Journal
of General Virology (2001), 82, 1855–1866. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Comparisons among the larger genome segments of six
nodaviruses and their encoded RNA replicases
Karyn N. Johnson,1 Kyle L. Johnson,1 Ranjit Dasgupta,2 Theresa Gratsch1† and L. Andrew Ball1
1
Department of Microbiology, University of Alabama at Birmingham, BBRB 373/17, 845 19th Street South, Birmingham,
AL 35294-2170, USA
2
Department of Animal Health and Biomedical Science, University of Wisconsin-Madison, Madison, WI 53706, USA
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 positivesense 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 5h and 3h 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
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 4n5 kb, nodavirus genomes are among the smallest
of all known animal viruses. Both genome segments are capped
at their 5h ends but lack poly(A) tails (Newman & Brown,
1976). The smaller segment (RNA2, 1n3–1n4 kb) encodes a
precursor to the capsid proteins, which are clearly homologous
Author for correspondence : Andrew Ball.
Fax j1 205 934 1636. e-mail andyb!uab.edu
† Present address : Department of Cell and Developmental Biology,
University of Michigan, Ann Arbor, MI 48109, USA.
The GenBank accession numbers of the sequences reported in this
paper are AF174533 (NoV RNA1) and AF329080 (BoV RNA1).
0001-7686 # 2001 SGM
within each genus but show only marginal sequence similarity
between the genera. Despite containing only 3n0–3n2 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.
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
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
BIFF
K. N. Johnson and others
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 l 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
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 (VR679, 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).
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
BIFG
isolate of FHV obtained from Paul Scotti. For BoV, an oligonucleotide
complementary to the 3h-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 5h end of BoV
RNA1 was determined by using 5hRACE (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. 5h-terminal cDNA clones were generated, as
described previously (Johnson et al., 2000), by 5hRACE. cDNA clones
representing the 3h-terminal 200 nt were generated by RT–PCR across
the 3h–5h 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 5h end of RNA1. The 3h–5h dimer
junction was amplified by PCR using a positive-sense primer that
annealed near to the 3h end of the adjacent RNA1 copy. RT–PCR
products representing the 3h–5h junctions were then cloned and
sequenced. Full-length cDNA of NoV RNA1 was synthesized by
RT–PCR using primers specific for the 5h- and 3h-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 SWISSMODEL at Glaxo–Wellcome Experimental research (http :\\www.
expasy.ch\spdbv\).
Results and Discussion
Comparisons of RNA sequences
The RNA1 sequences of NoV and BoV reported here bring
the total number of nodavirus RNA1s available to six and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
Nodavirus RNA1s and encoded RNA replicases
Table 1. Nodavirus RNA1 sequences : GenBank accession numbers, sizes of RNA1,
subgenomic RNA3 and ORFs encoded therein
For references see Johnson & Ball (1998, 2000) and Nagai & Nishizawa (1999). Recently, the RNA1 sequence
of Atlantic halibut nervous necrosis virus was released on GenBank (AJ401165) but is yet to be published.
Virus
BBV
FHV
BoV
NoV
PaV
SJNNV
Accession no.
RNA1 (nt)
RNA3 (nt)
ORF A
(aa)
ORF B1
(aa)
ORF B2
(aa)
X02396
X77156
AF329080
AF174533
AF171942
AB025018
3106
3107
3096
3204
3011
3081
387
387
" 390*
471
414
" 400*†
998
998
998
1042
973
983
102‡
102‡
No ORF
131
94
No ORF
106‡
106‡
106‡
137
90‡
75
* Precise termini not confirmed.
† While synthesis of RNA3 has not been investigated for SJNNV, it has been reported for the related fish
nodavirus Dicentrarchus labrax encephalitis virus (Delsert et al., 1997).
‡ Synthesis of protein has been confirmed experimentally (see text for references).
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.
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 3n0–3n2 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 3h 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 j1 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).
