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
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Abstract |
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Introduction |
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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, 1820 nm in diameter and 100150 nm or 250300 nm in length. The genome consists of three messenger-polarity single-stranded RNA molecules of ca. 6 (RNA 1), 3·2 (RNA 2) and 2·43·1 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.
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Methods |
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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).
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.
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Results and Discussion |
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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 10141021 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 16631665 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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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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Acknowledgments |
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References |
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Received 5 May 1999;
accepted 18 June 1999.