Department of Microbiology, University of Alabama at Birmingham, BBRB 373/17, 845 19th Street South, Birmingham, AL 35294-2170, USA1
Department of Animal Health and Biomedical Science, University of Wisconsin-Madison, Madison, WI 53706, USA2
Author for correspondence: Andrew Ball. Fax +1 205 934 1636. e-mail andyb{at}uab.edu
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Abstract |
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Introduction |
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Partly because nodaviruses provide a leading model system for understanding the structure and assembly of T=3 icosahedral virions, previous comparative studies of the Nodaviridae have focused mainly on the sequence and structure of the capsid protein (Kaesberg et al., 1990 ; Cheng et al., 1994
; Nishizawa et al., 1995
; Schneemann et al., 1998
; Johnson et al., 2000
). Furthermore, for several years the only known RNA1 sequences were those of FHV and BBV, which are so similar to one another that comparisons are uninformative. Recently, however, we determined the RNA1 sequence of Pariacoto virus (PaV), a distant relative of FHV and BBV (Johnson et al., 2000
), and Nagai & Nishizawa (1999)
published the RNA1 sequence of Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses. In this report, we add to these the RNA1 sequences of two more insect nodaviruses: Nodamura virus (NoV), the type species of the alphanodaviruses, and Boolarra virus (BoV). For the first time, these data allow us to compare the RNA1 segments of six diverse members of this virus family, to examine in detail the predicted sequences and structures of protein A and to compare the catalytic subunit of a nodavirus RdRp with the known structures of other viral RNA polymerases.
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Methods |
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cDNA synthesis, cloning and sequence determination.
Virion RNAs were extracted from purified viruses as described previously (Dasgupta et al., 1984 ; Dasmahapatra et al., 1985
; Ball et al., 1992
; Johnson et al., 2000
). The synthesis and cloning of full-length FHV cDNA (at that time called BBV-W17) has been described (Dasmahapatra et al., 1986
). Part of the sequence of RNA1 was confirmed using an independent isolate of FHV obtained from Paul Scotti. For BoV, an oligonucleotide complementary to the 3'-terminal sequence of FHV RNA1 (Dasmahapatra et al., 1985
) was used to prime first-strand cDNA synthesis on RNA1 on the assumption that there is close homology between these viruses. Second-strand cDNA was created using replacement synthesis and the nucleotide sequence of the cloned cDNA was determined by dideoxynucleotide chain-termination (Henikoff, 1984
; Kraft et al., 1988
). The sequence of 250 nt at the extreme 5' end of BoV RNA1 was determined by using 5'RACE (GibcoBRL; Frohman, 1990
). The sequence of subgenomic BoV RNA3 was determined previously by dideoxynucleotide sequencing of positive- and negative-sense RNA3 isolated from BoV-infected cells (Harper, 1994
).
For NoV, genomic RNA was used as a template for first-strand cDNA synthesis with random hexameric primers. NoV1-specific primers were then designed from the sequences of partial cDNA clones and used to generate larger cDNAs. 5'-terminal cDNA clones were generated, as described previously (Johnson et al., 2000 ), by 5'RACE. cDNA clones representing the 3'-terminal 200 nt were generated by RTPCR across the 3'5' junctions of dimers of RNA1 that arise naturally during RNA replication, as described previously (Johnson et al., 2000
). Such molecules have been detected during the replication of FHV, NoV and PaV RNAs (Ball et al., 1992
; Ball, 1994
; Johnson et al., 2000
). For this procedure, total cellular RNA from cells transfected with NoV virion RNA was used for RTPCR. First-strand cDNA was primed with a negative-sense oligonucleotide that annealed near to the 5' end of RNA1. The 3'5' dimer junction was amplified by PCR using a positive-sense primer that annealed near to the 3' end of the adjacent RNA1 copy. RTPCR products representing the 3'5' junctions were then cloned and sequenced. Full-length cDNA of NoV RNA1 was synthesized by RTPCR using primers specific for the 5'- and 3'-termini and ligated into plasmid TVT7R(0,0) (Johnson et al., 2000
). As for PaV, the cDNA inserts of plasmids whose transcripts replicated autonomously were sequenced completely along both strands and this sequence was deposited and used for all further analyses presented below.
Sequence manipulation.
