1 Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, UK
2 Forest Research Agency, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UK
Correspondence
Kenneth W. Buck
k.buck{at}imperial.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number of the sequence determined in this work is AJ877914.
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INTRODUCTION |
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METHODS |
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Extraction and fractionation of nucleic acids.
Nucleic acids were extracted from the mycelium of Phytophthora isolate P441 and dsRNA was isolated from the total nucleic acids by lithium chloride fractionation and purified by removal of traces of DNA and ssRNA by digestion with DNase and S1 nuclease, respectively, followed by electrophoresis through a 1 % agarose gel and extraction of the dsRNA band with an RNaid kit (Bio 101), as described by Hong et al. (1998a). dsRNA was also analysed by polyacrylamide gel electrophoresis as described by Rogers et al. (1986)
. Incubations with DNase and S1 nuclease were carried out as described by Hong et al. (1998b)
.
cDNA cloning, sequencing and sequence analysis.
cDNA libraries were constructed using gel-purified dsRNA either using random hexamer primers as described by Hong et al. (1998b) or by a novel RT-PCR method (M. J. Roossinck, personal communication). Nucleotide sequences were obtained by the Sanger chain-termination method (Sanger et al., 1977
) using dye-terminator cycle sequencing with AmpliTaq DNA polymerase FS (ABI Prism 377). cDNA clones between contigs were obtained using RT-PCR with sequence-specific primers. cDNA clones of the ends of the dsRNA were obtained by the rapid amplification of cDNA ends (RACE) procedure (Frohman et al., 1988
) using a Gibco-BRL 5' RACE system with the vector pGEM-T Easy (Promega) and by RNA ligase-mediated (RLM)-RACE (Schaefer, 1995
). Sequences were assembled and analysed using the University of Wisconsin Genetics Computer Group programs (Devereux et al., 1984
). BLASTP searches of the UniProt database were done using WU-Blast2 programs at http://www.ebi.ac.uk. TBLASTN searches of the P. ramorum and Phytophthora sojae genome databases were carried out at http://genome.jgi-psf.org. Multiple sequence alignments, construction of phylogenetic trees and bootstrap analysis were done with the CLUSTAL X program (Thompson et al., 1997
). Phylogenetic trees were displayed using TREEVIEW (Page, 1996
).
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RESULTS AND DISCUSSION |
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Phytophthora P441 dsRNA is related to plant viruses in the genus Endornavirus
A BLASTP search of the UniProt protein database using the complete amino acid sequence of the predicted P441 protein produced highly significant alignments with predicted polyprotein sequences encoded by two plant viruses in the genus Endornavirus (Gibbs et al., 2000, 2004
), Vicia faba endornavirus (VfEV) (Pfeiffer, 1998
) and Oryza sativa endornavirus (OsEV) (Moriyama et al., 1995
). Seven alignments totalling 1998 aa were obtained between corresponding regions spread across most of the P441 and VfEV polyproteins (Table 1
) with a composite P value of 3·3e139. Similarly, eight alignments totalling 1722 aa were obtained between corresponding regions spread across most of the P441 and OsEV polyproteins (Table 1
) with a composite P value of 1·7e117. There was also significant sequence similarity between the P441 polyprotein and those of two other endornaviruses, Oryza rufipogon endornavirus (OrEV) (Moriyama et al., 1999
) and Phaseolus vulgaris endornavirus (Wakarchuk & Hamilton, 1985
). For the latter virus, only a small amount of sequence (630 bp, 210 aa) was available in the GenBank/EMBL database (Wakarchuk & Hamilton, 1990
). Endornaviruses are endogenous dsRNA elements with genomes in the size range of 1418 kbp and contain a single ORF spanning most of the length of the RNA (Gibbs et al., 2004
). The size of the Phytophthora P441 dsRNA, the presence of a single ORF and the significant sequence similarity indicated that this dsRNA was related evolutionarily to the plant endornaviruses. We therefore propose that it is a novel member of the genus Endornavirus and suggest the name phytophthora endornavirus 1 (PEV1). This would be the first non-plant member of the genus.
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The large proteins encoded by the endornaviruses have been assumed to be polyproteins that are processed by virus-encoded proteinases by analogy with polyproteins encoded by other viruses, such as the picornaviruses (Seipelt et al., 1999). No proteinase motifs or proteolytic cleavage sites have yet been reported for the plant endornaviruses. We searched the PEV1 polyprotein sequence for conserved proteinase motifs, as described by Koonin & Dolja (1993)
, but were unable to identify any such motifs unequivocally. However, during the search we identified a cysteine-rich region that contained 19 cysteine residues in a sequence of 89 residues (aa 675763). This sequence could be aligned with previously undescribed cysteine-rich regions in the polyproteins of VfEV (14 cysteines), OsEV (nine cysteines) and OrEV (nine cysteines). Whether these regions have structural or enzymic roles is not known.
There was no significant sequence similarity between the 5'UTR of PEV1 plus-strand RNA and those of VfEV, OsEV or OrEV, irrespective of whether the first or second methionine codon was used as the translation initiation site. Also there was no similarity between the 3'UTR of the plus strand of PEV1 and that of VfEV. Although the 3'UTRs of OsEV and OrEV did not terminate in a run of C residues as found in PEV1, it was noted that the 3'UTRs of these RNAs were C-rich (CCCCTCCCAAACCCCGG and TCCCTCCCCAACCCCGG, respectively; Moriyama et al., 1995, 1999
).
