Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases

Karyn N. Johnson1, Kyle L. Johnson1, Ranjit Dasgupta2, Theresa Gratschb,1 and L. Andrew Ball1

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


   Abstract
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The Nodaviridae are a family of isometric RNA viruses that infect insects and fish. Their genomes, which are among the smallest known for animal viruses, consist of two co-encapsidated positive-sense RNA segments: RNA1 encodes the viral contribution to the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome, whereas RNA2 encodes the capsid protein precursor. In this study, the RNA1 sequences of two insect nodaviruses – Nodamura virus (the prototype of the genus) and Boolarra virus – are reported as well as detailed comparisons of their encoded RdRps with those of three other nodaviruses of insects and one of fish. Although the 5' and 3' untranslated regions did not reveal common features of RNA sequence or secondary structure, these divergent viruses showed similar genome organizations and encoded RdRps that had from 26 to 99% amino acid sequence identity. All six RdRp amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. A search for structural homologues in the protein structure database identified the poliovirus RdRp, 3Dpol, as the best template for homology modelling of the RNA polymerase domain of Pariacoto virus and allowed the construction of a congruent three-dimensional model. These results extend our understanding of the relationships among the RNA1 segments of nodaviruses and the predicted structures of their encoded RdRps.


   Introduction
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
The Nodaviridae are a family of small, non-enveloped, isometric viruses with bipartite positive-sense RNA genomes (Ball & Johnson, 1998 ). Two genera have been distinguished: the alphanodaviruses which primarily infect insects and the betanodaviruses which infect fish (Ball et al., 2000 ). Containing only about 4·5 kb, nodavirus genomes are among the smallest of all known animal viruses. Both genome segments are capped at their 5' ends but lack poly(A) tails (Newman & Brown, 1976 ). The smaller segment (RNA2, 1·3–1·4 kb) encodes a precursor to the capsid proteins, which are clearly homologous within each genus but show only marginal sequence similarity between the genera. Despite containing only 3·0–3·2 kb, the larger genome segment (RNA1) encodes the entire virus contribution to the RNA-dependent RNA polymerase (RdRp), which replicates both RNA1 and 2 (see Fig. 1; Ball & Johnson, 1998 ). Thus, the bipartite viral genome naturally segregates the genes involved in intracellular RNA replication from those involved in virion formation and the spread of infection.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of the nodavirus genome organization showing the two segments of the bipartite RNA genome (RNAs 1 and 2) and the subgenomic RNA3. ORFs are shown in white and grey boxes.

 
Since it functions as both mRNA and template for the viral RdRp, RNA1 of Flock house virus (FHV) can replicate autonomously in cells derived from insects, vertebrates, plants and even the yeast Saccharomyces cerevisiae (Gallagher et al., 1983 ; Selling et al., 1990 ; Ball et al., 1992 ; Price et al., 1996 ). Its small size and extreme genetic simplicity make RNA1 an accessible system for basic studies of RNA replication (Ball, 1995 ). Moreover, its robust capacity to synthesize high cytoplasmic levels of capped and functional mRNAs provides a promising approach to the amplification of heterologous RNAs expressed from vectors (Ball et al., 1994 ; Ball & Johnson, 1999 ). These considerations focus attention on the coding potential and template properties of nodavirus RNA1. Protein A, the product of the major open reading frame (ORF) of Black beetle virus (BBV) contains sequence motifs characteristic of an RdRp catalytic subunit (Dasmahapatra et al., 1985 ; Koonin, 1991 ), and an amino acid insertion among these motifs in FHV protein A (which is 99% identical in sequence to BBV) abolishes autonomous RNA replication (Ball, 1995 ). The C terminus of the protein A ORF overlaps an ORF for an 11 kDa protein (B2), which is translated from a subgenomic RNA (RNA3) made during RNA1 replication (Friesen & Rueckert, 1982 ; Guarino et al., 1984 ). Mutations that eliminate the expression of FHV RNA3 or protein B2 have little immediate effect on RNA1 replication but curtail its endurance (Ball, 1995 ). However, attempts to recover infectious B2- mutants of FHV succeeded only in isolating revertants that had restored B2 protein synthesis, suggesting that whatever its role, protein B2 was important for virus replication (Harper, 1994 ).

