Department of Entomology and Interdepartmental Program in Genetics, Iowa State University, Ames, Iowa 50011, USA
Correspondence
Bryony C. Bonning
bbonning{at}iastate.edu
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ABSTRACT |
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The GenBank accession number of the sequence reported in this paper is AY145471.
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INTRODUCTION |
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Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) was first identified in 1960 when it caused an epizootic in populations of the mint looper, Rachiplusia ou, in mint fields (Paschke & Hamm, 1961). Restriction mapping and nucleic acid hybridization studies revealed that RoMNPV is closely related to Autographa californica MNPV (AcMNPV), the type species for Nucleopolyhedrovirus (Jewell & Miller, 1980
; Smith & Summers, 1980
, 1982
). The sequence of the EcoRI-G restriction fragment of RoMNPV (R1 strain) confirmed that a high degree of nucleotide sequence identity exists between RoMNPV and AcMNPV in this region (Harrison & Bonning, 1999
). In addition, the RoMNPV EcoRI-G sequence was found to be almost completely identical to the sequence of the corresponding region in Anagrapha falcifera MNPV (AfMNPV; Federici & Hice, 1997
), which was first identified in 1985 (Hostetter & Puttler, 1991
). Restriction enzyme analysis and bioassays against three different host species demonstrated that AfMNPV and RoMNPV are isolates of the same virus (Harrison & Bonning, 1999
). RoMNPV and AfMNPV are characterized as variants of AcMNPV (Blissard et al., 2000
).
Both AcMNPV and RoMNPV/AfMNPV are known to infect a relatively large number of lepidopteran species (31 and 43 species, respectively; Granados & Williams, 1986; Payne, 1986
; Hostetter & Puttler, 1991
). Although the host ranges for these viruses overlap, there are significant differences in the abilities of AcMNPV and RoMNPV/AfMNPV to infect and kill several agricultural pest species. The corn earworm, Helicoverpa zea, is approximately 2·5- to 29-fold more susceptible to RoMNPV/AfMNPV than AcMNPV (Harrison & Bonning, 1999
; Hostetter & Puttler, 1991
). Ostrinia nubilalis, the European corn borer, is approximately 5- to 11-fold more susceptible to RoMNPV than AcMNPV (Harrison & Bonning, 1999
; Lewis & Johnson, 1982
). The navel orangeworm, Amyelois transitella, is non-permissive to AcMNPV but can be infected and killed with AfMNPV (Vail et al., 1993
; Cardenas et al., 1997
). The tobacco hornworm, Manduca sexta, a species that is highly refractory to AcMNPV (Washburn et al., 2000
), is susceptible to a dose of 100 polyhedra mm-2 diet surface of AfMNPV (Hostetter & Puttler, 1991
). The fall armyworm, Spodoptera frugipera, and the velvet bean caterpillar Anticarsia gemmatalis, are also more susceptible to AfMNPV than AcMNPV (Hostetter & Puttler, 1991
).
Given the high degree of sequence similarity between the genomes of these viruses, we reasoned that comparison of the genomes of RoMNPV and AcMNPV would reveal genetic differences that could account for differences in host range and virulence. Towards this end, we sequenced the RoMNPV genome. Here we present a comparison of the RoMNPV and AcMNPV genomes and an analysis of selection pressures on RoMNPV and AcMNPV genes.
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METHODS |
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Viral DNA isolation and cloning.
Sf9 or Sf21 cells were infected with RoMNPV-R1 at an m.o.i. of 1. Budded virus (BV) was harvested at 5 days post-infection (p.i.). BV was precipitated by overnight incubation on ice with an equal volume of 20 % polyethylene glycol and 1 M NaCl. After pelleting by centrifugation, the BV was resuspended in 10 mM Tris/HCl and 1 mM EDTA (pH 8·0) and incubated for 3 h at 37 °C with 1 % SDS and 1 mg proteinase K ml-1. Viral DNA was purified by phenol/chloroform extraction and ethanol precipitation. Alternatively, the BV pellet was resuspended in 1 ml DNAzol, a genomic DNA isolation reagent (Invitrogen/Life Technologies), and viral DNA was isolated following the manufacturer's instructions.
