Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus

Eugene I. Savenkov1, Maria Sandgren1 and Jari P. T. Valkonen1

Department of Plant Biology, Genetic Centre, SLU, PO Box 7080, S- 750 07 Uppsala, Sweden1

Author for correspondence: Eugene Savenkov.Fax +46 18 67 33 92. e-mail eugene.savenkov{at}vbiol.slu.se


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5'-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genusPomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5' and 3' termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3'-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3'-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3'-UTR of RNA 3, which confirms its presence in the 3'-UTRs of all three RNAs of PMTV.


   Introduction
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The fungus-transmitted rod-shaped viruses (commonly called furoviruses) contain several similar genomic structures and biological traits (Brunt, 1995 ). They are taxonomically grouped in the genera Furovirus, Pomovirus , Pecluvirus and Benyvirus (Pringle, 1999 ). The structural similarities include tubular virions, a multipartite genome possessing the triple gene block (except in the genus Furovirus), a tRNA-like structure at the 3'-end of the RNAs (except in the genus Benyvirus), and a readthrough domain expressed as a fusion with the coat protein (except in the genus Pecluvirus) (Torrance & Mayo, 1997 ). Biological similarities include transmission by fungal vectors in soil (Brunt, 1995 ).

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus (Torrance & Mayo, 1997 ). It is economically one of the most damaging viruses in the Nordic countries (Sandgren, 1996 ). PMTV is transmitted by motile zoospores of the protist Spongospora subterranea, which is an important potato pathogen causing powdery scab (Harrison, 1974 ). The particles of PMTV are tubular and rigid, 18–20 nm in diameter and 100–150 nm or 250–300 nm in length. The genome consists of three messenger-polarity single-stranded RNA molecules of ca. 6 (RNA 1), 3·2 (RNA 2) and 2·4–3·1 kb (RNA 3) (Torrance et al., 1992 ). The complete nucleotide sequences of PMTV RNA 2 (Scott et al., 1994 ) and RNA 3 (Kashiwazaki et al., 1995 ) are available from isolate PMTV-T from Scotland, UK, and, similar to several other fungus-transmitted viruses, do not contain a poly(A) sequence at the genome RNA 3' ends (Scott et al., 1994 ).

RNA 2 of PMTV encodes four putative proteins. The first three proteins [51, 13 and 21 kDa (`K')] form the triple gene block (TGB) found in several plant virus genera. Based on the data from other TGB-containing viruses (Morozov et al., 1991 ; Seppänen et al., 1997 ; Lauber et al., 1998 ; Solovyev et al., 1996 , 1999 ), the TGB of PMTV is probably involved in cell-to-cell movement of virus in host plants. The 51K protein may bind viral RNA, and the 13K and 21K proteins contain two hydrophobic regions and may be membrane-bound. The fourth protein (8K) contains an unusually large proportion of cysteine residues, has a unique sequence, and its function is unknown (Scott et al., 1994 ).

RNA 3 of PMTV encodes the coat protein (CP) (176 amino acids; 20K), which structurally resembles the CP of Tobacco mosaic virus (TMV, genusTobamovirus) (Pereira et al., 1994 ). CP of PMTV is encoded from the 5' end of RNA 3, which also encodes a readthrough protein of 47K with the CP at its N terminus (Kashiwazaki et al., 1995 ; Cowan et al ., 1997 ). In Beet necrotic yellow vein virus (BNYVV, genus Benyvirus) the corresponding readthrough protein is incorporated into virus particles and is involved in virion assembly and virus transmission by the fungal vector (Haeberle et al., 1994 ; Tamada et al., 1996 ). A recent study has suggested that deletions in the internal part of the readthrough protein of PMTV may be associated with loss of transmissibility by the vector (Reavy et al., 1998 ).