Nucleotide sequences were assembled and analysed using the University of Wisconsin Genetics Computer Group (GCG) programs (Devereux et al., 1984 ). Other nodavirus RNA1 sequences were retrieved from GenBank: BBV (X02396; Dasmahapatra et al., 1985
), FHV (X77156; R. Dasgupta, unpublished), PaV (AF171942; Johnson et al., 2000
) and SJNNV (AB025018; Nagai & Nishizawa, 1999
). Amino acid sequences were aligned using PILEUP with a gap weight of 3.
Search, prediction and modelling algorithms.
The amino acid sequences encoded by ORF A of each nodavirus were submitted to the PredictProtein website (http://www.embl-heidelberg.de/predictprotein/) and disseminated to all the linked websites for computer protein analysis. The most informative results were obtained from the University of California-Santa Cruz Sequence Alignment and Modeling Software system (http://www.cse.ucsc.edu/research/compbio/sam.html), the Brunel Bioinformatics Group (http://insulin.brunel.ac.uk/psipred/), the San Diego Supercomputer Protein Structure Homology Modeling server (http://cl.sdsc.edu/hm.html) and SWISS-MODEL at GlaxoWellcome Experimental research (http://www.expasy.ch/spdbv/).
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Results and Discussion |
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For BoV and SJNNV the situation is less clear. The final BoV RNA1 sequence was compiled from nt 1250 of the consensus sequence obtained from the 5'RACE clones, nt 2513088 of a BoV RNA1 cDNA and nt 30893096 as determined from RNA3 by Harper (1994) . However, the sequence of the extreme 3' end of BoV RNA1 is less certain. The description of the primer used for first-strand cDNA synthesis does not match the sequence reported for the resulting clone; however, direct sequencing from gel-purified RNA confirmed the reported sequence (Harper, 1994
; Fig. 2
). Stocks of infectious BoV are no longer available so we are presently unable to resolve this ambiguity. Similarly, although the published SJNNV RNA1 sequence may contain the complete termini, the methods described by Nagai & Nishizawa (1999)
do not guarantee this.
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Synthesis of RNA3 during replication opens the ORFs for one or two proteins (B1 and B2). In BBV, protein B2 is expressed only from RNA3 rather than from RNA1 (Friesen & Rueckert, 1982 ; Guarino et al., 1984
), and in view of the 3' proximal position of this ORF in RNA1, it is probable that RNA3 alone directs B protein synthesis for all nodaviruses. For the six viruses compared here, ORF B2 overlaps ORF A in the +1 reading frame, but whereas it extends one to seven codons beyond ORF A in BBV, FHV, BoV and NoV, it terminates 17 or 16 codons, respectively, before ORF A in PaV and SJNNV. In RNA3 of BBV and FHV, ORF B2 is preceded by ORF B1, which corresponds to the 3' end of ORF A, and the encoded B1 protein has been detected in infected cells (Harper, 1994
; unpublished results). Despite being encoded in the 5'-proximal ORF in RNA3, protein B1 is made in low amounts relative to B2, perhaps because the initiating AUG lies only 8 nt from the 5' end of RNA3 and has a sub-optimal context for initiation, i.e....CCAAUGU...(Cavener & Ray, 1991
; Kozak, 1999
). However, protein B1 is not essential for FHV replication and the B1 ORF is absent from BoV and SJNNV RNA3 (Table 1
; Harper, 1994
; Nagai & Nishizawa, 1999
). NoV and PaV RNA3s contain potential B1 ORFs of 131 and 94 codons, respectively, but in contrast to the situation in BBV and FHV, they start downstream of the B2 ORFs and are therefore less likely to be expressed. For both NoV and PaV, single B proteins, which are most likely translation products of the B2 ORFs, have been detected (data not shown).
Comparisons of RNA termini
The 5' and 3' untranslated regions (UTRs) of the RNA1 segments are shown in Fig. 2. For the five insect nodaviruses, the 5' UTRs vary from 21 nt for NoV to 39 nt for FHV (Fig. 2a
). In contrast, the 5' UTR of SJNNV is at least 64 nt long and may be longer (Nagai & Nishizawa, 1999
). Even among the insect nodaviruses, the 5' UTRs have dissimilar sequences, although all are AU-rich. Like RNA3, most RNA1s start 5' GU..., although PaV RNAs 1 and 2 start 5' AUG...(Johnson et al., 2000
). ORF A starts at the first AUG in every virus except PaV, where it starts at the second AUG; the 5' terminal AUG of PaV RNA1 opens an ORF of only two codons.
