1 Department of Entomology and Interdepartmental Graduate Program in Genetics, University of California, Riverside, CA 92521, USA
2 California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504-3297, USA
3 Laboratoire d'Etude des Parasites Génétiques, FRE CNRS 2535, Université François Rabelais, UFR des Sciences et Techniques, Parc de Grandmont, 37200 Tours, France
Correspondence
Yves Bigot
(at Université François Rabelais)
bigot{at}univ-tours.fr
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ABSTRACT |
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The sequences reported here have been deposited in the DDBJ/EMBL/GenBank sequence database under accession nos AJ292546AJ292551.
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INTRODUCTION |
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MGFs also occur in insect dsDNA viruses with large genomes. One of these families, the Baculoviridae, contains viruses in which repeated genes called baculovirus repeated open reading frames (bro) occur commonly among different nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) (Goto et al., 1998; Ahrens et al., 1999
; Gomi et al., 1999
; Kang et al., 1999
; Kuzio et al., 1999
; Iyer et al., 2002
). Baculovirus bro genes vary in number and length from one virus to another, even among closely related viruses. For example, only one copy of a bro gene (orf2) is present in Autographa californica multicapsid NPV (AcMNPV) (Ayres et al., 1994
), whereas three, five, sixteen and seven are present in, respectively, the baculoviruses from Orgyia pseudotsugata (OpMNPV; Ahrens et al., 1999
), Bombyx mori (BmNPV; Gomi et al., 1999
), Lymantria dispar (LdMNPV; Kuzio et al., 1999
) and Xestia c-nigrum (XcGV; Goto et al., 1998
; Hayakawa et al., 1999
). Though prevalent among many baculoviruses, bro genes are absent in Plutella xylostella (Px) GV (Hashimoto et al., 2000
) and in Anagrapha falcifera MNPV (Federici & Hice, 1997
) and Rachiplusia ou MNPV (Harrison & Bonning, 1999
), both of which are closely related to AcMNPV. Homologues of bro genes have also been reported from more distantly related baculoviruses, such as the Culex nigripalpus NPV, which contains six bro genes (Afonso et al., 2001
), but are absent in the Hz-1 virus (Cheng et al., 2002
) and the shrimp white spot bacilliform virus (Yang et al., 2001
).
Although reported originally from baculoviruses, homologues of bro genes have been identified in other insect dsDNA viruses, including the entomopoxviruses (subfamily Entomopoxvirinae) of Amsacta moorei (AmEPV) and Melanoplus sanguinipes (MsEPV), where they are referred to as the ALI family (Bawden et al., 2000; Afonso et al., 1999
). They have also been reported in Chilo iridescent virus (CIV), family Iridoviridae, an iridovirus from the lepidopteran Chilo suppressalis (Jakob et al., 2001
). Only a few invertebrate dsDNA viral genomes have been fully sequenced, but the occurrence of bro genes in baculovirus, entomopoxvirus and entomoiridovirus genomes suggests that these genes may be widespread among insect dsDNA viruses. Interestingly, bro genes have been reported to show homology with genes in bacteriophages, probacteriophages and in the phycodnavirus Ectocarpus siliculosus virus (ESV) (Afonso et al., 1999
; Kang et al., 1999
; Iyer et al., 2002
). Here we refer to these homologues as bro-like (bro-l) to distinguish them from baculovirus bro genes.
With respect to length, BRO and BRO-like (BRO-l) proteins vary from about 88 to 450 amino acid residues. A characteristic of these proteins is that the first 100150 N-terminal residues are highly conserved and contain a nucleic acid binding domain, BRO-N (Zemskov et al., 2000; Iyer et al., 2002
). The BRO-N domain is widely distributed, being found alone or in conjunction with domains in proteins encoded by eukaryotic and prokaryotic viruses (Iyer et al., 2002
). A less conserved C-terminal domain, BRO-C, is also present in BRO proteins, but this domain appears to be restricted to baculoviruses and viruses that constitute the recently identified nucleo-cytoplasmic large DNA viruses (NCLDV), a monophyletic clade of eukaryotic viruses, which includes poxviruses, phycodnaviruses, asfarviruses and iridoviruses (Iyer et al., 2001
, 2002
). Due to high levels of divergence in the C-terminal regions, the major criterion therefore required for identifying BRO and BRO-l proteins is the presence of the BRO-N domain.