RoMNPV-R1 DNA was digested with EcoRI, HindIII, PstI, BglII and XbaI. Restriction fragments were ligated into the vectors pGEM-9Zf(-) (Promega), pUC18 and pUC19M (Clontech), a variant of pUC19 in which the EcoRI site has been replaced with an EcoRV site. Ligation products were transformed into competent Escherichia coli JM109. Plasmid DNA for clones with RoMNPV inserts was prepared using Qiagen columns (Qiagen).
DNA sequencing and sequence analysis.
A primer walking strategy was used to sequence selected RoMNPV restriction fragments from both ends. Automated dideoxy sequencing was performed at the Iowa State University DNA Sequencing and Synthesis Facility. Reactions were set up using the Applied Biosystems Prism BigDye Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase and electrophoresed on an Applied Biosystems Prism 377 DNA sequencer.
To confirm the order of some RoMNPV-R1 restriction fragments, regions encompassing restriction fragment junctions were amplified from viral DNA by PCR, purified with Qiagen columns and sequenced. The sequences of selected AcMNPV strain C6 ORFs were also re-determined by amplifying the ORFs from an AcMNPV-C6 stock and sequencing the amplification products.
DNA sequence data were compiled and analysed with the software of the Wisconsin package (version 10.0, Genetics Computer Group) and the Lasergene suite (DNASTAR). ORFs greater than 50 codons in length that did not overlap larger ORFs by more than 75 nt were selected for further characterization. Predicted amino acid sequence identities were obtained from the results of protein database searches using the standard proteinprotein BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/).
To assess the relationship of the AcMNPV and RoMNPV polyhedrins to the polyhedrins of other baculoviruses, phylogenetic analysis of 35 baculovirus occlusion matrix proteins was carried out. Amino acid sequences in this data set were aligned with CLUSTAL W (Thompson et al., 1994) using Gonnet matrices with a gap penalty of 15 and a gap extension penalty of 0·3 and adjusted manually. Phylogenetic inferences were performed with MEGA, version 2.1 (Kumar et al., 2001
) using minimum-evolution (ME) and maximum-parsimony (MP) methods (Nei & Kumar, 2000
). ME and MP trees were sought using a close-neighbour-interchange heuristic search, starting with one initial tree generated by the neighbour-joining method (for ME) or 10 initial trees generated by random addition of sequences (for MP). For ME, evolutionary distance was estimated using the gamma distance model with the gamma shape parameter set to 2·25. The reliability of the trees was tested with the bootstrap resampling strategy using 1000 replicates. For comparison, ME trees of NPV dnapol and p10 predicted amino acid sequences were constructed in the same way using alignments assembled with a gap penalty of 10 and a gap extension penalty of 0·2.
Analysis of selection pressures on individual genes.
PAML software (Yang, 1997; http://abacus.gene.ucl.ac.uk/software/paml.html) was used to investigate selection pressures on the genes of AcMNPV and RoMNPV. This software uses a maximum-likelihood approach to determine the numbers of non-synonymous (amino acid changing) substitutions per non-synonymous site (dN) and of synonymous (silent) substitutions per synonymous site (dS). The ratio of dN to dS,
, is a measure of the selective pressure on a gene. Genes with
=1 are undergoing neutral evolution, in which there is no effect of non-synonymous mutations on fitness. Genes with
<1 are undergoing negative or purifying selection, in which non-synonymous mutations are deleterious and are eliminated at a faster rate than synonymous mutations. Genes with
>1 are undergoing positive or diversifying selection, in which non-synonymous mutations are favourable and are fixed at a faster rate than synonymous mutations.