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.


   Methods
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Provenance and propagation of viruses.
FHV was provided by Paul Scotti (Scotti et al., 1983 ), BoV by the late Carl Reinganum (Reinganum et al., 1985 ) and by Paul Scotti, and PaV by Jean-Louis Zeddam (Zeddam et al., 1999 ). NoV was obtained from the ATCC (VR-679, strain Mag 115; Ball et al., 1992 ). FHV and BoV were isolated after three successive cycles of plaque purification on monolayers of Drosophila melanogaster cells, as described previously (Gallagher et al., 1983 ). NoV was purified from infected Galleria mellonella larvae (Johnson et al., 2000 ).

{blacksquare} 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 RT–PCR 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 RT–PCR. 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. RT–PCR products representing the 3'–5' junctions were then cloned and sequenced. Full-length cDNA of NoV RNA1 was synthesized by RT–PCR 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.

{blacksquare} 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.

{blacksquare} 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 Glaxo–Wellcome Experimental research (http://www.expasy.ch/spdbv/).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Comparisons of RNA sequences
The RNA1 sequences of NoV and BoV reported here bring the total number of nodavirus RNA1s available to six and allow for the first time a thorough examination of their commonalities. Until recently, the only known RNA1 sequences were those of FHV and BBV, which are almost identical (Dasmahapatra et al., 1985 ; Dasgupta, unpublished results). Those of PaV (Johnson et al., 2000 ) and SJNNV (Nagai & Nishizawa, 1999 ) were reported without a thorough comparative analysis across the Nodaviridae. Table 1 shows the GenBank accession numbers and lengths of the six RNA1s and their encoded ORFs. Each RNA1 contains 3·0–3·2 kb and encodes a large ORF of 973–1042 amino acids (ORF A). During replication, each RNA1 synthesizes a subgenomic RNA (RNA3, 387–471 nt) that corresponds to the 3' end of RNA1 and encodes one or two ORFs of about 100 amino acids each (ORFs B1 and B2). Where present, ORF B1 corresponds (by definition) to the C terminus of ORF A and is overlapped in the +1 reading frame by ORF B2. All six viruses reflect the general nodavirus genome organization, which was originally based on studies of BBV (Fig. 1) (reviewed in Ball & Johnson, 1998 ).


View this table:
[in this window]
[in a new window]
 
Table 1. Nodavirus RNA1 sequences: GenBank accession numbers, sizes of RNA1, subgenomic RNA3 and ORFs encoded therein

 
Since authentic termini are important for efficient RNA replication (Ball & Li, 1993 ; Ball, 1995 ), we were particularly interested to establish the terminal sequences of the RNA1 segments. Sequence data on the termini of BBV RNA1 are dependable because they were determined by direct RNA sequencing (Dasmahapatra et al., 1985 ). In the case of FHV, NoV and PaV, several lines of evidence indicate that the ends of the deposited cDNA sequences accurately reflect the complete termini of the authentic RNAs. These lines of evidence include primer-extension mapping and 5'RACE on positive-sense RNA for FHV, NoV and PaV; 5'RACE on negative-sense RNA for PaV; RT–PCR across the 3'–5' junctions of naturally occurring head-to-tail RNA1 dimers for FHV, NoV and PaV; self-directed replication of full-length cDNA transcripts for FHV, NoV and PaV; and the recovery of infectious virus from cDNA clones for FHV, NoV and PaV (Dasmahapatra et al., 1985 ; Ball, 1995 ; Johnson et al., 2000 ; unpublished results).

For BoV and SJNNV the situation is less clear. The final BoV RNA1 sequence was compiled from nt 1–250 of the consensus sequence obtained from the 5'RACE clones, nt 251–3088 of a BoV RNA1 cDNA and nt 3089–3096 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.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. Nucleotide sequences of the UTRs of six nodavirus RNA1 segments. (a) 5' UTRs aligned at the initiation codon for ORF A. (b) 3' UTRs aligned at the termination codon for ORF A. Nucleotides in the B2 ORFs are shown in italics. The sequences shown for the BoV 3' UTR and both UTRs of SJNNV may not represent the complete RNA termini and, as such, are shown in parentheses (see text).

