Department of Plant Pathology, University of Kentucky, 201F Plant Science Building, 1405 Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA
Correspondence
Said A. Ghabrial
saghab00{at}uky.edu
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ABSTRACT |
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Present address: Department of Plant Protection, Huazhong Agricultural University, Wuhan 430070, Hubei Province, People's Republic of China.
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INTRODUCTION |
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PcV and the related Penicillium viruses Penicillium brevicompactum virus (PbV) and Penicillium cyaneo-fulvum virus (Pc-fV) have isometric particles, 3540 nm in diameter, and contain four separately encapsidated dsRNA segments (Wood et al., 1971; Wood & Bozarth, 1972
; Buck & Girvan, 1977
; Edmondson et al., 1984
; Castón et al., 2003
). As they have not been previously characterized at the molecular level, these three viruses (grouped under the genus Chrysovirus) were provisionally placed in the family Partitiviridae (Ghabrial et al., 2000
) with the assumption that their genomes are bipartite, with dsRNA1 encoding the RNA-dependent RNA polymerase (RDRP) and dsRNA2 encoding the major capsid protein (CP). The additional dsRNAs (dsRNAs 3 and 4), like those of some partitiviruses, were presumed to be defective or satellite dsRNAs (Ghabrial et al., 2000
; Ghabrial, 2002
). In the present study, we report the complete nucleotide sequence and genome organization of each of the four monocistronic dsRNA segments associated with PcV virions and discuss similarities to viruses with multipartite and multicomponent RNA genomes. Based on the consistent co-presence of their four dsRNA segments, the existence of extended regions of highly conserved terminal sequences at both ends of all four segments, sequence comparisons and phylogenetic analysis, PcV and related viruses should not be classified with the family Partitiviridae. Our results support the creation of a new family of fungal viruses with multipartite genomes.
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METHODS |
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Extraction of viral dsRNA and Northern hybridization analysis.
Viral dsRNA was isolated from purified virions (100 µg virions resuspended in 100 µl DEPC-treated water) by SDS/phenol extraction and ethanol precipitation. The pellet was washed twice with 70 % ethanol and resuspended in TE buffer, pH 8·0. For Northern hybridization analysis, dsRNAs were separated on a 1·5 % agarose gel for 67 h at 70 V. The gel was then soaked for 20 min in 0·1 M NaOH, followed by soaking in 0·1 M Tris/HCl, pH 8·0, for 20 min, and the RNA was transferred by capillary action to a Hybond-N+ nylon membrane (Amersham) in 10x SSC buffer. Prehybridization and hybridization were performed under high stringency conditions in a hybridization solution (5x Denhardt's reagent, 6x SSC, 20 mM Tris/HCl, pH 7·5, 0·5 % SDS) containing 50 % formamide for 1416 h at 42 °C. The RNA blots were probed with 32P-labelled probes prepared by random-primer labelling of cloned cDNA to the PcV dsRNAs.
cDNA synthesis and molecular cloning.
cDNA clones of the viral dsRNA were synthesized using the TimeSaver cDNA synthesis kit (Amersham Pharmacia Biotech). Virion dsRNA (2 µg) was mixed with 0·74 µg random hexamers and incubated in 90 % DMSO at 65 °C for 30 min, ethanol-precipitated and resuspended in 20 µl DEPC-treated water. First and second strand cDNA synthesis and addition of EcoRINotI adaptors were performed according to the manufacturer's instructions. The cDNAs were inserted into the EcoRI and NotI sites of a Bluescript vector using T4 DNA ligase and recombinant plasmids were transformed into E. coli strain DH5. Clones corresponding to the different dsRNA segments were identified by Northern hybridization analysis of viral dsRNA using the cloned cDNAs as probes.