All ORFs common to AcMNPV and RoMNPV were analysed initially using a pairwise comparison method that assumes a single value of for all codon sites in an ORF. A subset of these ORFs that are present in multiple genomes or known to be expressed was analysed further using models that allow for heterogeneous values of
among codon sites (Yang et al., 2000
). With this second analysis, ORF alignments were fitted to six models: (1) M0 assumes one
value for all codons; (2) M1 divides codons into an invariant category p0, where
is set at zero (purifying selection), and a neutral category p1, where
is set at one (neutral evolution); (3) M2 includes p0 and p1 from M1 and adds a third category p2, where
is estimated from the underlying data and can be greater than one; (4) M3 divides codons among three categories of sites (p0, p1 and p2).
is estimated independently for all three categories and can be greater than one; (5) M7 features 10 categories modelled with a discrete
distribution. The shape of the distribution is determined by parameters p and q, and
values for these categories cannot be greater than one; and (6) M8 includes the 10 categories of M7 (collectively referred to as p0), and uses an additional category p1, where
can be greater than one.
Models M0 and M1 are nested with models M2 and M3, and model M7 is nested with M8. Models that are nested together can be compared statistically using a likelihood ratio test, in which twice the difference between the log-likelihood values for two models is compared with a 2 distribution table with the degrees of freedom equal to the difference in the number of parameters between the two models. This comparison supplies a P value for the probability that the null hypothesis (no positive selection, embodied in models M0, M1 and M7) is an equally good or better fit for the data when compared to the nested models that indicate positive selection. Positive selection can be inferred from this analysis when: (1) models M2, M3 or M7 indicate a group of codons with an
ratio greater than 1; and (2) the likelihood of the positive selection model is significantly higher than that of the nested null hypothesis model (at P<0·05). An empirical Bayes procedure calculates the probabilities for individual codons belonging to each of the site categories and can be used to infer which codons are under positive selection.
For selection pressure analysis, predicted amino acid sequences of AcMNPV and RoMNPV ORFs were aligned using CLUSTAL W, as described for phylogenetic analysis of occlusion matrix proteins. The sequences in the alignment were then converted back to the original nucleotide sequences. The CODEML and CODEMLSITES programs of PAML were run with the nucleotide sequence alignments. For pairwise analysis, codon frequency bias was accounted for using the F61 model of codon frequency, in which the frequency of each codon is used as a free parameter. Analysis with models allowing to vary used the F3x4 model, in which codon frequencies are calculated from average nucleotide frequencies at the three codon positions. In all analyses, the transition/transversion ratio (
) was estimated from the underlying data.
Alignments and output files from these analyses can be downloaded from http://www.ent.iastate.edu/dept/faculty/bonningb/selection_pressure.zip.
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RESULTS AND DISCUSSION |
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As reported previously (Federici & Hice, 1997; Harrison & Bonning, 1999
), RoMNPV is missing a 1275 bp region, which, in AcMNPV, contains ac2 (baculovirus repeated ORF, bro) and ac3 (conotoxin-like gene, ctl). No other homologues of these genes were detected elsewhere in the RoMNPV genome. The ctl ORF has also been found in the genomes of Orgyia pseudotsugata MNPV (OpMNPV; Ahrens et al., 1997
), McNPV-90/2 and -96B (Li et al., 2002a
, b
), Lymantria dispar MNPV (LdMNPV; Kuzio et al., 1999
) and Xestia c-nigrum granulovirus (XecnGV; Hayakawa et al., 1999
). Deletion of ctl from AcMNPV had no effect on replication in tissue culture (Eldridge et al., 1992
) and the function of ctl is unknown. Homologues of the bro ORF are present in multiple copies in other baculovirus genomes. The BRO proteins of BmNPV are associated with nucleoprotein complexes and bind nucleic acids (Zemskov et al., 2000
). Kang et al. (1999)
were unable to produce a viable BmNPV bro-d single deletion mutant or a bro-a/bro-c double deletion mutant, suggesting that these ORFs may be essential for BmNPV replication in cell culture (Kang et al., 1999
). bro genes are also absent from Spodoptera exigua NPV (SeNPV; IJkel et al., 1999
) and Plutella xylostella GV (PxGV; Hashimoto et al., 2000
).
