.1% with those of PSV-Mi [5]. Another taxonomically uncertain PSV isolate is PSV-Ro, originally named Robinia mosaic virus. Recently PSV-Ro was placed with in PSV subgroup I, based on Western blot analysis, but it could not be classified within either subgroup based on Northern hybridization results [11]. These results indicate that within the

᭤ Fig. 1. Phylogenetic analysis of 1a, 2a, 3a and cp gene nt sequences of Cucumoviruses. Numbers on nodes indicate bootstrap values. The bar on each tree corresponds toa5or10% difference

1210 L. Y.Yan et al.

Cucumovirus genus, PSV is genetically the most diverse, followed by CMV, whereas TAV is least variable. Despite having more genetic diversity, PSV has a relatively narrow host range (mainly legumes), in contrast to the wide host range of CMV.Due to their economic importance, CMV strains have been most extensively studied in terms of genetic diversity and the complete nucleotide sequence of 22 CMV isolates and partial sequences of 190 CMV isolates have been deposited in the GenBank. However, the complete nucleotide sequences of only 3 PSV isolates and partial sequences of a further 2 PSV isolates have been deposited in GenBank to date [15]. We believe a clearer picture of genetic diversity of PSV strains will emerge as the genomic sequences of more PSV strains are determined and analyzed. Sequencing the PSV-Mi strain as a representative member of Chinese PSV strains and the type member of the novel subgroup III is a ﬁrst step in that direction.

Acknowledgements This research was supported by the PhD project from Wageningen University (WU)-Chinese Academy of Agricultural Sciences (CAAS) joint program, the project (30170606) from National Natural Science Foundation of China and Short-Time Corporation Grand of United Nations Educational, Scientiﬁc and Cultural Organization.

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