Gaps between non-overlapping cDNA clones were connected using RT-PCR and sequence-specific primers. Viral dsRNA (510 µg) was denatured by incubation in 90 % DMSO at 90 °C for 5 min and then chilled on ice for 5 min. The denatured dsRNA was reverse transcribed using Superscript II reverse transcriptase (BRL) and a sequence-specific reverse primer. The reaction mixture was incubated for 60 min at 45 °C followed by treatment with RNase H (2 U, at 37 °C for 20 min) and 5 % of the reaction volume (1 µl) was used for PCR amplification by using the pertinent sequence-specific forward and reverse primers. PCR was carried out by using Platinum Pfx DNA polymerase (BRL) and cycling parameters for touch-down PCR were 94 °C for 4 min; 94 °C for 1 min, 6550 °C (0·5 °C per cycle) for 2 min, 72 °C for 3 min, 30 cycles; 94 °C for 1 min, 50 °C for 2 min, 72 °C for 3 min, 10 cycles; 72 °C for 12 min. PCR products were purified with QIAquick gel extraction kit (Qiagen), A-tailed with Taq DNA polymerase at 70 °C for 3045 min and cloned into the pGEM-T Easy cloning vector (Promega).
In addition, clones for terminal sequences of the four dsRNAs were generated by T4 RNA ligase oligo-mediated amplification. T4 polynucleotide kinase-phosphorylated oligo-x-lig [5'-CCATATGCGGCCGCGTCGACGAATTCAGATCTTAAGGCGAC-(NH2)-3'] was ligated to the 3' ends of each strand of the viral dsRNAs using T4 RNA ligase (NEB) in 1x RNA ligase buffer, at 4 °C for 18 h. The unligated oligonucleotide was removed from the RNA ligation reaction by membrane filtration using a Microcon YM-100 (Amicon; Millipore). The oligo-ligated dsRNA was denatured by incubation in 90 % DMSO at 90 °C for 5 min and then quickly chilled on ice for 5 min. An aliquot equivalent to 5 % of the reaction mixture was used for the reverse transcription reaction with Superscript II reverse transcriptase (BRL) in 1x first strand buffer, 10 mM DTT, 0·5 mM dNTPs in a total reaction volume of 20 µl at 48 °C for 50 min and 5 pmol of a primer with sequence complementary to the oligonucleotide used for RNA ligation (oligo-REV, 5'-TCGGCCTTAAGATCTGAATTC-3'). The reaction mixture was then treated with RNase H (2 U, at 37 °C for 20 min) and a sample equivalent to 5 % of the reaction mixture was used for PCR amplification with the oligo-REV primer and a sequence-specific primer corresponding to dsRNA1, 2, 3 or 4. PCR conditions were as described above by using Platinum Pfx DNA polymerase (BRL) and cycling parameters for touch-down PCR. The PCR products were gel-purified using a gel extraction kit (Qiagen), A-tailed with Taq DNA polymerase (5 U, in 1x reaction buffer, 1·5 mM MgCl2, BRL) and dATP (0·2 mM) and then cloned into pGEM-T Easy.
Generation of full-length cDNA clones and in vitro translation.
Full-length cDNA clones of the four dsRNAs were produced using sequence-specific primers and the RT-PCR protocol described above. Forward and reverse primers of the four dsRNAs corresponding to the 5'- and 3'-terminal nucleotide sequences were designed and used for RT-PCR. The PCR products were gel-purified using a gel extraction purification kit (Qiagen) and cloned into pGEM-T Easy.
In vitro translation was performed using the TNT T7 Quick coupled transcriptiontranslation kit (rabbit reticulocyte lysates; Promega) in the presence of [35S]methionine (Redivue; Amersham). Recombinant plasmids were linearized with the pertinent restriction enzymes (SpeI for dsRNA1; SalI for dsRNAs 2 and 3; NdeI for dsRNA4) and purified with the Concert Rapid PCR purification system (Gibco-BRL, Life Technologies) and approximately 1 µg template DNA was used for the in vitro translation reaction. The in vitro translation products were separated by SDS-PAGE on an 8 % polyacrylamide gel and analysed by autoradiography using a PhosphorImager (Molecular Dynamics).
Protein sequence analysis.
For sequencing internal peptides of PcV CP, gradient-purified PcV virions were digested with sequencing-grade trypsin (Promega) at 37 °C for 18 h and the digestion products were separated by reverse-phase HPLC on an analytical C18 column (Vydak). Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation.
Nucleotide sequencing and sequence analysis.
