Comparative analysis of the genomes of Rachiplusia ou and Autographa californica multiple nucleopolyhedroviruses

Robert L. Harrison and Bryony C. Bonning

Department of Entomology and Interdepartmental Program in Genetics, Iowa State University, Ames, Iowa 50011, USA

Correspondence
Bryony C. Bonning
bbonning{at}iastate.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) is a variant of Autographa californica MNPV (AcMNPV) but is significantly more virulent against several major agricultural pests. The genome sequence of the R1 strain of RoMNPV was determined and compared to that of AcMNPV strain C6. The RoMNPV genome is approximately 131·5 kbp with a G+C content of 39·1 %. The homologous repeat regions (hrs) described for AcMNPV-C6 are present in RoMNPV-R1 but the hrs of RoMNPV have fewer palindromic repeats. The RoMNPV-R1 nucleotide sequence is almost completely collinear with the sequence of AcMNPV-C6 and contains homologues of 150 of the 155 ORFs described for AcMNPV-C6. Deletions, insertions and substitutions have resulted in the loss of homologues for AcMNPV ORFs ac2 (bro), ac3 (ctl), ac97, ac121 and ac140 from the RoMNPV genome. The average amino acid sequence identity between RoMNPV and AcMNPV ORFs is 96·1 % and there are differences in promoter motif composition for 23 of these ORFs. Maximum-likelihood analysis of selection pressures on AcMNPV and RoMNPV ORFs indicate that ORFs ro18/ac20-ac21 (arif-1) and ro135/ac143 (odv-e18) have undergone positive selection.

The GenBank accession number of the sequence reported in this paper is AY145471.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Baculoviruses are invertebrate-specific viruses with large, double-stranded DNA genomes contained within enveloped, rod-shaped virions. The baculoviruses are grouped into one family, the Baculoviridae, with two genera, Nucleopolyhedrovirus and Granulovirus, which are distinguished by the morphology of the virus occlusion bodies. Members of the Baculoviridae have been isolated from insects mainly within the order Lepidoptera (Adams & McClintock, 1991; Blissard et al., 2000). Because nucleopolyhedroviruses (NPVs) are effective against many lepidopteran pests and do not infect non-target organisms, they have been the subject of intensive study as environmentally benign insecticides. Wild-type baculoviruses have been effective when deployed against agricultural and forestry pests (Cunningham, 1995; Moscardi, 1999). Genetic engineering of NPVs to shorten the survival time of infected hosts has improved the performance of NPV-based insecticides in the field (Treacy & All, 1996; Smith et al., 2000; Treacy et al., 2000). Further studies on the molecular mechanisms of baculovirus infection, replication and host specificity are essential for additional augmentation of insecticidal potential.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Virus and cells.
RoMNPV-R1 (Smith & Summers, 1980) was propagated in S. frugiperda (Sf) cell lines (Vaughn et al., 1977) and titrated by plaque assay. Sf21 cells were grown in Ex-Cell 405 medium (JRH Biosciences) supplemented with 3 % FBS (Intergen) and antibiotics (1 U penicillin ml-1 and 1 µg streptomycin ml-1; Sigma). Sf9 cells were grown in TNM-FH medium (JRH Biosciences) supplemented with 3 % FBS, antibiotics and 0·1 % Pluronic F-68 (JRH Biosciences).

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 protein–protein 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, {omega}, is a measure of the selective pressure on a gene. Genes with {omega}=1 are undergoing neutral evolution, in which there is no effect of non-synonymous mutations on fitness. Genes with {omega}<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 {omega}>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 {omega} 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 {omega} among codon sites (Yang et al., 2000). With this second analysis, ORF alignments were fitted to six models: (1) M0 assumes one {omega} value for all codons; (2) M1 divides codons into an invariant category p0, where {omega} is set at zero (purifying selection), and a neutral category p1, where {omega} is set at one (neutral evolution); (3) M2 includes p0 and p1 from M1 and adds a third category p2, where {omega} 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). {omega} is estimated independently for all three categories and can be greater than one; (5) M7 features 10 categories modelled with a discrete {beta} distribution. The shape of the distribution is determined by parameters p and q, and {omega} 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 {omega} 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 {chi}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 {omega} 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 {omega} 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 ({kappa}) 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.