 
All members of the Nodaviridae family examined to date synthesize a subgenomic RNA3 that corresponds to the 3' end of RNA1 (Delsert et al., 1997 ; Ball & Johnson, 1998 ). The 5' end of NoV RNA3 was mapped by primer extension to nt 2734 of NoV RNA1, yielding a 471 nt RNA3 that started 5' GUAUU...after the cap (Table 1). Harper (1994) cloned and sequenced BoV RNA3 and mapped its 5' end to nt 2708 of RNA1, suggesting that it contained 389 nt and started 5' UAUUA...However, in comparison with the 5' end of BBV and FHV RNA3, which starts 5' GUUAC..., it seems likely that the terminus of BoV RNA3 corresponds to nt 2706 in RNA1, which would yield a length of 391 nt and the homologous terminal sequence 5' GUUAU...Due to the uncertainties at both ends of BoV RNA3, we have listed its length as approximately 390 nt in Table 1.

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 stem–loops (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, 22–24 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 41–44% 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.


View this table:
[in this window]
[in a new window]
 
Table 2. Percentage amino acid sequence identities between nodavirus protein A sequences

 
The pattern of protein A relationships differs from that among the capsid proteins (Kaesberg et al., 1990 ; Johnson et al., 2000 ). For example, the BBV and FHV capsid proteins are only 87% identical and share only 54 and 51% amino acid identity with BoV, indicating a far greater divergence than for protein A of these viruses. This is unremarkable because virus structural proteins generally diverge more rapidly than their non-structural proteins, presumably in response to the greater environmental variations they encounter. Unusually, however, FHV and NoV capsid proteins are more similar than their protein A sequences (51% versus 44% identity). This may be because NoV is the only nodavirus known to infect warm-blooded animals and its RdRp is significantly more thermostable than that of FHV (Ball et al., 1992 ). Strikingly, even though protein A of PaV is closer to that of the fish nodavirus SJNNV than it is to the other insect nodaviruses, the SJNNV capsid protein shares only marginal sequence similarity with any of the insect viruses (Nishizawa et al., 1995 ; Johnson et al., 2000 ). These data suggest a possible discontinuity in the evolution of these two viral genes, consistent with reassortment of genome segments during the evolution of the Nodaviridae family. The demonstration that RNAs 1 and 2 of BBV, FHV and BoV produce viable reassortant viruses in the laboratory (Gallagher, 1987 ) shows that lateral transfer of genome segments among members of the Nodaviridae is feasible.

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.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3. Variations in the level of sequence conservation along protein A. Protein sequences were aligned using PILEUP and the plurality of the consensus sequence, as calculated by PLOTSIMILARITY (Devereux et al., 1984 ), is plotted as a function of the amino acid position. Notice that the numbering of the consensus sequence includes gaps introduced during alignment and therefore does not correspond to any of the individual sequences. The location of the conserved GDD motif in the consensus sequence is shown. The scale on the left indicates the number of sequences out of six that were identical. Note a non-integral value on the left scale indicates that there is more than one group of identical amino acids at that position in the alignment (e.g. 2/6 are amino acid X and 2/6 are amino acid Y). The scale on the right indicates the identity score, as calculated by the program PLOTSIMILARITY.

 


View larger version (133K):
[in this window]
[in a new window]
 
Fig. 4. Alignment of the amino acid sequences encoded in ORF A of the six nodavirus RNA1 segments. Sequences were aligned using PILEUP with a gap weight of 3. Positions where four or more of the aligned sequences match are highlighted. The core residues of the RdRp signature sequence are boxed. Sequences are numbered individually ignoring gaps introduced during alignment.