All sequencing was performed by dideoxy chain termination sequencing using the Big Dye terminator sequencing kit (ABI) and an ABI 310 system for automated sequencing. Sequencing was carried out bidirectionally on both strands using either M13 universal primers or sequence-specific walking primers. At least two independent clones, usually three to five clones, were analysed for sequence determination at all nucleotide positions. Paired and multiple sequence alignments were performed with the programs BESTFIT, GAP and PILEUP (university of wisconsin GCG software package version 10). Sequence similarity searches of GenBank, Swissprot and EMBL databases were conducted using the BLAST program (Altschul et al., 1997). Sequence alignments and phylogenetic analysis were performed using the programs CLUSTAL X and PAUP* (Thompson et al., 1997
; Swofford, 2000
). Searches for amino acid signatures and protein motifs were conducted using the programs included in the ExPASy proteomics tools (http://www.expasy.org/tools/).
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RESULTS |
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BLAST searches of the deduced amino acid sequences of the proteins encoded by PcV dsRNAs 3 and 4 (designated hereafter as p3 and p4, respectively) showed high similarity to the corresponding proteins encoded by the chrysovirus Hv145SV (AF297178 and AF297179; A. Soldevila and S. A. Ghabrial, unpublished). The identity and similarity scores between p3 encoded by PcV and Hv145SV, as calculated by the BESTFIT program, were, respectively, 23 and 32 %. The corresponding values for p4 were 30 and 40 %. Evaluation of the pairwise sequence alignments of p3 and p4 of these two chrysoviruses, using the BESTFIT program, indicated that they were significant to highly significant; quality scores of 16 and 600 standard deviations above the mean of random alignments were obtained for p3 and p4, respectively. Interestingly, BLAST searches of the amino acid sequences of p3 also revealed significant similarity (37 % similarity; BLAST hit of 4e12) between an N-terminal region of p3 (aa positions 102 to 447) and comparable N-terminal regions of the putative RDRP encoded by dsRNA1 of PcV (aa positions 84454) and Hv145SV(aa positions 60370). These regions in the dsRNA1-encoded proteins with high similarity to p3 occur upstream of the eight highly conserved motifs characteristic of RDRPs. The significance of this sequence similarity to the function of p3 is not clear at present. It is of interest that ProDom database searches (Servant et al., 2002) revealed that the p3 sequence from aa positions 104 to 116 (GVVMPMGHGKTTLA) also shares a phytoreo S7 domain with a family consisting of several phytoreovirus S7 proteins thought to be viral core proteins.
The finding that the amino acid sequence of a tryptic peptide (KFTINGFR) derived from PcV capsid matched perfectly the amino acid sequence deduced from dsRNA4 cDNA sequence at nt positions 357380 (Fig. 6, boxed) is of considerable interest as it suggests that p4 is virion-associated as a minor protein. Although no minor proteins were discernible when purified PcV virions, loaded at 5 µg per well, were subjected to SDS-PAGE analysis (Fig. 2
, lane PcV), a faint band of the predicted size of p4 (95 kDa) was observed when 20 µg purified PcV was used (data not shown). It is evident that this observation must be confirmed when a p4-specific antiserum becomes available. Furthermore, p4 contains a consensus signature of the glycosyl hydrolases family 10 (Fig. 6
; underlined). This conserved region is centred on a conserved glutamic acid (aa position 709; Fig. 6
, boxed), which was demonstrated for one member of the glycosyl hydrolases family to be directly involved in glycosidic bond cleavage by acting as a nucleophile (Tull et al., 1991
). The significance of a putative glycosyl hydrolase activity of p4 to the PcV life-cycle is not known at present.
5' and 3' UTRs
The 5' UTRs of PcV dsRNAs are relatively long, between 144 and 162 nt in length. Direct comparison of the nucleotide sequences of the 5' UTR of the four dsRNA segments revealed regions of high sequence similarity (Fig. 7a). In addition to the absolutely conserved 5' termini (positions 1 to 10), a 50 nt region with high sequence identity is present within the 5' UTR of all four dsRNAs. Fifty-two of the 5'-terminal 60 nt are identical in at least three of the four dsRNAs. A second region of strong sequence similarity is positioned immediately upstream of the AUG initiator codon (indicated in bold, Fig. 7a
). This region consists of a stretch of 5075 nt containing reiteration of the sequence CAA (underlined, Fig. 6a
). The (CAA)n repeats are similar to the translational enhancer elements present at the 5' UTRs of tobamoviruses (Gallie & Walbot, 1992
). A highly conserved region (a stretch of 36 nt) with sequence identity above 80 % is present within the 3' UTR of all four PcV dsRNAs (Fig. 7b
). The 14 nt at the 3' end are strictly conserved among the four genomic dsRNAs (shaded area, Fig. 7b
) and 44 out of the 3'-terminal 46 nt are identical in at least three of the four dsRNAs.