The sequence from RNA 1 of PMTV is not available. It is estimated to constitute approximately half of the PMTV genome (Torrance et al ., 1992 ). RNA 1 encodes an RNA-dependent RNA polymerase (RdRp) in the two other known pomoviruses, Broad bean necrosis virus (BBNV) (Lu et al., 1998 ) and Beet soil-borne virus (BSBV) (Koenig & Loss, 1997 ). RdRp sequences are among the most important features used in molecular taxonomy of viruses (Koonin & Dolja, 1993 ; Mayo & Pringle, 1998 ). Therefore, it is of intrinsic scientific importance to characterize the RNA 1 sequence from PMTV, the type member of the genus. In this study, the complete nucleotide sequence of RNA 1 from PMTV was determined. The data show that it encodes a predicted RdRp that most closely resembles that of BSBV. The 3'-untranslated region (UTR) of RNA 1 contains a tRNA- like structure which has previously been found only in RNA 2 of PMTV, but which was found also in RNA 3 in this study.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} cDNA synthesis, cloning and sequencing.
Isolate PMTV-Sw was obtained from soil from a potato field in Halland, southern Sweden, using Nicotiana debneyi as a bait plant. Root extracts from these plants were used to obtain cDNA and partial clones from RNA 1 by immunocapture (IC)–RT–PCR, essentially as described by Koenig et al. (1995) . Primers used for cloning are listed in Table 1 and the cloning strategy is presented in Fig. 1. The first strand of cDNA was synthesized using primer 1, complementary to the 3'-terminal highly conserved region of RNA 2 and RNA 3 of PMTV-T (Kashiwazaki et al., 1995 ). Subsequently, specific primers 2 and 4, corresponding to the internal conserved regions of RNA 1 from fungal- transmitted viruses, were used to clone the 3'-proximal and 5'-proximal parts of RNA 1. These primers were designed by comparison of the helicase (primer 2) and methyltransferase motifs (primer 4) in the RNA 1 of BBNV (Lu et al., 1998 ), BSBV (Koenig & Loss, 1997 ), Peanut clump virus (PCV, genus Pecluvirus) (Herzog et al., 1994 ) and Soil-borne wheat mosaic virus (SBWMV, genus Furovirus) (Shirako & Wilson, 1993 ). Most of the RNA 1 sequence was amplified using the primer combinations 2/1 or 4/3 (Fig. 1; Table 1).


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Table 1. Deoxyoligonucleotides used for cloning RNA 1 (primers 1–7) and RNA 3 (primer 8) from PMTV-Sw

 


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Fig. 1. Genetic organization of RNA 1 in PMTV-Sw and cloning strategy used for obtaining the complete nucleotide sequence of RNA 1. The large rectangle represents the ORFs encoding the 148K and 206K proteins. Shaded regions within these ORFs indicate the location of the methyltransferase (MetT), helicase (Hel) and polymerase (Pol) domains. {bigtriangledown}, The opal (UGA) stop codon which interrupts internally the ORF for the 206K protein and terminates the coding region for the 148K protein; Var, region variable among the viruses transmitted by fungi; RT, readthrough protein.

 
The extreme 5'-terminal regions of PMTV RNA 1 and the extreme 3'-terminal regions of RNA 1 and RNA 3 were cloned from the total RNA isolated from N. debneyi infected with PMTV-Sw using the Rnease Plant Mini kit (Qiagen). For the 5'-terminal region, cDNA was synthesized using primer 5 and the 5'RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Life Technologies), according to the manufacturer's instructions. To clone the extreme 3'-terminal regions, the total RNA was 3'- polyadenylated by poly(A)polymerase (Pharmacia) and amplified by RT–PCR using primers 6 and 7 (RNA 1) or 8 and 7 (RNA 3) ( Table 1).

The PCR products were cloned into TA vector pCR2.1 (Invitrogen). Both strands were sequenced using an automated ABI Prism 377 DNA sequencer (Perkin Elmer) with the Thermo Sequenase Dye Terminator Cycle Sequencing Pre-mix kit (Amersham). To sequence long cDNA inserts, sets of unidirectional deletions were generated with exonuclease III and S1 nuclease using the Erase-a-Base system (Promega).

{blacksquare} Sequence analysis.
The data were analysed with the sequence analysis software of the Wisconsin package version 9.1 (Genetics Computer Group, Madison, WI, USA). Sequences from type members and other tubular viruses from databases were included for comparison. Multiple sequence alignments were subjected to phylogenetic analyses using different methods such as the PileUp program and the Distances program, the Kimura distance matrixes, the neighbour-joining method, Jotun Hein method and Clustal method.