A single break has been reported in the plus strand of OsEV, OrEV and VfEV at nt 1211 (or 1256), 1197 and 2735 from the 5' end, respectively, while the minus strand contained no break (Fukuhara et al., 1995; Moriyama et al., 1999
; Pfeiffer, 1998
). We examined the plus and minus strands of PEV1 dsRNA for breaks or discontinuities using RLM-RACE. An oligonucleotide was ligated to 3'-OH groups in denatured PEV1 dsRNA. There should be three types of 3'-OH group, two from the 3' termini of the dsRNA and the third from any internal breaks in the RNA. RLM-RACE had been used to determine the sequences of the 3' termini of the RNA. When RLM-RACE was used in conjunction with a forward primer close to the 5' end of the plus strand together with a reverse primer complementary to the ligated oligonucleotide, three independently obtained clones indicated a break in the plus strand at nt 1215. Additionally, two independent clones indicated a break at nt 926, three clones indicated a break at nt 930 and two clones indicated a break at nt 933. It is noteworthy that the break at nt 1215 is similar in position to breaks in the plus strand reported for OsEV (nt 1211 and 1256) (Fukuhara et al., 1995
) and OrEV (nt 1197) (Moriyama et al., 1999
). Attempts to determine whether all or only a proportion of the PEV1 dsRNA molecules contained the breaks by denaturing gel electrophoresis were unsuccessful due to difficulties in completely denaturing the dsRNA.
PEV1 polyprotein contains RNA-dependent RNA polymerase-like and helicase-like regions
The region of the PEV1 polyprotein with the highest similarity to the VfEV and OsEV polyproteins, identified by the BLASTP search (Table 1), was located near the C terminus (aa 40524542). Inspection of this sequence showed that it contained sequence motifs, labelled AE, characteristic of RNA-dependent RNA polymerases (RdRps) (Fig. 2a
). Motifs AD corresponded to those described by Poch et al. (1989)
; similar motifs have been described by Habili & Symons (1989)
, Bruenn (1991)
and Koonin (1991)
. Motif E was similar to motifs described by Bruenn (1993)
and Koonin (1991)
. When the region of the PEV1 polyprotein defined by motifs AE was used in a BLASTP search of the UniProt database, as expected the two highest scores were single homologous regions from the VfEF polyprotein (49 % identity, 65 % similarity, P value 3·8e54) and OsEV polyprotein (45 % identity, 61 % similarity, P value 5·2e48), previously identified as containing RdRp domains (Pfeiffer, 1998
; Moriyama et al., 1995
). The next most similar alignments were with regions of RdRps of several closteroviruses, tobamoviruses, a cucumovirus and hepatitis E virus (HEV) (P values in the range of 9·4e8 to 9·6e5). A neighbour-joining phylogenetic tree constructed from motifs AE of a range of fungal and plant RNA viruses and HEV showed that PEV1 clustered with the three members of the genus Endornavirus with 100 % bootstrap support (Fig. 2b
). Another branch with moderate (50·1 %) bootstrap support clustered PEV1 and the endornaviruses with tobamoviruses, closteroviruses, potexviruses, a cucumovirus (Bromoviridae) and HEV.
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PEV1 contains a putative UDP glycosyltransferase gene
A BLASTP search of the UniProt database using aa 27753299 of the PEV1 polyprotein produced significant alignments with many bacterial and fungal UDP-glucose : sterol glucosyltransferases. The highest score was produced from the UDP-glucose : sterol glucosyltransferase of Ustilago maydis, which aligned as two fragments with a composite P value of 5·1x104. The shorter fragment (42 % identity, 66 % similarity), which was located at the N terminus of the PEV1 region (aa 27752794), contained an 8 aa sequence motif found in UDP-glucose : sterol glucosyltransferases from bacteria, fungi and plants (Warnecke et al., 1999). The longer fragment (23 % identity, 39 % similarity) was located near the C terminus of this region (aa 30403272) and contained a 39 aa sequence motif characteristic of all UDP glycosyltransferases (UGTs) and suggested to be a UDP-sugar-binding domain (Mackenzie et al., 1997
). A multiple alignment of these two motifs, designated A and B, from UDP-glucose : sterol glucosyltransferases of bacteria, fungi, plants and protists is shown in Fig. 4
. Out of 47 residues compared, there were 13 residues that were invariant in the 16 sequences compared and 40 of the residues in the PEV1 protein were found in at least one of the other sequences. This constitutes good evidence that PEV1 contains a UGT gene, which is probably a UDP-glucose : sterol glucosyltransferase gene.
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It is noteworthy that the PEV1 UGT sequence was more similar to those of cellular UGTs than to the UGTs of OrEV and OsEV. The RdRps that replicate RNA viruses lack proofreading mechanisms (reviewed by Buck, 1996) and therefore a UGT gene incorporated into an RNA virus has the potential to evolve more rapidly than cellular UGT genes. Hence, if both the PEV1 and OrEV (and OsEV) UGT genes have been evolving independently since the separation of protists from plants, it is likely that they would have become more divergent from each other than from the host genes. It is possible that VfEV has originated from the same UGT-containing ancestral virus as PEV1, OrEV and OsEV and that the UGT gene has been lost. This could have occurred if the UGT gene did not have a useful function to the virus in its bean host. The PEV1 UGT amino acid sequence was only very distantly related to that of baculovirus ecdysteroid UGTs, which lack motif A. The cellular sterol UGTs and baculovirus ecdysteroid UGTs have different substrate specificities and belong to different UGT families (Mackenzie et al., 1997
). Hence, PEV1 and baculoviruses have most likely acquired their UGT genes after the divergence of these two families of cellular UGT genes.
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ACKNOWLEDGEMENTS |
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Received 6 December 2004;
accepted 7 February 2005.
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