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DISCUSSION |
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The dsRNA pattern of PcV virions isolated from different strains of Penicillium chrysogenum [e.g. strain Wis. Q176 (ATCC 10002) and a number of strains derived from strain NRRL 1951 (ATCC 9480)] has remained unchanged throughout the years since PcV was first isolated (Banks et al., 1969; Buck et al., 1971
; Border et al., 1972
; Wood & Bozarth, 1972
; Nash et al., 1973
; Buck & Girvan, 1977
; Edmondson et al., 1984
; this study). This is also true for other chrysoviruses isolated from different Penicillium species (Buck & Girvan, 1977
; Wood et al., 1971
) and from various strains of Helminthosporium victoriae (Sanderlin & Ghabrial, 1978
; Soldevila et al., 2000
; Ghabrial et al., 2002
). The co-presence of all four segments in different fungal species and strains harbouring chrysoviruses and the stability of the dsRNA patterns support the contention that all four segments are essential for infection and that none is defective or satellite in nature (see below for additional discussion). As defective and satellite dsRNAs are dispensable for virus infection, they are subject to loss during repeated subculturing and single spore isolation. This is well exemplified by the different dsRNA patterns in various strains of Ustilago maydis infected with the totivirus UmV-H and associated defective and satellite dsRNAs (Koltin, 1988
). To provide unequivocal evidence that all four dsRNA segments of PcV are necessary for infection, however, would require performing infectivity assays. Unfortunately, such infectivity assays are not presently available for dsRNA mycoviruses (Ghabrial, 1998
).
Results from molecular cloning, sequencing and Northern hybridization analysis of PcV dsRNAs (this study) provided conclusive evidence that none of the PcV dsRNA segments is defective in nature. The possibility that dsRNAs 3 and 4 of PcV and related viruses are satellites can be dismissed, considering their consistent and stable co-presence with dsRNAs 1 and 2 in all fungal strains and species known to harbour chrysoviruses, and the existence in all four dsRNA segments of extended regions of high sequence similarity at their 5' and 3' UTRs. These properties are typical of RNA viruses with multipartite and multicomponent genomes, as discussed above, and are unlike those of satellite RNAs. Satellite RNAs, which are generally significantly smaller in size than the genomic RNAs of the helper virus, are known to share very little or no sequence similarity with their helper viruses (Mayo et al., 2000; Bruening, 2002
). PcV dsRNAs 3 and 4 (as well as those of the chrysovirus Hv145SV; Ghabrial et al., 2002
; A. Soldevila and S. A. Ghabrial, unpublished) are of comparable size to dsRNAs 1 and 2 and all four dsRNAs share extended regions (5075 nt) of high sequence similarity (>80 %) at both of their termini. Furthermore, all four segments of PcV and Hv145SV contain tobamovirus-like enhancer elements, (CAA)n repeats, upstream of their AUG initiator codons. We are not aware of any satellite RNAs (ssRNA or dsRNA; messenger or non-messenger type) that share extended sequence similarity at the 5' and 3' UTR with their helper viruses (Bruening, 2002
). Like other satellite RNAs, the known satellite dsRNAs that are associated with totiviruses and partitiviruses share only 25 nt at their termini with the helper genomic dsRNAs (Esteban et al., 1989
; Wickner, 1996
; Rong et al., 2002
). Although the terminal sequence at the 3' end of the well-characterized M1-dsRNA, a satellite dsRNA associated with the totivirus Saccharomyces cerevisiae virus L-A (Esteban et al., 1989
; Wickner, 1996
), is substantially different from its helper virus at the level of primary nucleotide sequence, it shares similar RNA secondary structures (stemloop structures) at the 3' terminus and at an internal site with its helper virus. These structures have been shown to serve as cis-acting signals for replication and packaging (Wickner, 1996
). Moreover, the extreme 5' and 3' ends of the sense strand of all four dsRNAs from PcV and Hv145SV have the same sequence, 5'-GAUAAAAA ... UAAGUGU-3' (Ghabrial et al., 2002
; this study). The property of having regions of terminal sequences conserved within a genus is characteristic of viruses with multipartite dsRNA genomes, as exemplified by members of the family Reoviridae that include the orthoreoviruses, phytoreoviruses, orbiviruses, rotaviruses and cypoviruses (Mertens et al., 2000
). Furthermore, direct evidence based on amino acid sequencing of a tryptic peptide derived from purified PcV capsid showed that p4, encoded by dsRNA4, is virion-associated as a minor component, a characteristic of some gene products of viruses with multipartite genomes (Mertens et al., 2000
), but unknown for satellite RNA-encoded proteins (Bruening, 2002
). Collectively, these findings are consistent with the conclusion that each of the four dsRNAs of PcV and related viruses represents a component of a multipartite (multicomponent) viral genome and that none is satellite or defective dsRNA.