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 5hRACE on
positive-sense RNA for FHV, NoV and PaV ; 5hRACE on
negative-sense RNA for PaV ; RT–PCR across the 3h–5h
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 5hRACE 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 3h 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.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
BIFH
K. N. Johnson and others
(a)
(b)
Fig. 2. Nucleotide sequences of the UTRs of six nodavirus RNA1 segments. (a) 5h UTRs aligned at the initiation codon for ORF
A. (b) 3h UTRs aligned at the termination codon for ORF A. Nucleotides in the B2 ORFs are shown in italics. The sequences
shown for the BoV 3h 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 3h end
of RNA1 (Delsert et al., 1997 ; Ball & Johnson, 1998). The 5h
end of NoV RNA3 was mapped by primer extension to nt
2734 of NoV RNA1, yielding a 471 nt RNA3 that started 5h
GUAUU … after the cap (Table 1). Harper (1994) cloned and
sequenced BoV RNA3 and mapped its 5h end to nt 2708 of
RNA1, suggesting that it contained 389 nt and started 5h
UAUUA … However, in comparison with the 5h end of BBV
and FHV RNA3, which starts 5h 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 5h 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 3h
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
j1 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 3h end of ORF A, and the encoded B1
protein has been detected in infected cells (Harper, 1994 ; unpublished results). Despite being encoded in the 5h-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
5h end of RNA3 and has a sub-optimal context for initiation,
BIFI
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 5h and 3h untranslated regions (UTRs) of the RNA1
segments are shown in Fig. 2. For the five insect nodaviruses,
the 5h UTRs vary from 21 nt for NoV to 39 nt for FHV (Fig.
2 a). In contrast, the 5h UTR of SJNNV is at least 64 nt long and
may be longer (Nagai & Nishizawa, 1999). Even among the
insect nodaviruses, the 5h UTRs have dissimilar sequences,
although all are AU-rich. Like RNA3, most RNA1s start 5h
GU …, although PaV RNAs 1 and 2 start 5h 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 5h terminal
AUG of PaV RNA1 opens an ORF of only two codons.
Fig. 2 (b) shows the 3h 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 3h 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 3h ends because nodavirus RNAs are not
polyadenylated (Newman & Brown, 1976). The 3h UTRs of
BBV and FHV RNA1 are identical in length and sequence (Fig.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
Nodavirus RNA1s and encoded RNA replicases
Table 2. Percentage amino acid sequence identities
between nodavirus protein A sequences
Virus
FHV
BoV
NoV
PaV
SJNNV
BBV
FHV
BoV
NoV
PaV
99
–
–
–
–
84
84
–
–
–
44
44
41
–
–
26
26
26
29
–
28
28
27
29
31
2 b) ; 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 3h 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 3h UTRs of SJNNV, NoV and PaV,
nor between any of these viruses and BBV, FHV and BoV.
Previous analysis of the 3h 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 3h 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 3h 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 5h CCCAUCU 3h 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 3h 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 3h UTR of BoV RNA1. Evidently the cisacting 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 3h 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.
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 nonstructural 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 warmblooded 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
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
BIFJ
K. N. Johnson and others
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.
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.
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
DX (FYWLCA)X – DXn(STM)GX TX (NE)Xn(GS)DD (Koo$ $
$
!"
nin & 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 NBIGA
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 aboveaverage 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 wellrecognized 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) : 5h-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 N( 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 N(
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
Nodavirus RNA1s and encoded RNA replicases
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.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
BIGB
K. N. Johnson and others
Fig. 5. Secondary structures predicted from the six protein A sequences by the program PSIPRED (Jones, 1999). Predicted
α-helices of four or more residues are shown in red, β-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.
position before m(GMP 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 H)! in BMV 1a protein
abolished guanylyltransferase activity and destroyed its ability
to form a covalent adduct with m(GMP (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
m(GMP 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 (H*$) is also
conserved for BBV, FHV, BoV and NoV, albeit in the context
of an NXH sequence as found in the alphavirus nsP1 proteins.
A canonical HXH motif occurs in BBV, FHV, BoV and NoV
protein A at position 134–136 (BBV numbering), but it did not
align with a similar sequence in the PaV and SJNNV proteins.
We consider BBV H*$ 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
BIGC
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 threedimensional 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).
This analysis revealed common features among the six
protein A molecules that were not readily apparent from the
primary sequence comparisons. Predicted β-strands were
particularly abundant in the N-terminal third of each molecule
with a well-conserved α–β–α–β–β–β–β arrangement between
residues 150–220, whereas predicted α-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
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
Nodavirus RNA1s and encoded RNA replicases
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 90m.
(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.
every case. Curiously, the predicted secondary structures
flanking the GDD sequence differed among the proteins. Only
in PaV was this motif surrounded by β-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
CPHmodels\).