   RESULTS AND DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
General characteristics of the RoMNPV genome
The size of the RoMNPV-R1 genome is 131 526 bp, 2368 bp smaller than the AcMNPV-C6 genome (Fig. 1). This size difference is accounted for almost entirely by the absence of the ac2 (bro)-ac3 (ctl) region from RoMNPV (Harrison & Bonning, 1999) and the reduced size of RoMNPV homologous repeat regions (hrs). The RoMNPV G+C content (39·1 %) is slightly less than that of AcMNPV (41 %) and Bombyx mori NPV (BmNPV; 40 %). The RoMNPV genome is almost completely collinear with the AcMNPV-C6 sequence and the overall nucleotide sequence identity between RoMNPV and AcMNPV is approximately 96 %. For ease of comparison with the AcMNPV-C6 genome, nucleotide position #1 of RoMNPV was set to the nucleotide that aligned with the first nucleotide of the AcMNPV-C6 sequence. RoMNPV has no extra sequences that are not also present in the AcMNPV genome. Table 1 lists RoMNPV homologues of 150 expressed or potentially expressed AcMNPV-C6 ORFs described by Ayres et al. (1994). These homologues are in the same relative positions and orientations as the AcMNPV ORFs. In addition, Table 1 lists three RoMNPV homologues of ORFs previously undocumented in AcMNPV (ac15a, ac100a and ac139a). These ORFs overlap larger, adjacent ORFs by less than 75 bp. They are not preceded by baculovirus early or late gene promoter motifs in either virus and do not contain any recognizable conserved domains in their predicted amino acid sequences. With the exception of ro13a/ac15a (which has 55·4 % sequence identity with BmNPV small ORF7a; Gomi et al., 1999), these ORFs share no significant sequence identity with other viral or cellular genes.



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Fig. 1. Circular map of the RoMNPV genome (R1 strain). Locations for HindIII and EcoRI are shown on the inner and outer rings, respectively. The positions for the 149 ORFs listed in Table 1 are presented as arrowheads, with the direction of the arrowhead indicating the orientation of the ORF. The locations for the nine homologous repeat regions (hrs) are indicated.

 

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Table 1. Potentially expressed ORFs of RoMNPV (R1 strain)

Of the 149 ORFs listed, 146 are homologues of AcMNPV ORFs described previously. Four RoMNPV ORFs are homologues of AcMNPV ORFs that are split in two in the original C6 sequence. These RoMNPV ORFs are, therefore, homologous to two AcMNPV ORFs each in the original C6 sequence. Hence, for the 155 ORFs described for C6, there are 150 (146+4) homologues present in RoMNPV.

 
RoMNPV hrs
hrs are sequences that function as enhancers of gene expression and origins of DNA replication (Possee & Rohrmann, 1997). RoMNPV has nine hrs consisting of imperfect 30 bp palindromic repeats similar to those described for AcMNPV hrs (Fig. 2). The RoMNPV hrs occupy the same positions on the genome with respect to surrounding ORFs as the AcMNPV hrs (Fig. 1) and are numbered in the same order as the AcMNPV hrs. Each of the RoMNPV hrs has at least one less palindromic repeat than the corresponding AcMNPV hrs (Fig. 3). RoMNPV hr2 displays the greatest reduction in size, with four less repeats than AcMNPV hr2. The spacing between the palindromic repeats of RoMNPV and AcMNPV hrs is variable (Fig. 3) and the alignments of AcMNPV and RoMNPV hrs require multiple gaps, suggesting that numerous deletions and insertions have occurred in the regions between the palindromic repeats in the hrs of these viruses. A similar pattern of deletions and insertions has been observed upon comparison of the hrs of Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) and Helicoverpa zea SNPV (HzSNPV; Chen et al., 2002), and Mamestra configurata NPV (McNPV) isolates 90/2 and 96B (Li et al., 2002a). These observations confirm previous suggestions that hrs are sites of frequent recombination and rearrangement in baculovirus genomes.



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Fig. 2. Alignment of the consensus sequences of the 30 bp palindromic repeats for each hr of RoMNPV-R1 (RoR1) and AcMNPV-C6 (AcC6). Black boxes indicate sequence mismatches. DNA codes: K=G or T; R=G or A; M=A or C; and Y=C or T.