Fig. 2(b) shows the 3' UTRs, which vary in length from 51 nt for NoV to 71 nt for BBV and FHV, and maybe more for BoV. As described above, it is unclear whether the 3' sequences of BoV and SJNNV RNA1 shown in Fig. 2
reflect the authentic RNA termini, but in the other cases, the sequences shown represent the 3' ends because nodavirus RNAs are not polyadenylated (Newman & Brown, 1976
). The 3' UTRs of BBV and FHV RNA1 are identical in length and sequence (Fig. 2b
); indeed the sequences of these RNAs are identical for the last 569 nt and differ by only 1% overall. This is an extraordinary level of similarity between two distinct virus species and it raises the possibility that BBV-W17, the virus from which the FHV RNA1 sequence was determined, might be an inadvertent laboratory reassortant between FHV RNA2 and BBV RNA1. However, this possibility was eliminated when we obtained another sample of the original isolate of FHV directly from Paul Scotti and found that its RNA1 sequence was almost identical to that of the BBV-W17 isolate of FHV. Nevertheless, the high level of RNA1 sequence identity suggests that BBV and FHV, which were isolated about 600 km apart in New Zealand, shared a common RNA1 ancestor relatively recently. The 3' UTR of BoV RNA1 has clear similarities to BBV and FHV and overall the RNAs are 78% identical in sequence. However, there is little sequence conservation among the 3' UTRs of SJNNV, NoV and PaV, nor between any of these viruses and BBV, FHV and BoV.
Previous analysis of the 3' UTR of RNA2 from BBV, FHV, BoV and NoV and RNA1 from BBV showed that these sequences contained a conserved C-rich motif [CCCC(X)nCGC] followed by two predicted stemloops (Kaesberg et al., 1990 ). The first of the loops contained a UUA triplet in all of the sequences examined, except NoV RNA2. Although there is little primary sequence conservation at the 3' end among the RNA1 sequences of the six viruses analysed in this study, all of the sequences are GC-rich in this region. The C-rich motif, as described by Kaesberg et al. (1990)
, was identified in BBV, FHV and BoV. PaV and SJNNV lack the conserved motif but have GC-tracts of 7 and 10 nt, respectively, 2224 nt downstream of the ORF A stop codon. The RNA1 3' UTRs were analysed using MFOLD (Zuker, 1989
) and the predicted secondary structures were examined to see if any general features could be identified. Although there were some intriguing commonalities, such as the 7 nt sequence 5' CCCAUCU 3' located in a loop for NoV RNAs 1 and 2 (Kaesberg et al., 1990
), a similar pattern was not found for any of the other RNA1/RNA2 combinations. Furthermore, no common pattern of secondary structure was identified among the six RNA1 3' UTRs using MFOLD. Also, the UUA loop motif identified previously by Kaesberg et al. (1990)
for RNA2 of BBV, FHV and BoV was not conserved for BoV RNA1. Indeed, the sequence UUA does not occur anywhere in the 3' UTR of BoV RNA1. Evidently the cis-acting elements in nodavirus RNAs that are recognized by the RNA replicase are not obvious in either the primary sequences or the predicted secondary structures of their 3' ends.
Comparisons of protein A sequences
The levels of identity among the six nodavirus protein A sequences were determined by pairwise comparisons using the GCG program GAP (Table 2). The FHV and BBV protein A sequences are nearly indistinguishable with 99% identity and they closely resemble BoV protein A, which shares 84% identity with each. NoV protein A has the next nearest sequence, sharing 4144% identity with BBV, FHV and BoV. PaV and SJNNV protein A sequences are 31% identical to one another but each shares less than 30% identity with the other viruses (Table 2
). Thus, both in the sequence of protein A and in the truncation of the B2 ORF, the insect virus PaV resembles the fish virus SJNNV more closely than it resembles the other insect viruses. However, RNA sequences from close relatives of PaV and from additional fish nodaviruses will be necessary to define the overall phylogenetic relationships among members of the Nodaviridae family.
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The six protein A sequences were aligned using PILEUP. The variation in the level of conservation along the protein is presented quantitatively in Fig. 3 as the plurality of the consensus sequence derived from the alignment shown in Fig. 4
. The alignment illustrates the high level of similarity among BBV, FHV and BoV, but in order to highlight sequence conservation that extends beyond these three viruses, only those residues that are identical in four or more of the proteins are shaded in Fig. 4
.
