Department of Biological Sciences, University of Maine, Orono, ME 04469-0102, USA1
Author for correspondence: Stellos Tavantzis. Fax +1 207 581 2969. e-mail stellos{at}umit.maine.edu
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Abstract |
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Introduction |
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Although progress has been made, we have no knowledge of the genetic content of a large number of dsRNAs found in field isolates of R. solani (Zanzinger et al., 1984 ; Hyakumachi et al., 1985
; Kousik et al., 1994
). The high degree of genetic diversity among dsRNA elements found in natural populations of R. solani (Bharathan & Tavantzis, 1990
, 1991
) suggests that it is reasonable to assume that the genetic information carried by a dsRNA is more important in phenotype determination than the mere presence of any dsRNA in a R. solani isolate. We have previously described the physico-chemical properties of a dsRNA mycovirus from isolate Rhs 717, which belongs to anastomosis group (AG) 2 of R. solani. The virus consists of icosahedral virions, 33 nm in diameter, containing dsRNAs of 2·4 kb (dsRNA 1) and 2·2 kb (dsRNA 2), and an RNA-dependent RNA polymerase (RDRP) (Tavantzis & Bandy, 1988
). We report here the complete sequence of both genomic segments of this virus. Genetic information on dsRNA 1 includes a putative RDRP, whereas that on dsRNA 2 includes a putative capsid protein (CP). Both segments have a high degree of sequence similarity with partitiviruses from the ascomycetes Atkinsonella hypoxylon (Oh & Hillman, 1995
) and Fusarium poae (Compel et al., 1999
).
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Methods |
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Virus purification.
Purification of the R. solani 717 virus, and subsequent isolation of the virion RNA, were carried out as previously described (Tavantzis & Bandy, 1988 ).
Cloning of viral RNA.
cDNA cloning was as described by Lakshman & Tavantzis (1994) using virion dsRNAs as templates.
Total RNA extraction.
Total RNA was extracted from mycelium according to Logemann et al. (1987) , with minor modifications.
dsRNA extraction.
The procedure for extraction of dsRNA from fungal mycelium using CF-11 cellulose column chromatography was as described by Morris & Dodds (1979) with minor modifications (Lakshman & Tavantzis, 1994
). Contaminating DNA and single-stranded RNA were removed as described previously (Hoch et al., 1985
).
Northern blot hybridization analysis.
Total RNA was denatured in formalin/formaldehyde and fractionated on 1·2% agarose gels containing 2·2 M formaldehyde (Sambrook et al., 1989 ). dsRNAs were denatured by immersing gels in 50 mM NaOH for 30 min at room temperature (Lakshman & Tavantzis, 1994
). RNA was transferred to Hybond-N (Amersham) membranes by capillary transfer, and Northern blots were prehybridized, hybridized and washed as described by Lakshman et al. (1998)
.
Sequencing.
cDNA sequences were determined by the Sanger dideoxy method using cycle sequencing and an Applied Biosystems 373A automated DNA sequencer. Ambiguous sequences were confirmed by direct sequencing of the products of RTPCR reactions, which where carried out using purified viral RNA as templates. The sequences of the extreme ends of both segments were determined by amplification using 5' RACE (GIBCO-BRL), followed by direct sequencing of the amplified products.
Sequence analysis.
DNA sequences were analysed using PC Gene (Intelligenetics), the GCG sequence analysis package (Genetics Computer Group) and the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990 ). Multiple sequence alignment was performed with CLUSTAL W (Thompson et al., 1994
), and the tree topology was obtained using the exhaustive search strategy in PAUP* (Swofford, 1998
).
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Results |
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The 3' end of the coding strand of dsRNA 1 has an interrupted poly(A) tail. Thirty-three of the 47 3' terminal bases are adenosines. This poly(A) region is broken up into four stretches of As, with the longest uninterrupted stretch being 14 bases long (data not shown).
Direct comparison of the sequences of the two dsRNAs of the Rhs 717 virus revealed a stretch of similarity between the 5' ends of the coding strands. This region has 100% identity over 47 bases, with a 6 base gap introduced into the dsRNA 2 sequence, and 65% identity over the next 40 bases (Fig. 2). Within the 47 base identical region, there is a short inverted repeat sequence which could allow for the formation of a stemloop structure having a 6 base stem and a 24 base loop in dsRNA 1, and a stemloop structure consisting of a 6 base stem and a 19 base loop in dsRNA 2 (data not shown). BLAST searches (Altschul et al., 1990
) were performed comparing the core region with the complete GenBank DNA sequence database, but no significant homologies with other sequences were observed.
A BLAST search of the GenBank DNA database using the nucleic acid sequence of dsRNA 1 revealed a phylogenetic relationship (p score 8·2e-62) between this dsRNA and the first genomic segment of Atkinsonella hypoxylon 2H partitivirus (Oh & Hillman, 1995 ). A slightly higher homology (p score 4·4e-136) was observed between dsRNA 1 of the Rhs 717 virus and dsRNA 2 of Fusarium poae virus 1 (Compel et al., 1999
).
Analysis of the coding potential of each of the two dsRNAs revealed that dsRNA 1 could potentially code for a 730 aa protein (bases 862275) (Table 1). Similarly, dsRNA 2 contains an ORF that could code for a protein of 683 aa (bases 792130) (Table 1
). The estimated molecular masses of the two proteins are 85·8 and 76·4 kDa, respectively.
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Discussion |
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An RNA moiety migrating slower than the respective genome-sized band was visible in autoradiograms from Northern blots of denaturing gels (Fig. 1), but inverse RTPCR failed to show that dsRNA 1 or dsRNA 2 occur as covalent circles, head-to-tail, head-to-head or tail-to-tail concatemers in vivo (data not shown). In contrast, the M2 dsRNA from R. solani has been shown to have a circular form (Lakshman et al., 1998
). The absence of subgenomic RNAs can be attributed to the fact that ORF 1 and ORF 2 span the entire coding capacity of dsRNA 1 and dsRNA 2, respectively.