 
Polymerase motifs in protein A
All RdRps examined to date share a set of conserved sequence motifs (Kamer & Argos, 1984 ; Poch et al., 1989 ; Koonin, 1991 ; Koonin & Dolja, 1993 ). Koonin (1991) and Koonin & Dolja (1993) identified eight motifs (I–VIII) in the RdRps of positive-stranded RNA viruses, although only three (IV, V and VI) showed complete conservation among all RdRps, which overall share very little sequence identity. These motifs define an RdRp signature sequence as DX3(FYWLCA)X0–1DXn(STM)GX3TX3(NE)Xn(GS)DD (Koonin & Dolja, 1993 ), which is matched precisely by each of the six protein A sequences in the most highly conserved region of their alignment between residues 600–900 (Figs 3 and 4). The location of the GDD motif is shown in Fig. 3 and the conserved core residues of the RdRp signature sequence are boxed in Fig. 4. This observation identifies protein A as the catalytic subunit of the viral RNA replicase and clearly defines the heart of its polymerase domain. On the basis of phylogenetic analyses, Koonin (1991) further divided viral RdRps into three supergroups and tentatively assigned BBV protein A to supergroup 1. The pattern of conserved motifs in the six protein A sequences supports this assignment.

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 70–80 amino acids, as demonstrated by the many gaps in the alignment, but between residues 80–250 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 91–93 (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 134–136 (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 ).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Secondary structures predicted from the six protein A sequences by the program PSIPRED (Jones, 1999 ). Predicted {alpha}-helices of four or more residues are shown in red, {beta}-strands of four or more residues in blue and random coils or turns as thin black lines. Black circles indicate the positions of the conserved GDD sequences.

 
This analysis revealed common features among the six protein A molecules that were not readily apparent from the primary sequence comparisons. Predicted {beta}-strands were particularly abundant in the N-terminal third of each molecule with a well-conserved {alpha}{beta}{alpha}{beta}{beta}{beta}{beta} arrangement between residues 150–220, whereas predicted {alpha}-helices dominated the C-terminal halves (Fig. 5). Despite being poorly conserved in sequence, the C-terminal 100 or so residues of protein A overlapping ORF B2 were strikingly devoid of structure in every case. Curiously, the predicted secondary structures flanking the GDD sequence differed among the proteins. Only in PaV was this motif surrounded by {beta}-strands, similar to that found in the X-ray crystal structures of the poliovirus and hepatitis C virus RdRps (Hansen et al., 1997 ; Ago et al., 1999 ; Bressanelli et al., 1999 ; Lesburg et al., 1999 ). It seems unlikely that RdRp structures differ substantially in the polymerase domain, so this diversity may reflect mistakes in the structural predictions, which have an error-rate of about 22% (Jones, 1999 ). Nevertheless, the secondary structure predictions suggested additional commonalities among this family of proteins and lent further support to the notion that the N-terminal third of the molecule might constitute a distinct structural and functional domain.

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 344–803 of PaV protein A and residues 12–461 of poliovirus 3Dpol (Hansen et al., 1997 ). The alignment was trimmed to the centre of the polymerase domain encompassing structural motifs A–E (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.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6. Homology modelling of the centre of the polymerase domain of PaV protein A onto the X-ray crystal structure of poliovirus 3Dpol (Hansen et al., 1997 ). (a) Sequence alignment of poliovirus 3Dpol (residues 233–269 and 291–391) with PaV protein A (residues 564–602 and 628–725). The characteristic polymerase motifs are highlighted as follows: A, red; B, green; C, blue; D, purple; E, orange. (b) The X-ray crystal structure of poliovirus 3Dpol, as determined by Hansen et al. (1997) , is shown. The region corresponding to the partial sequence in (a) is shown in yellow and the five motif colours, whereas the rest of the molecule is shown in grey. The inset portrays poliovirus 3Dpol (coloured similarly) in its conventional orientation showing the ‘fingers’, ‘palm’ and ‘thumb’ domains. The molecule (b, c) has been rotated towards the viewer by about 90°. (c) Three-dimensional structure of the corresponding region of PaV protein A as predicted by SWISS-MODEL (Guex & Peitsch, 1997 ). The five characteristic polymerase motifs are coloured as in the other panels.