   Results and Discussion
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Abstract
Introduction
Methods
Results and Discussion
References
 
Three clones of about 2·8 kb each covering most of the RNA 1 and ten additional 5'-terminal and 3'-terminal overlapping clones were sequenced and aligned to provide the complete 6043 nt sequence of RNA 1 of PMTV-Sw (Fig. 1; EMBL Sequence Database accession no. AJ238607). A search for potential open reading frames (ORFs) revealed the presence of two ORFs (Fig. 1). ORF1 (3909 nt) starts at the first AUG codon at position 115 and terminates with a UGA stop codon at position 4024. The putative polypeptide encoded by this ORF contains 1303 amino acid residues (calculated molecular mass 148 kDa). An in-frame coding sequence extends immediately after the opal codon and creates ORF 2. It ends at a UGA (5551) and has a polypeptide coding capacity of 509 amino acids (molecular mass 58 kDa). The predicted translational fusion protein from ORF 1 and 2 has a molecular mass of 206 kDa. No additional ORFs that could encode proteins larger than 4 kDa were identified in the RNA 1 sequence, in either sense or antisense orientation. The 5'- and 3'-UTR of RNA 1 are 114 and 489 nt long, respectively. Thus, ORFs 1 and 2 account for 89·9% of the RNA 1 sequence.

Analysis of the deduced amino acid sequence of the 206K protein revealed the presence of a methyltransferase (Rozanov et al., 1992 ), a helicase (Gorbalenya & Koonin, 1989 ; Habili & Symons, 1989 ) and a polymerase (Karmer & Argos, 1984 ; Koonin, 1991 ) motif, respectively, resembling those of other positive-sense RNA viruses (Fig. 1). Evidence of the presence of a cap structure on the PMTV genomic RNAs is not available, but the methyltransferase encoded by RNA 1 suggests that the PMTV RNAs may be capped. The NTPase/helicase motif GXXXXGKS/T (Gorbalenya et al., 1988 ) was found at amino acid positions 1014–1021 at the C-terminal part of the 148K protein. The RdRp motif Gly-Asp-Asp (GDD) (Kamer & Argos, 1984 ) is located at amino acid positions 1663–1665 at the C-proximal part of the predicted 209K readthrough protein (Fig. 1). The arrangement of the conserved motifs and ORFs in RNA 1 of PMTV was similar to the two previously characterized pomoviruses, BSBV (Koenig & Loss, 1997 ) and BBNV (Lu et al., 1998 ).

Multiple alignments and phylogenetic analyses of the RdRp sequences from PMTV, BBNV, BSBV and other representatives of tubular viruses revealed significantly greater sequence similarity between pomoviruses and SBWMV, the only furovirus from which sequence data are available, than between pomoviruses and the other viruses analysed (Fig. 2). These findings indicated that the RdRps of pomoviruses and furoviruses are closely related, forming a distinct cluster (Fig. 2). On the other hand, the RdRps of hordeiviruses are closely related to those of pecluviruses, forming another cluster (Fig. 2). These data are consistent with the previous reports that PCV is more closely related to the hordeiviruses than to the pomoviruses based on comparison of the RdRps, TGB proteins, CPs and cysteine-rich proteins (Herzog et al., 1994 ; Solovyev et al., 1996 ; Lu et al., 1998 ; Savenkov et al., 1998 ).



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Fig. 2. Phylogenetic analysis of the deduced protein sequences of RdRps from different tubular viruses (EMBL database accession numbers in parentheses). PCV, Peanut clump virus , RNA 1 (X78602); LRSV, Lychnis ringspot virus, RNA{alpha} (Z46630), RNA{gamma} (Z46353); PSLV, Poa semilatent virus , RNA{alpha} (Z46352), RNA{gamma} (M81487); BSMV, Barley stripe mosaic virus, RNA{alpha} (U35767), RNA{gamma} (U13917); SBWMV, Soil-borne wheat mosaic virus, RNA 1 (L07937); BBNV, Broad bean necrosis virus, RNA 1 (D86636); BSBV, Beet soil- borne virus, RNA 1 (Z97873); TRV, Tobacco rattle virus , RNA 1 (Z36974); TMV, Tobacco mosaic virus (Z92909).

 
Sequencing of six independent 5'-terminal clones of RNA 1 from PMTV-Sw indicated that the 5'-terminal base of RNA 1 is a guanosine (G). The starting sequence GUAUUU in RNA 1 is also found in RNA 2 and RNA 3 of PMTV, as well in the RNAs of many other tubular viruses that start with the sequence GUA (Goelet et al., 1982 ; Gustafson & Armour, 1986 ; Gustafson et al., 1987 , 1989 ; Manohar et al., 1993 ; Shirako & Wilson, 1993 ; Herzog et al., 1994 ; Scott et al., 1994 ; Kashiwazaki et al., 1995 ; Koenig et al., 1996 ; 1997 ).