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On the C-terminal side of the polymerase domain, the protein A sequence alignment deteriorates and the level of conservation decreases, although the C terminus itself is rich in proline and glycine in all six proteins. Its sequence complexity diminishes in the region of overlap with ORF B2. The N-termini are also poorly conserved for the first 7080 amino acids, as demonstrated by the many gaps in the alignment, but between residues 80250 there is a region with an above-average level of conservation among all six proteins (Fig. 3; see below for further discussion). This is followed by a stretch of polypeptide between amino acid 250 and the start of the RdRp signature sequence that is well conserved among BBV, FHV, BoV and NoV but much less so between this group and the PaV and SJNNV sequences.
Other sequence motifs in protein A
Since nodavirus RNAs are capped and RNA replication occurs in the cytoplasm, protein A is expected also to have RNA guanylyl- and methyltransferase activities (Dasgupta et al., 1984 ; Dasmahapatra et al., 1985
), but none of the well-recognized RNA guanylyl- or methyltransferase motifs (Koonin, 1993
; Shuman & Schwer, 1995
; Luongo et al., 2000
) was found in the nodavirus protein A sequences. No motifs characteristic of RNA helicases were detected (Koonin & Dolja, 1993
; Kadare & Haenni, 1997
), nor were there any other identifiable conserved amino acid sequence motifs.
The standard mechanism of RNA capping in eukaryotic cells involves three enzymatic reactions (Shuman & Schwer, 1995 ; Bisaillon & Lemay, 1997
): 5'-triphosphate cleavage by an RNA triphosphatase to yield a diphosphorylated RNA terminus, which is then capped with GMP by RNA guanylyltransferase and methylated at the N7 position of the terminal guanosine by RNA methyltransferase. The guanylyltransferase reaction proceeds via a covalent guanylylated enzyme intermediate in which the GMP residue is linked to the lysine residue of the conserved KXDG motif. However, in some members of the alphavirus family, such as Sindbis virus (SIN) and Semliki Forest virus (SFV), capping proceeds by an alternative pathway in which GTP is first methylated at the N7 position before m7GMP is transferred to RNA, again via a covalent enzyme intermediate. Both reactions are catalysed by the viral non-structural protein nsP1 (Mi et al., 1989
; Ahola & Kaariainen, 1995
). Brome mosaic virus (BMV) and Bamboo mosaic virus (BaMV), which belong to the alphavirus-like supergroup, also use the alternative pathway catalysed by the viral 1a and ORF1 proteins, respectively (Ahola & Ahlquist, 1999
; Kong et al., 1999
; Li et al., 2001
).
Mutational analyses have implicated a conserved histidine residue as the site of guanylylation of SIN and SFV nsP1 and BMV 1a proteins (Wang et al., 1996 ; Ahola et al., 1997
; Ahola & Ahlquist, 1999
), in contrast to the lysine residue found in several of the other capping enzymes (Shuman & Schwer, 1995
). Specifically, mutation of residue H80 in BMV 1a protein abolished guanylyltransferase activity and destroyed its ability to form a covalent adduct with m7GMP (Ahola & Ahlquist, 1999
; Kong et al., 1999
; Ahola et al., 2000
). The ORF1 protein of BaMV also contains a conserved histidine residue that aligns with those of SIN, SFV and BMV (Li et al., 2001
). The Gag protein of the double-stranded RNA-containing L-A virus of yeast catalyses a similar reaction during the decapping of host mRNAs by forming a covalent phosphoamide linkage with m7GMP via a histidine residue (Blanc et al., 1992
, 1994
). Galactose-1-phosphate uridylyltransferase also catalyses nucleotidyl transfer via a conserved HPH motif (Lima et al., 1997
). Interestingly, the conserved histidine residue in BaMV ORF1 protein is contained in an HTH motif, although for the alphavirus nsP1 proteins, the corresponding sequence is NDH, and for BMV 1a, it is APH (Li et al., 2001
).