 
Conclusions
These comparisons substantiate some of the relationships that exist among the RNA1 segments of the six nodaviruses and their encoded polypeptides, and they enrich our understanding of the larger of the two viral genome segments. The evidence for some of the conserved features is compelling. For example, with only minor differences, the viral genes are arranged and expressed similarly (Fig. 1). The ORF A sequences encode a family of proteins whose homology, unlike that of the capsid proteins, extends persuasively across both genera of the Nodaviridae (Figs 3 and 4; Table 2). Each protein A contains a canonical RdRp signature that leaves little doubt as to its role as the catalytic subunit of the RNA replicase (Fig. 4). Indeed, PaV protein A is sufficiently similar to poliovirus 3Dpol that the centre of its polymerase domain can be modelled onto the crystal structure of the latter protein (Fig. 6).

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.


   Acknowledgments
 
We thank Dr Thomas A. Harper for providing the cDNA clones of BoV RNA; Dr Paul Scotti (HortResearch, Auckland, New Zealand) for providing samples of BoV and FHV; Dr Elliott Lefkowitz (University of Alabama at Birmingham, USA) for assistance with the GCG programs; Dr Stewart Shuman (Sloan–Kettering Institute, New York, USA) for information on guanylyltransferases; Fenglan Li, Bin Ye and the UAB Microbiology Department core DNA sequencing facility for excellent technical assistance; and members of the laboratories of Drs Gail Wertz and Andrew Ball for critical appraisal of the manuscript. This work was supported by Public Health Service grant R01 AI18270 from the National Institute for Allergy and Infectious Diseases, and R41 GM58998 from the National Institute for General Medical Sciences.


   Footnotes
 
The GenBank accession numbers of the sequences reported in this paper are AF174533 (NoV RNA1) and AF329080 (BoV RNA1).

b Present address: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.


   References
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K. & Miyano, M. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 7, 1417-1426.[Medline]

Ahola, T. & Kaariainen, L. (1995). Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proceedings of the National Academy of Sciences, USA 92, 507-511.[Abstract]

Ahola, T. & Ahlquist, P. (1999). Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a. Journal of Virology 73, 10061-10069.[Abstract/Free Full Text]

Ahola, T., Laakkonen, P., Vihinen, H. & Kaariainen, L. (1997). Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. Journal of Virology 71, 392-397.[Abstract]

Ahola, T., den Boon, J. A. & Ahlquist, P. (2000). Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping. Journal of Virology 74, 8803-8811.[Abstract/Free Full Text]

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389-3402.[Abstract/Free Full Text]

Ball, L. A. (1994). Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts. Proceedings of the National Academy of Sciences, USA 91, 12443-12447.[Abstract/Free Full Text]

Ball, L. A. (1995). Requirements for the self-directed replication of flock house virus RNA 1. Journal of Virology 69, 720-727.[Abstract]

Ball, L. A. & Li, Y. (1993). cis-Acting requirements for the replication of flock house virus RNA 2. Journal of Virology 67, 3544-3551.[Abstract]

Ball, L. A. & Johnson, K. L. (1998). Nodaviruses of insects. In The Insect Viruses , pp. 225-267. Edited by L. K. Miller & L. A. Ball. New York:Plenum.

Ball, L. A. & Johnson, K. L. (1999). Reverse genetics of nodaviruses. Advances in Virus Research 53, 229-244.[Medline]

Ball, L. A., Amann, J. M. & Garrett, B. K. (1992). Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture. Journal of Virology 66, 2326-2334.[Abstract]

Ball, L. A., Wolhrab, B. & Li, Y. (1994). Nodavirus RNA replication: mechanism and harnessing to vaccinia virus recombinants. Third International Symposium on Positive Strand RNA Viruses. Archives of Virology9, 407–416.

Ball, L. A., Hendry, D. A., Johnson, J. E., Rueckert, R. R. & Scotti, P. D. (2000). Family Nodaviridae. In Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses , pp. 747-755. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.