Three small (60 codons) AcMNPV ORFs are also missing from the RoMNPV genome due to mutations that reduce the size of the ORFs below 50 codons (Fig. 4
a). For ac97, a G
A substitution in the RoMNPV homologue results in the appearance of a stop codon, terminating the ORF after three codons. The region containing ac97 is missing from the BmNPV genome sequence (Gomi et al., 1999
). For ac121 and ac140, single nucleotide insertions in the RoMNPV homologues result in frameshifts that lead to premature stop codons. ORFs ac97, ac121 and ac140 are not present in the genomes of any other lepidopteran NPVs and GVs. The presence of ac97, ac121 and ac140 in AcMNPV-C6 was confirmed by amplifying and sequencing the regions of these ORFs from our laboratory stock of AcMNPV-C6. Because of the small size of these ORFs and their truncation in RoMNPV, these ORFs may not be expressed in AcMNPV.
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Four pairs of adjacent AcMNPV ORFs that are in the same orientation (ac20/ac21, ac58/ac59, ac106/ac107 and ac112/ac113) are fused into a single ORF in RoMNPV. Where these ORFs occur in other baculovirus genomes, they are also fused into a single ORF. To confirm that these ORF pairs exist as separate ORFs in AcMNPV-C6, the sequences containing these ORFs were amplified from our laboratory stock of AcMNPV-C6 and subjected to DNA sequence analysis. In all four cases, the ORF pairs occurred as a single ORF in our stock of AcMNPV-C6. With respect to ac20/ac21, this finding is consistent with the sequence results obtained by Roncarati & Knebel-Mörsdorf (1997). It is not clear if these or other differences between the original AcMNPV-C6 genome sequence and our re-determined sequences represent errors in the original sequence or sequence properties unique to the AcMNPV-C6 stock sequenced by Ayres et al. (1994)
.
Of the ORFs that RoMNPV and AcMNPV have in common, the average predicted amino acid sequence identity (with one SD) is 96·1±3·12 %. Twelve RoMNPV ORFs (v-ubi, ro54, ro57, ro72, ro73, ro82, vp15, cg30, ro89, p6·9, ro96a and odv-ec27) are completely identical in amino acid sequence (100 %) with their AcMNPV homologues along their entire length (Table 1). The most divergent ORF is hcf-1 (host cell factor-1), with an amino acid sequence identity of 84·1 % between the AcMNPV and RoMNPV homologues. Mutations to eliminate expression of hcf-1 during AcMNPV infection resulted in impairment of virus replication in two cells lines derived from Trichoplusia ni but not in a Sf cell line (Lu & Miller, 1996
). hcf-1 mutants killed T. ni more slowly. A reduction in the infectivity of hcf-1 mutant BV, but not virus occlusions, towards T. ni larvae was also seen. In contrast, mutations in hcf-1 had no effect on the dose of virus or the time required to kill S. frugiperda larvae (Lu & Miller, 1996
). The relatively large degree of sequence divergence between AcMNPV and RoMNPV hcf-1 and the species-specific effects of hcf-1 mutation suggest that hcf-1 may account for the different host range and virulence characteristics of AcMNPV and RoMNPV. However, hcf-1 is required for optimal replication in T. ni, a species that is equally susceptible to both AcMNPV and AfMNPV, but not in S. frugiperda, which is more susceptible to AfMNPV than AcMNPV. It is not known if hcf-1 influences virus replication in species other than T. ni.