Amino acid sequence analysis of the protein encoded by PcV dsRNA1 revealed the presence of the eight conserved motifs characteristic of RDRPs of dsRNA viruses of simple eukaryotes (Bruenn, 1993; Ghabrial, 1998
). Comparison of the conserved motifs of PcV RDRP with those of viruses in the families Partitiviridae and Totiviridae indicated that it is most closely related to that of another chrysovirus, Hv145SV (Ghabrial et al., 2002
), and is more closely related to the totiviruses than to the partitiviruses. This conclusion is also supported by phylogenetic analysis of the conserved motifs and flanking sequences of viruses in the families Totiviridae and Partitiviridae in comparison with the chrysoviruses (Fig. 3
). Furthermore, whereas the amino acid positions corresponding to motifs 1 and 2 are clear for PcV and Hv145SV, those for partitivirus RDRPs are not well defined (Fig. 3
). Interestingly, phylogenetic analysis of the RDRP conserved motifs also revealed that the previously unclassified Agaricus bisporus virus 1 (AbV1; Van der Lende et al., 1996
) forms a sister clade to the chrysoviruses PcV and Hv145SV (this study), suggesting its placement as a tentative member of the genus Chrysovirus. Additionally, purified preparations of PcV as well as of other members of the genus Chrysovirus consistently contained four dsRNA segments, as discussed above. In contrast, members of the genus Partitivirus, known to support satellite dsRNA, may contain none, one or two satellite dsRNAs besides their two genomic segments (Oh & Hillman, 1995
; Rong et al., 2002
). Thus, the number of dsRNA segments associated with partitivirus infection may vary from two to four segments among members within the same genus. Taken together, these attributes of PcV and similar viruses suggest that they should not be classified with the family Partitiviridae, which includes viruses with bipartite genomes. A proposal, which was based on the properties of the chrysoviruses so far characterized at the molecular level (PcV and Hv145SV; Ghabrial et al., 2002
; this study), to establish a new family of isometric mycoviruses with multipartite dsRNA genomes was recently accepted by the International Committee on Taxonomy of Viruses (ICTV) and the new family was named Chrysoviridae. The genus Chrysovirus, which was removed from the family Partitiviridae, was designated the type genus of the new family and PcV as the type species of the genus (see http://www.danforthcenter.org/iltab/ictvnet under taxonomic proposals accepted by ICTV).
The three-dimensional structure of PcV was recently determined and it showed that PcV has an authentic T=1 capsid with 60 equivalent protein subunits. This is in contrast to the ubiquitous T=2 capsid, with 120 copies of the structural protein, typical of dsRNA viruses including viruses in the family Totiviridae (Castón et al., 2003). This structural study, which utilized cryo-electron microscopy combined with three-dimensional reconstruction techniques, described major conformational differences between full and empty PcV particles and suggested that a number of interactions between the inner surface of the protein shell and the genomic RNA take place. Furthermore, a mechanism for transcript release from transcribing particles was proposed based on the structure of empty particles. The availability of the complete nucleotide sequences of the PcV genome and full-length cDNA clones of viral dsRNAs and the elucidation of the common terminal structural features shared by all PcV dsRNA segments (this study) should prove valuable in future structural studies on proteinRNA interactions, mechanisms of RNA packaging and release and the role of dsRNA in the conformation of the structural subunits.
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ACKNOWLEDGEMENTS |
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Received 24 November 2003;
accepted 17 February 2004.