Bisaillon, M. & Lemay, G. (1997). Viral and cellular enzymes involved in synthesis of mRNA cap structure. Virology 236, 1-7.[Medline]

Blanc, A., Goyer, C. & Sonenberg, N. (1992). The coat protein of the yeast double-stranded RNA virus L-A attaches covalently to the cap structure of eukaryotic mRNA. Molecular and Cellular Biology 12, 3390-3398.[Abstract]

Blanc, A., Ribas, J. C., Wickner, R. B. & Sonenberg, N. (1994). His-154 is involved in the linkage of the Saccharomyces cerevisiae L-A double-stranded RNA virus Gag protein to the cap structure of mRNAs and is essential for M1 satellite virus expression. Molecular and Cellular Biology 14, 2664-2674.[Abstract]

Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., De Francesco, R. & Rey, F. A. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proceedings of the National Academy of Sciences, USA 96, 13034-13039.[Abstract/Free Full Text]

Cavener, D. R. & Ray, S. C. (1991). Eukaryotic start and stop translation sites. Nucleic Acids Research 19, 3185-3192.[Abstract]

Cheng, R. H., Reddy, V. S., Olson, N. H., Fisher, A. J., Baker, T. S. & Johnson, J. E. (1994). Functional implications of quasi-equivalence in a T=3 icosahedral animal virus established by cryo-electron microscopy and X-ray crystallography. Structure 2, 271-282.[Medline]

Dasgupta, R., Ghosh, A., Dasmahapatra, B., Guarino, L. A. & Kaesberg, P. (1984). Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor. Nucleic Acids Research 12, 7215-7223.[Abstract]

Dasmahapatra, B., Dasgupta, R., Ghosh, A. & Kaesberg, P. (1985). Structure of the black beetle virus genome and its functional implications. Journal of Molecular Biology 182, 183-189.[Medline]

Dasmahapatra, B., Dasgupta, R., Saunders, K., Selling, B., Gallagher, T. & Kaesberg, P. (1986). Infectious RNA derived from transcription from cloned cDNA copies of the genomic RNA of an insect virus. Proceedings of the National Academy of Sciences, USA 83, 63-66.[Abstract]

Delsert, C., Morin, N. & Comps, M. (1997). Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures. Journal of Virology 71, 5673-5677.[Abstract]

Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387-395.[Abstract]

Friesen, P. D. & Rueckert, R. R. (1982). Black beetle virus: messenger RNA for protein B is a subgenomic viral RNA. Journal of Virology 42, 986-995.

Frohman, M. A. (1990). RACE: Rapid amplification of cDNA ends. In PCR Protocols: A Guide to Methods and Applications , pp. 28-38. Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White. San Diego:Academic Press.

Gallagher, T. M. (1987). Synthesis and assembly of nodaviruses. PhD thesis, University of Wisconsin, Madison, USA.

Gallagher, T. M., Friesen, P. D. & Rueckert, R. R. (1983). Autonomous replication and expression of RNA1 from black beetle virus. Journal of Virology 46, 481-489.

Guarino, L. A., Ghosh, A., Dasmahapatra, B., Dasgupta, R. & Kaesberg, P. (1984). Sequence of the black beetle virus subgenomic RNA and its location in the viral genome. Virology 139, 199-203.[Medline]

Guex, N. & Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714-2723.[Medline]

Hansen, J. L., Long, A. M. & Schultz, S. C. (1997). Structure of the RNA-dependent RNA polymerase of poliovirus. Structure 5, 1109-1122.[Medline]

Harper, T. A. (1994). Characterization of the proteins encoded from the nodaviral subgenomic RNA. PhD thesis, University of Wisconsin, Madison, USA.

Henikoff, S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351-359.[Medline]

Johnson, K. N., Zeddam, J. & Ball, L. A. (2000). Characterization and construction of functional cDNA clones of Pariacoto virus, the first Alphanodavirus isolated outside Australasia. Journal of Virology 74, 5123-5132.[Abstract/Free Full Text]

Jones, D. T. (1999). Protein secondary structure prediction based on position-specific scoring matrices. Journal of Molecular Biology 292, 195-202.[Medline]

Kadare, G. & Haenni, A. L. (1997). Virus-encoded RNA helicases. Journal of Virology 71, 2583-2590.[Free Full Text]

Kaesberg, P., Dasgupta, R., Sgro, J.-Y., Wery, J.-P., Selling, B. H., Hosur, M. V. & Johnson, J. E. (1990). Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography. Journal of Molecular Biology 214, 423-435.[Medline]

Kamer, G. & Argos, P. (1984). Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Research 12, 7269-7282.[Abstract]