(http :\\www.cbs.dtu.dk\services\
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 ;
OhReilly & 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. 6 c) compared
with the crystal structure of poliovirus 3Dpol (Fig. 6 b), as
determined by Hansen et al. (1997). In the Ramachandran plot
of the PaV model (data not shown), only 4 of the 137 modelled
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
BIGD
K. N. Johnson and others
residues lie outside preferred or allowable regions : E&(%, W&(&,
H&*# and Q&*$, all of which lie near gaps in the sequence
alignment (Fig. 6 a). While the true protein A structure can be
determined only by rigorous experimental methods, the results
of modelling suggest that the core of the PaV RdRp domain
closely resembles that of poliovirus 3Dpol. This model will be
used to guide and interpret mutational analysis of protein A.
Conclusions
These comparisons substantiate some of the relationships
that exist among the RNA1 segments of the six nodaviruses
and their encoded polypeptides, and they enrich our understanding of the larger of the two viral genome segments. The
evidence for some of the conserved features is compelling. For
example, with only minor differences, the viral genes are
arranged and expressed similarly (Fig. 1). The ORF A sequences
encode a family of proteins whose homology, unlike that of the
capsid proteins, extends persuasively across both genera of the
Nodaviridae (Figs 3 and 4 ; Table 2). Each protein A contains a
canonical RdRp signature that leaves little doubt as to its role
as the catalytic subunit of the RNA replicase (Fig. 4). Indeed,
PaV protein A is sufficiently similar to poliovirus 3Dpol that
the centre of its polymerase domain can be modelled onto the
crystal structure of the latter protein (Fig. 6).
Other common features are more tenuous. The location of
the active sites of the capping enzyme remains to be
established, although several observations suggest that they
may lie in the N-terminal third of protein A, with H*$ 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 5h and 3h 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.
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.
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.
BIGE
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.
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.
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.
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.
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.
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.
Ball, L. A. (1995). Requirements for the self-directed replication of flock
house virus RNA 1. Journal of Virology 69, 720–727.
Ball, L. A. & Li, Y. (1993). cis-Acting requirements for the replication of
flock house virus RNA 2. Journal of Virology 67, 3544–3551.
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.
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.
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
Virology 9, 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.
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.
Blanc, A., Ribas, J. C., Wickner, R. B. & Sonenberg, N. (1994). His-154
is involved in the linkage of the Saccharomyces cerevisiae L-A doublestranded 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.
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.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
Nodavirus RNA1s and encoded RNA replicases
Cavener, D. R. & Ray, S. C. (1991). Eukaryotic start and stop translation
sites. Nucleic Acids Research 19, 3185–3192.
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 l 3 icosahedral animal virus established by cryo-electron microscopy
and X-ray crystallography. Structure 2, 271–282.
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.
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.
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.
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.
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set
of sequence analysis programs for the VAX. Nucleic Acids Research 12,
387–395.
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.
Guex, N. & Peitsch, M. C. (1997). SWISS-MODEL and the SwissPdbViewer : an environment for comparative protein modeling. Electrophoresis 18, 2714–2723.
Hansen, J. L., Long, A. M. & Schultz, S. C. (1997). Structure of the
RNA-dependent RNA polymerase of poliovirus. Structure 5, 1109–1122.
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.
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.
Jones, D. T. (1999). Protein secondary structure prediction based on
position-specific scoring matrices. Journal of Molecular Biology 292,
195–202.
Kadare, G. & Haenni, A. L. (1997). Virus-encoded RNA helicases.
Journal of Virology 71, 2583–2590.
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.
Kamer, G. & Argos, P. (1984). Primary structural comparison of RNAdependent polymerases from plant, animal and bacterial viruses. Nucleic
Acids Research 12, 7269–7282.
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.
Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA
polymerases of positive-strand RNA viruses. Journal of General Virology
72, 2197–2206.
Koonin, E. V. (1993). Computer-assisted identification of a putative
methyltransferase domain in NS5 protein of flaviviruses and λ2 protein
of reovirus. Journal of General Virology 74, 733–740.
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.
Kozak, M. (1999). Initiation of translation in prokaryotes and eukaryotes.
Gene 234, 187–208.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34
BIGF
K. N. Johnson and others
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.
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.
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.
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.
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.
Shindyalov, I. N. & Bourne, P. E. (2000). Improving alignments in HM
protocol with intermediate sequences. Fourth Meeting on the Critical
BIGG
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.
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.
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.
Zuker, M. (1989). On finding all suboptimal foldings of an RNA
molecule. Science 244, 48–52.
Received 13 February 2001 ; Accepted 3 April 2001
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:03:34