All RoMNPV ORFs, except for ro6 (polyhedrin, polh) and ro76, possess the greatest degree of amino acid sequence identity with AcMNPV homologues. The ro76 ORF shows 96·2 % amino acid sequence identity with the BmNPV homologue bm65 and 95·2 % identity with ac79. RoMNPV polyhedrin shows the greatest degree of amino acid sequence identity (98 %) with the predicted polyhedrin sequence of Thysanoplusia orichalcea MNPV. To examine the relationships of AcMNPV and RoMNPV polyhedrins to other baculovirus polyhedrins, phylogenetic trees of polyhedrin amino acid sequences were produced by two different methods. Both trees place AcMNPV polyhedrin on a branch outside of the clade containing the other group 1 NPVs (Fig. 5). The RoMNPV polyhedrin is found among group 1 NPV polyhedrins with a clade containing the polyhedrins from NPVs of Thysanoplusia orichalcea, Antheraea pernyi and Attacus ricini. In contrast, phylograms of NPV dnapol and p10 predicted amino acid sequences place the AcMNPV and RoMNPV sequences together within the group 1 NPV clade (Fig. 6
). These analyses suggest that AcMNPV acquired its polh gene by recombination with another virus that is not closely related to other group 1 NPVs.
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Analysis of selection pressure on RoMNPV and AcMNPV genes
Selection pressure analysis of vertebrate virus genes has identified positively selected sites that map to regions involved in host immune recognition and receptor binding (Woelk & Holmes, 2001; Woelk et al., 2001
; Holmes et al., 2002
; Twiddy et al., 2002
). Analysis of selection pressure on viral genes can potentially identify genes involved in virulence or in crossing of species barriers, even without prior knowledge of the mechanisms governing host range and virulence.
To detect instances of positive selection among AcMNPV and RoMNPV genes, was calculated for all of the RoMNPV and AcMNPV ORFs listed in Table 1
using the pairwise method available in PAML (Yang, 1997
). The average value of
from this analysis was 0·23, suggesting that, in general, negative selection pressure has been the dominant force in the evolution of most of the genes in the lineage containing RoMNPV and AcMNPV. Three ORFs were found to possess an
value greater than 1: ro65/ac68 (
=1·20), ro110/ac116 (
=3·50) and ro132a/ac139a (
=1·89). Because function and protein expression has not been demonstrated for these ORFs, their values of dN and dS may be the product of random drift rather than selection at the protein level. The ro110/ac116 and ro132a/ac139a ORFs have not been found in other baculovirus genomes. In contrast, ro65/ac68 is present in all NPV and GV genomes sequenced previously, suggesting that its gene product plays an important role in the baculovirus life cycle. The value of
for this ORF suggests that it is subject to a slight degree of positive selection pressure.
The pairwise method used to obtain the values shown in Table 1
calculates an average
value for the entire ORF. Because a large number of amino acids in a protein are invariant (
=0) due to functional constraints, it is difficult to detect positive selection using this approach. To overcome this problem, a selection of RoMNPV and AcMNPV ORFs was analysed with models that allow for heterogeneous
values among different codon sites (Yang et al., 2000
; see Methods). The ORFs analysed consisted of 63 genes present in nine other baculovirus genomes (Herniou et al., 2001
) and other ORFs for which protein expression or functional activity had been demonstrated for AcMNPV. Models M2, M3 and M7 identified positively selected sites in several ORFs. However, the null hypothesis models (M0, M1 and/or M7) could be rejected at the P<0·05 level only for ro18/ac20-ac21 (arif-1) and ro135/ac143 (odv-e18) (Table 2
). Re-sequencing of the ac20-ac21 and ac143 ORFs confirmed that the results from analysis of selection pressures on these genes were not based on AcMNPV sequences containing potential errors. The arif-1 ORF encodes the 48 kDa actin rearrangement-inducing factor, a protein that localizes to vesicular structures at the plasma membrane of infected cells (Roncarati & Knebel-Mörsdorf, 1997
). ARIF-1 mediates the dissociation of the host cell actin network and of virus-induced actin cables that form early during infection, as well as subsequent formation of actin aggregates at the plasma membrane (Dreschers et al., 2001
). Mutations in AcMNPV arif-1 had no effect upon replication in S. frugiperda or T. ni cells in vitro (Roncarati & Knebel-Mörsdorf, 1997
; Dreschers et al., 2001
). ODV-E18 migrates as a potential dimer on protein gels and is located in virus-associated intranuclear membranes and envelopes of occlusion-derived virus (Braunagel et al., 1996
). The odv-e18 ORF also appears to encode the N-terminal portion of a larger occluded virus envelope protein, ODV-E35 (Braunagel et al., 1996
).