Against this background, we searched the nodavirus protein A sequences for (HNA)XH motifs. In PaV and SJNNV, HXH occurs at position 9193 (numbered relative to the BBV sequence; Fig. 4). The second histidine residue (H93) is also conserved for BBV, FHV, BoV and NoV, albeit in the context of an NXH sequence as found in the alphavirus nsP1 proteins. A canonical HXH motif occurs in BBV, FHV, BoV and NoV protein A at position 134136 (BBV numbering), but it did not align with a similar sequence in the PaV and SJNNV proteins. We consider BBV H93 and its homologues in the other protein A sequences to be good candidates for the site of guanylylation during capping, particularly since they occur in a domain of the protein with an above-average level of overall conservation and no other known function. Studies to test this proposal by mutagenesis are in progress.
Comparisons of protein B2 sequences
The amino acid sequences of protein B2 were also compared in an attempt to discern its function, which is presently unknown. However, SJNNV B2 was so dissimilar that it could not be aligned with the other five sequences, and even the NoV and PaV B2 proteins showed only marginal sequence similarity with B2 of BBV, FHV and BoV. We were unable to identify any conserved sequence motifs within these proteins that might have suggested their function and none of the individual B2 proteins showed significant sequence similarity with any other proteins currently in the GenBank database. Therefore, the amino acid sequences of protein B2 provide no suggestion of its function at present.
Predicted secondary structures of protein A
Secondary structures were predicted for each of the protein A sequences using the six algorithms available through the PredictProtein website. Despite minor differences in their predictions, the different methods agreed well with one another overall. However, the program PSIPRED (version 2.0) predicted secondary structural features for the polymerase domain of PaV protein A that agreed most closely with the three-dimensional model independently generated by homology modelling (see below) and so the secondary structures predicted by PSIPRED are presented in Fig. 5. PSIPRED (Jones, 1999
) is a secondary structure prediction method that incorporates two feed-forward neural networks to analyse the output from PSI-BLAST (Position-Specific Iterated-BLAST) (Altschul et al., 1997
).
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Homology modelling of PaV protein A
The following three programs were used to search the database of known protein structures for templates that were sufficiently similar to the six protein A sequences to allow a three-dimensional structure for protein A to be predicted by homology modelling:
(i)SAM-T99 HMM (http://www.cse.ucsc.edu/research/compbio/HMM-apps/)
(ii)SWISS-MODEL (http://www.expasy.ch/spdbv/)
(iii)CPH-MODELS (http://www.cbs.dtu.dk/services/CPHmodels/).
Only one of eighteen attempts was successful: SAM-T99 HMM (Shindyalov & Bourne, 2000 ), run by the San Diego Supercomputer Protein Structure Homology Modeling server (http://cl.sdsc.edu/hm.html), found structural homology between residues 344803 of PaV protein A and residues 12461 of poliovirus 3Dpol (Hansen et al., 1997
). The alignment was trimmed to the centre of the polymerase domain encompassing structural motifs AE (Poch et al., 1989
; Hansen et al., 1997
; O'Reilly & Kao, 1998
) and used as input for SWISS-MODEL, an interactive homology modelling program (Guex & Peitsch, 1997
). Fig. 6
shows the resulting three-dimensional model of the polymerase domain of PaV protein A (Fig. 6c
) compared with the crystal structure of poliovirus 3Dpol (Fig. 6b
), as determined by Hansen et al. (1997)
. In the Ramachandran plot of the PaV model (data not shown), only 4 of the 137 modelled residues lie outside preferred or allowable regions: E574, W575, H592 and Q593, all of which lie near gaps in the sequence alignment (Fig. 6a
). While the true protein A structure can be determined only by rigorous experimental methods, the results of modelling suggest that the core of the PaV RdRp domain closely resembles that of poliovirus 3Dpol. This model will be used to guide and interpret mutational analysis of protein A.
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Other common features are more tenuous. The location of the active sites of the capping enzyme remains to be established, although several observations suggest that they may lie in the N-terminal third of protein A, with H93 as a leading candidate for covalent guanylylation. The function of protein B2 remains elusive and intriguing, particularly in view of the complex phenotype of mutants that cannot express this protein (Ball, 1995 ). Finally, comparisons of the 5' and 3' UTRs fail to reveal the cis-acting RNA signals that mediate specific recognition by the cognate RdRp during RNA replication and transcription. More work on this powerful and accessible experimental system will be required to address these questions directly.
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Acknowledgments |
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Footnotes |
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b Present address: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
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References |
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Received 13 February 2001;
accepted 3 April 2001.