Kong, F., Sivakumaran, K. & Kao, C. (1999). The N-terminal half of the brome mosaic virus 1a protein has RNA capping-associated activities: specificity for GTP and S-adenosylmethionine. Virology 259, 200-210.[Medline]

Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. Journal of General Virology 72, 2197-2206.[Abstract]

Koonin, E. V. (1993). Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and {lambda}2 protein of reovirus. Journal of General Virology 74, 733-740.[Abstract]

Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28, 375-430.[Abstract]

Kozak, M. (1999). Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187-208.[Medline]

Kraft, R., Tardiff, J., Krauter, K. S. & Leinwand, L. A. (1988). Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques 6, 544-546.[Medline]

Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F. & Weber, P. C. (1999). Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nature Structural Biology 6, 937-943.[Medline]

Li, Y. I., Chen, Y. J., Hsu, Y. H. & Meng, M. (2001). Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. Journal of Virology 75, 782-788.[Abstract/Free Full Text]

Lima, C. D., Klein, M. G. & Hendrickson, W. A. (1997). Structure-based analysis of catalysis and substrate definition in the HIT protein family. Science 278, 286-290.[Abstract/Free Full Text]

Luongo, C. L., Reinisch, K. M., Harrison, S. C. & Nibert, M. L. (2000). Identification of the guanylyltransferase region and active site in reovirus mRNA capping protein lambda 2. Journal of Biological Chemistry 275, 2804-2810.[Abstract/Free Full Text]

Mi, S., Durbin, R., Huang, H. V., Rice, C. M. & Stollar, V. (1989). Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1. Virology 170, 385-391.[Medline]

Nagai, T. & Nishizawa, T. (1999). Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus. Journal of General Virology 80, 3019-3022.[Abstract/Free Full Text]

Newman, J. F. E. & Brown, F. (1976). Absence of poly(A) from the infective RNA of nodamura virus. Journal of General Virology 30, 137-140.[Abstract]

Nishizawa, T., Mori, K.-i., Furuhashi, M., Nakai, T., Furusawa, I. & Muroga, K. (1995). Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish. Journal of General Virology 76, 1563-1569.[Abstract]

O’Reilly, E. K. & Kao, C. C. (1998). Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology 252, 287-303.[Medline]

Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO Journal 8, 3867-3874.[Abstract]

Price, B. D., Rueckert, R. R. & Ahlquist, P. (1996). Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, USA 93, 9465-9470.[Abstract/Free Full Text]

Reinganum, C., Bashiruddin, J. B. & Cross, G. F. (1985). Boolarra virus: a member of the Nodaviridae isolated from Oncopera intricoides (Lepidoptera: Hepialidae). Intervirology 24, 10-17.[Medline]

Schneemann, A., Reddy, V. & Johnson, J. E. (1998). The structure and function of nodavirus particles: a paradigm for understanding chemical biology. Advances in Virus Research 50, 381-446.[Medline]

Scotti, P. D., Dearing, S. & Mossop, D. W. (1983). Flock house virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae). Archives of Virology 75, 181-189.[Medline]

Selling, B. H., Allison, R. F. & Kaesberg, P. (1990). Genomic RNA of an insect virus directs synthesis of infectious virions in plants. Proceedings of the National Academy of Sciences, USA 87, 434-438.[Abstract]

Shindyalov, I. N. & Bourne, P. E. (2000). Improving alignments in HM protocol with intermediate sequences. Fourth Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. (Asilomar, California, USA, December 2000). Abstract A-92 (http://predictioncenter.llnl.gov/casp4/).

Shuman, S. & Schwer, B. (1995). RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Molecular Microbiology 17, 405-410.[Medline]

Wang, H. L., O’Rear, J. & Stollar, V. (1996). Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity. Virology 217, 527-531.[Medline]

Zeddam, J. L., Rodriguez, J. L., Ravallec, M. & Lagnaoui, A. (1999). A noda-like virus isolated from the sweetpotato pest Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae). Journal of Invertebrate Pathology 74, 267-274.[Medline]

Zuker, M. (1989). On finding all suboptimal foldings of an RNA molecule. Science 244, 48-52.[Medline]

Received 13 February 2001; accepted 3 April 2001.