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In addition to the cap site and TATA box in the upstream regions of both AcMNPV and RoMNPV arif-1, the RoMNPV gene also contains a copy of the late gene promoter motif. It is unclear if the potential late phase transcription of RoMNPV arif-1 would have any impact on the function of ARIF-1 during infection that would influence its host range. AcMNPV ARIF-1 protein is detectable as late as 48 h p.i, but after 12 h p.i., it is phosphorylated and found solely in cytoplasmic vacuoles. Dreschers et al. (2001) speculate that ARIF-1 is rendered non-functional by phosphorylation. Also, the formation of filamentous actin in the nucleus of infected cells that takes place during the late phase of infection does not appear to involve ARIF-1 (Roncarati & Knebel-Mörsdorf, 1997
; Ohkawa et al., 2002
). Although mutations in arif-1 had no effect upon infection and replication in vitro, ARIF-1 may be required for the in vivo replication cycle.
The evidence for positive selection of odv-e18 was much stronger than for arif-1. Models M2, M3 and M8 all identified positively selected codon categories consisting of the same 10 sites (positions 52, 5561, 65 and 66) at P values well below 0·05. values were very high for all three models (99·00), indicating a very strong degree of positive selection. Bayesian analysis yielded probabilities greater than 0·95 for all 10 sites, except position 55 in model M3. Seven of the positively selected sites occurred contiguously in a region of high nucleotide and amino acid sequence divergence. Oddly, one of the positively selected sites (position 56) encodes a serine in both AcMNPV and RoMNPV genes. The codon positions at this site (TCG in ac143 and AGC in ro135) differ by transversions at every position. The codon-based substitution models implemented in PAML assume that only one codon position changes at a time (Yang, 2001
). To change from TCG to AGC, one position at a time, at least two non-synonymous substitutions must first occur, which may account for why position 56 was identified as a positively selected site. Because of its location, ODV-E18 may influence host range at the level of midgut cell binding and internalization of occluded virus.
Models M0, M2, M3 and M8 all calculated an of 1·14 for ro65/ac68, indicating a weak degree of positive selection for this ORF, but the null hypothesis models could not be rejected at P<0·05. Hence, the inference of selection pressure on this ORF should be treated with caution.
The power of the likelihood ratio test is defined as the probability of rejecting the null hypothesis when it is wrong and when the alternative hypothesis (in this case, the inference of positive selection) is correct. This probability decreases with decreasing number of sequences per data set but increases with increasing strength of positive selection (Anisimova et al., 2001). Hence, it is not surprising that, with data sets consisting of only two sequences (one RoMNPV and one AcMNPV gene), we were only able to reject the null hypothesis models in two instances where the strength of selection (the value of
) was very high. Selection pressure analysis that includes sequences from other NPV genomes may identify other genes undergoing positive selection.
Because of the genetic similarity between AcMNPV and RoMNPV, study of these viruses may enhance our knowledge of the genetic bases of baculovirus host range. In addition to genes identified as undergoing positive selection, ORFs that differ in size, exhibit a relatively large degree of amino acid sequence divergence or show differences in timing or level of gene expression due to differences in promoter organization may also play a role in host range and virulence. This comparison of the genomes of RoMNPV and AcMNPV will serve as the foundation for empirical study of the molecular basis of host range and virulence differences between these two viruses.
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ACKNOWLEDGEMENTS |
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Received 5 February 2003;
accepted 20 March 2003.