 


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Fig. 3. Structure of AcMNPV-C6 (AcC6) and RoMNPV-R1 (RoR1) hrs showing the number of 30 bp palindromic repeats (open boxes) in each hr. The spacing between each repeat is indicated in nucleotides.

 
Comparison of RoMNPV and AcMNPV ORFs
There were five fewer potentially expressed ORFs identified in RoMNPV than in AcMNPV. This reduced ORF count is due to the truncation of three ORFs and the complete deletion of two ORFs.

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. 4a). 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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Fig. 4. (a) AcMNPV ORFs that are truncated in RoMNPV. (b) Comparison of AcMNPV and RoMNPV ORFs that differ significantly in size. Shaded (AcMNPV) and black (RoMNPV) arrows represent homologous ORFs. Substitutions, insertions and deletions in the RoMNPV ORFs resulting in their termination or expansion are indicated. The hatched region of ro10 and the ac140 homologue indicates non-homologous amino acid sequences that result from frameshifts.

 
Forty-eight ORFs in RoMNPV differ in size from their AcMNPV homologues as a consequence of insertions, deletions and changes in stop codons. Of these, 26 ORFs differ in length by a single codon. Some RoMNPV ORFs are altered significantly in size (Fig. 4b). In ro10/ac12, a 5 nt insertion of 16 codons into the RoMNPV ORF causes a frameshift leading to a stop codon. An additional initiation codon occurs downstream and, after four codons, a single nucleotide deletion restores the original frame, resulting in an ac12 homologue that is shortened by 25 codons. With ro81/ac84, a G->A substitution in the start codon results in an N-terminal truncation of the ORF by 24 codons. With ro101/ac105, deletions and insertions result in N-terminal extensions of 19 codons. Four additional ORFs (ro15/ac17, ro49/ac52, ro135/ac143 and ro137/ac145) were determined initially to differ significantly in size, but PCR amplification and sequence analysis of these four ORFs from our laboratory stock of AcMNPV-C6 revealed that the AcMNPV ORFs were the same size as their RoMNPV homologues.

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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Fig. 5. Phylogenetic analysis of amino acid sequences from 34 polyhedrin genes and 1 granulin gene. (a) ME phylogram. Bootstrap values >=50 % (n=1000 replicates) are shown at interior branches where they occur. (b) MP cladogram. The topology of the tree is a majority consensus of 45 equally parsimonious trees. The percentage of trees displaying the given branching pattern at each node is shown. AcMNPV, Autographa californica MNPV (Ayres et al., 1994); AgMNPV, Anticarsia gemmatalis MNPV (Zanotto et al., 1992); AmblNPV, Amsacta albistriga NPV (accession no. AF118850); ApNPV, Antheraea pernyi NPV (accession no. AB062454); ArceNPV, Archips cerasivoranus NPV (accession no. U40834); AsNPV, Agrotis segetum NPV (Kozlov et al., 1992); AtriNPV, Attacus ricini NPV (accession no. S68462); BmNPV, Bombyx mori NPV (Gomi et al., 1999); BusuNPV, Buzuria suppressaria NPV (Hu et al., 1993); EcobNPV, Ecotropis oliqua NPV (accession no. U95014); EppoMNPV, Epiphyas postvittana MNPV (Hyink et al., 2002); HaSNPV, Helicoverpa armigera SNPV (Chen et al., 2001); HzSNPV, Helicoverpa zea SNPV (Chen et al., 2002); HycuNPV, Hyphantria cunea NPV (accession no. D14573); LdMNPV, Lymantria dispar MNPV (Kuzio et al., 1999); LeseNPV, Leucania separata NPV (accession no. AAB47865); LoobMNPV, Lonomia obliqua MNPV (accession no. AAF98122); MacoNPV-A, Mamestra configurata NPV strain 90/2 (Li et al., 2002b); MacoNPV-B, Mamestra configurata NPV strain 96B (Li et al., 2002a); MadiNPV, Malacosoma disstria NPV (accession no. AAD00095); ManeNPV, Malacosoma neustria NPV (accession no. AAB31529); MbMNPV, Mamestra brassicae MNPV (Cameron & Possee, 1989); OpMNPV, Orgyia pseudotsugata MNPV (Ahrens et al., 1997); OpSNPV, Orgyia pseudotsugata SNPV (Leisy et al., 1986); PaflMNPV, Panolis flammea MNPV (Oakey et al., 1989); PenuNPV, Perina nuda NPV (Chou et al., 1996); RoMNPV, Rachiplusia ou MNPV (Harrison & Bonning, 1999); SeMNPV, Spodoptera exigua MNPV (IJkel et al., 1999); SfMNPV, Spodoptera frugiperda MNPV (Gonzalez et al., 1989); SpltMNPV, Spodoptera litura MNPV (Pang et al., 2001); ThorMNPV, Thysanoplusia orichalcea MNPV (accession no. AF169480); ThorSNPV, Thysanoplusia orichalcea SNPV (Cheng et al., 1998); TnSNPV, Trichoplusia ni SNPV (Fielding & Davison, 1999); WisiNPV, Wiseana signata NPV (accession no. AF016916); XecnGV, Xestia c-nigrum granulovirus (Hayakawa et al., 1999).