Comparative analyses of the 5'-UTRs in the three RNAs of PMTV resulted in two significant observations. Firstly, the first 42 nt in RNA 1 are extremely similar to the first 38 nt of RNA 2, but there was little sequence similarity with the 5'-UTR of RNA 3. The remainder (72 nt) of the 5'-UTR in RNA 1 showed no significant similarity with 5'-UTRs of RNA 2 or RNA 3. Secondly, comparison of the 5'-UTRs of RNA 3 from PMTV-Sw and PMTV-T revealed that the PMTV-Sw sequence is longer, due to an insert of 25 nt. It is very unlikely that this insert is an artefact of cloning, PCR-amplification or sequencing, since the 5'-terminal sequence of PMTV-Sw RNA3 was determined and verified from seven independent clones.

The 3'-UTR of RNA 1 is 489 nt long in PMTV-Sw. The 80 3'-terminal nucleotides can be folded into a tRNA-like structure. Such a structure has previously been reported for the 3'-UTR of RNA 2 but not for RNA 3 of another PMTV isolate, PMTV-T (Scott et al ., 1994 ; Kashiwazaki et al., 1995 ). It is therefore significant that a tRNA-like structure was found also in the 3'-UTR from RNA 3 in PMTV-Sw in this study. The reason why no tRNA-like structure has been previously found in the 3'-UTR of PMTV RNA 3 may be associated with differences in maintenance of the two PMTV isolates. The RNA of PMTV-Sw was obtained directly from an infected bait plant, whereas PMTV-T had been serially propagated in test plants prior to analysis (Kashiwazaki et al ., 1995 ), which could result in deletions in viral genomic RNA (Reavy et al., 1998 ).

The tRNA-like structures of RNA 1 and RNA 3 (PMTV-Sw; this study) and RNA 2 (PMTV-T; Scott et al., 1994 ) are of equal length and are identical in nucleotide sequence. This is unusual because, for example, the tRNA-like structure of BSBV RNA 1 has six nucleotide changes in comparison with the tRNA-like structure in RNA 2 and 17 changes in comparison with the tRNA-like structure in RNA 3 (Koenig & Loss, 1997 ). The tRNA-like structures of PMTV, like in those other fungal-transmitted viruses analysed by Goodwin & Dreher (1998) , have an anticodon (GAC) for valine and are capable of efficient valylation. In contrast, the tRNA- like structures in hordeiviruses have an anticodon sequence for tyrosine and are capable of tyrosylation (Agranovsky et al., 1981 , 1992 ; Goodwin & Dreher, 1998 ). Interestingly, the difference in the type of the anticodon was consistent with the grouping of the viruses based on the phylogenetic analysis of polymerase sequences (Fig. 2). Although the tRNA-like structures in all these virus genera, including PMTV, show functional tRNA mimicry (Goodwin & Dreher, 1998 ) and the presence of a certain type of tRNA-like structure is correlated with a certain type of polymerase, the significance of these observations for the virus life-cycle remains to be resolved.

The four 3'-UTR sequences available from PMTV (excluding the tRNA-like structures) revealed high sequence similarity (Fig. 3). The 3'-UTR of RNA 1 (PMTV-Sw) was more similar to the 3'-UTR of RNA 2 (PMTV-T) than to the 3'-UTR of RNA 3 (PMTV-Sw and PMTV-T) (Fig. 3). This situation is different from BSBV, where the highest similarity has been observed between the 3' termini of RNA 2 and RNA 3 (Koenig et al., 1996 , 1997 ).



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Fig. 3. Sequence alignment of the 3'-UTRs in RNA 1 (PMTV-Sw; this study), RNA 2 from PMTV-T (Scott et al., 1994 ) and RNA 3 from PMTV-Sw (this study), and PMTV-T (Kashiwazaki et al., 1995 ). The elements involved in predicted pseudoknots (A–D) are marked below the nucleotide sequences (Scott et al., 1994 ; Kashiwazaki et al., 1995 ). The position of the tRNA-like structure is indicated, except in the 3'-UTR of RNA 3 from PMTV-T which is shorter and has been reported to lack a tRNA-like structure (Kashiwazaki et al., 1995 ). The nucleotide position for the first nucleotide of the 3'-UTR is indicated at the beginning of the sequence alignment. ***, Possible anticodon for Val.

 

   Acknowledgments
 
We thank Minna Rajamäki for help in computer-assisted analysis of sequences. Financial support from the Swedish Forestry and Agricultural Research Council (SJFR) (grant 30.0645/97) is gratefully acknowledged.


   References
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 5 May 1999; accepted 18 June 1999.