In addition to the Rhs 717 virus, other partitiviruses have been reported to have interrupted poly(A) tails at their 3' ends. The first dsRNA of A. hypoxylon 2H partitivirus has 13 As out of 21 bases whereas the second segment has 36 As out of 48 bases. The third segment of the 2H virus has an interrupted poly(U) tail (31 Us out of 58 bases) (Oh & Hillman, 1995 ). dsRNA 2 of F. poae virus 1 also has an interrupted poly(U) tail (Compel et al., 1999
). dsRNA 2 of the Rhs 717 virus has no poly(A)- or poly(U)-rich sequences, while neither dsRNA 1 nor dsRNA 2 has a eukaryotic polyadenylation signal (AAUAAA).
The 5' ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved and contain inverted repeats capable of forming stemloop structures (data not shown). Several multipartite viruses have 5' end sequences that are conserved among their genomic segments (Matthews, 1991 ; Anzola et al., 1987
; Wickner, 1993
; Oh & Hillman, 1995
), and some satellite RNAs show sequence similarity with their helper viruses at the 5' and/or 3' ends (Simon, 1988
). Conserved sequences at the 3' ends of the L-A and L-BC dsRNAs of Saccharomyces cerevisiae are involved in replication (Ribas & Wickner, 1996
), whereas small stemloops are present in dsRNA replication/packaging recognition sequences (Wickner, 1993
; Mindich et al., 1994
; Schuppli et al., 1994
; Ribas & Wickner, 1996
).
Each of the genomic segments has four CAA repeats within the respective 5'-end conserved regions, but downstream of the 47 base core region (Fig. 2). CAA repeats, located in the 5'-untranslated region of the tobacco mosaic virus genomic RNA, have been associated with initiation and enhancement of translation (Zaccomer et al., 1995
). It appears that the 5'-end conserved regions are involved in replication and packaging of the respective dsRNAs as well as translation of the RDRP and CP genes.
The deduced protein sequence encoded by ORF 1 of dsRNA 1 showed homology with the putative RDRP of F. poae virus 1 (Compel et al., 1999 ) (46% identities and 13% conservative substitutions over 681 aa) and the corresponding RDRP of dsRNA 1 of A. hypoxylon 2H partitivirus (39% identities, and 15% conservative substitutions over 641 aa) (Oh & Hillman, 1995
). Homologies were much higher within the core motifs of these polymerases (Fig. 3
). Homology (24% identities and 15% conservative substitutions over 357 aa) was also observed between ORF 1 of dsRNA 1 (aa 275625) and the putative RDRP core motif region (aa 149468) of the FusoV virus of F. solani (Nogawa et al., 1996
). Sequence similarity between the RDRP ORF (dsRNA 1) of the Rhs 717 partitivirus and the RDRP of plant partitiviruses (Cryptoviridae), such as beet cryptic virus 3 RNA 2, was lower (19·6% identities, 11·5% conservative substitutions over 480 aa) (data not shown) than that observed among fungal partitiviruses (Fig. 4
). The phylogenetic tree of Fig. 4
shows that when only partitivirus RDRP amino acid sequences are compared, fungal and plant partitiviruses form distinct branches sharing a relatively low homology. Similar relationships were observed when other virus groups were included in the comparison (Hong et al., 1998
), although there was no grouping into fungal and plant partitiviruses.
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The 683 aa (76·4 kDa) putative polypeptide encoded by ORF 2 of dsRNA 2 exhibited a moderate degree of phylogeny (22% identities and 16% conservative substitutions over 583 aa) with the putative capsid proteins of F. poae virus 1 (Compel et al., 1999 ) and A. hypoxylon 2H partitivirus (20% identities and 15% conservative substitutions over 632 aa) (data not shown) (Oh & Hillman, 1995
). Tavantzis & Bandy (1988)
showed that in vitro translation of the Rhs 717 virion dsRNA yielded two major polypeptides of 77 and 71 kDa, which were immunoprecipitated by antibodies raised against purified Rhs 717 virions. The deduced size (76·4 kDa) of the polypeptide encoded by ORF 2 (dsRNA 2) is very similar to that of the largest in vitro translation product. Thus, it appears that ORF 2 encodes the CP of the Rhs 717 partitivirus. This hypothesis is supported by the homology observed between this ORF and the putative CPs of both A. hypoxylon 2H virus (Oh & Hillman, 1995
) and F. poae virus 1 (Compel et al., 1999
). The immunoprecipitation data (Tavantzis & Bandy, 1988
) in conjunction with the sequence data suggest that the 71 kDa polypeptide is either an incomplete in vitro translation product or a result of proteolytic activity on the 77 kDa CP, a phenomenon observed with CPs of partially purified plant viruses (Tavantzis, 1980
).
R. solani 717 partitivirus is closely related to F. poae virus 1 and the 2H partitivirus from A. hypoxylon. dsRNAs viruses evolve and diverge rapidly, and thus RDRPs are identified by a small set of weakly conserved motifs (Koonin, 1992 ; Bruenn, 1993
; Koonin & Dolja, 1993
). It is significant in this regard that the RDRP genes of three dsRNA viruses, one found in a basidiomycete (R. solani) and two occurring in ascomycetes (F. poae and A. hypoxylon), retain a relatively strong homology both at the nucleic acid (see Results) and the amino acid level (Fig. 3
).
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Footnotes |
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b Present address: Promega Co., 2800 Woods Hollow Road, Madison, WI 53711-5399, USA.
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Received 1 July 1999;
accepted 18 October 1999.