 


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Fig. 6. Phylogenetic analysis of predicted amino acid sequences of NPV dnapol (a) and p10 (b) genes. Both trees are phylograms produced by ME with bootstrap values >=50 % (n=1000 replicates) displayed at interior branches where they occur. NPV abbreviations are as indicated for Fig. 5, with the addition of CfMNPV (Choristoneura fumiferana MNPV). The sources of dnapol and p10 sequences for AcMNPV, BmNPV, EppoMNPV, HaSNPV, HzSNPV, LdMNPV, MacoNPV-A, MacoNPV-B, OpMNPV, SeMNPV and SpltMNPV are as described for Fig. 5. CfMNPV dnapol, Liu & Carstens (1995); SpliMNPV dnapol, accession no. AF215639; AgMNPV p10, accession no. AY055828; BusuNPV p10, van Oers et al. (1998); CfMNPV p10, Wilson et al. (1995); PenuNPV p10, accession no. U50411; SpliMNPV p10, Faktor et al. (1997); TnSNPV p10, accession no. AF358416.

 
A comparison of promoter motifs upstream of RoMNPV and AcMNPV ORFs (Table 1) revealed differences in the presence of early and late gene promoter motifs in 23 ORFs. Differences in promoter element composition may lead to alterations in the timing or level of transcription of these ORFs.

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, {omega} 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 {omega} 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 {omega} value greater than 1: ro65/ac68 ({omega}=1·20), ro110/ac116 ({omega}=3·50) and ro132a/ac139a ({omega}=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 {omega} for this ORF suggests that it is subject to a slight degree of positive selection pressure.

The pairwise method used to obtain the {omega} values shown in Table 1 calculates an average {omega} value for the entire ORF. Because a large number of amino acids in a protein are invariant ({omega}=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 {omega} 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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Table 2. Positive selection analysis of ro18/ac20-ac21 and ro135/ac143

 
For arif-1, models M2 and M3 identified categories of codons consisting of approximately 1 % of all codon sites and possessing very high {omega} values ({omega}=80·55 for M2 and {omega}=59·44 for M3; Table 2), indicating a strong degree of positive selection. In likelihood ratio tests, the M0 and M1 models could be rejected in favour of the M2 model at P<0·05, but only the M0 model could be rejected in favour of the M3 model. Model M8 identified a positively selected codon category consisting of a larger number of sites (9·6 % of all codons) but possessing a much lower {omega} value (3·130). Null hypothesis model M7 could not be rejected in favour of M8 (P=0·336). Models M2 and M3 identified codon positions 94, 365 and 390 in arif-1 as being in the positively selected category, although only positions 365 and 390 were identified with a probability greater than 0·90 in both models. For all three sites, substitutions had taken place at two positions within the codons with at least one transversion. Positions 365 and 390 are located in a region of the protein localized to the cytoplasmic side of the plasma membrane (Dreschers et al., 2001).

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, 55–61, 65 and 66) at P values well below 0·05. {omega} 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 {omega} 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 {omega}) 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.


   ACKNOWLEDGEMENTS
 
We thank Michael Hensel for assistance with cloning restriction fragments, Ziheng Yang (Department of Biology, University College London, UK) and Gavin Naylor (Department of Zoology and Genetics, Iowa State University, USA) for helpful discussions, and Gary Polking and John Mlocek of the Iowa State University DNA Synthesis and Sequencing Facility. This study of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3301 was supported by Hatch Act and State of Iowa funds.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 5 February 2003; accepted 20 March 2003.