Despite their common occurrence among insect dsDNA viruses, little is known about the factors influencing the expression of bro genes or the function(s) of BRO proteins. Kang et al. (1999) showed that the five bro genes (bro ae) in BmNPV are expressed early, about 24 h after initiation of virus replication, and that transcription initiates 5070 nucleotides upstream from the translation start codon at the characteristic baculovirus early gene promoter motif, (C/T)AGT. In addition, no significant differences in pathobiology were observed for wild-type virus or certain bro deletion mutants grown in B. mori cells (BmN-4) or larvae (Kang et al., 1999
). Nevertheless, Kang et al. (1999)
were unable to isolate mutants deficient in bro-d or mutants that contained double deletions in bro-a and bro-c, which suggests that these genes could play significant roles in BmNPV pathogenesis. More recently, Zemskov et al. (2000)
showed that BRO-a, BRO-c and BRO-d are associated with the histone H1 fraction from the BmN-4 cell line and provided evidence that about 80 residues in the conserved N-terminal region are required for a non-specific nucleic acid binding activity. They proposed that BRO-a and BRO-c could function as DNA binding proteins that influence host DNA replication and/or transcription by regulating chromatin structure in the host chromosomes (Zemskov et al., 2000
; Iyer et al., 2002
).
The presence of bro and bro-l genes in different insect dsDNA virus families, along with evidence that BRO proteins could potentially play a role in virus replication, has suggested that the bro MGF may be larger and more extensively distributed than currently realized. Here we have reported the identification and sequences of bro genes from a crustacean virus belonging to the family Iridoviridae, as well as among viruses of the Ascoviridae (Federici et al., 2000). We have also shown that bro-l genes are not restricted to bacteriophages, but occur in certain bacterial transposable elements belonging to the IS3 and IS5 families. At least eight BRO lineages were identified by phylogenetic analysis using the sequences of 114 BRO and BRO-l N-terminal domains. These data suggest that the bro MGF has evolved by genetic processes of gene duplication and loss and horizontal transfer among viruses belonging to different families. Lastly, we have shown that the unique AcMNPV orf2 bro gene is not required for infection or replication of this virus in lepidopteran cells or larvae, although it may enhance replication during the occlusion phase of reproduction.
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METHODS |
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Genomic DNA libraries.
Fifty µg of SfAV1, HvAV3c and IIV DNA were sheared by sonication (20 W for 2·5 min with 1 s pulses) to produce fragments ranging in size from 0·5 to 3 kbp. DNA fragments were blunted with SI nuclease and T4 DNA polymerase (New England Biolabs) and EcoRI linkers were ligated at both ends. Fragments of approximately 0·851·1 kbp were purified from agarose gel using a QIAquick gel extraction kit (Qiagen) and ligated to the EcoRI site in pUC18.
DNA sequencing.
Plasmids were isolated and purified by standard protocols (Ausubel et al., 1994). Nucleotide sequences were determined by dideoxy-nucleotide sequencing (Sanger et al., 1977
) using the Sequitherm long-read cycle sequencing kit with universal and reverse IRD800 fluorescent-labelled primers (Epicentre Technologies). DNAs were amplified by PCR (25 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 15 s and polymerization at 70 °C for 1 min). Nucleotide sequences for both strands were generated using a DNA Sequencer Long Reader model 4200 (Li-cor). Sequences were determined for 500 cloned fragments of SfAV1a, 50 of HvAV3c and 50 of iridovirus IIV.
Database searches and sequence analyses.
The Infobiogen facilities were used for database searches (GenBank release 132, updated 10/15/2001; Swissprot release 40 and TrEMBL 21, both updated 12/06/2002), sequence alignments and calculations. Due to the presence of numerous deletions and insertions (1250 amino acids) between the regions conserved between BRO and BRO-l proteins, the alignment of their amino acid sequences was performed in three steps. First, 12 groups of related sequences identified from BLAST searches were aligned using CLUSTAL W (Thompson et al., 1994). Taking into account data described previously (Iyer et al., 2002
), the 12 alignments were then aligned to each other. Finally, ambiguities were identified by pair sequence alignments using Kanehisa's program for sequence comparison, and the quality of the sequence alignment of the C-terminal domain of the BRO and BRO-l was verified by comparison with their structural profiles determined by hydrophobic cluster analysis (HCA) (http://smi.snv.jussieu.fr/hca/hca-seq.html). At each step, the sequences were manually adjusted to facilitate the quality of alignment. The aligned sequences have been deposited in DDBJ/EMBL/GenBank (DS43784). Phylogenetic analyses were performed using the PHYLIP package, version 3.5c (Felsenstein, 1993
).
DNA probes and Southern blot hybridization.
DNA probes (SfAV1a-bro-12, HvAV3c-bro-l1 and orf2, chlr and tetr; see below) were prepared using a Dig DNA labelling and detection kit (Boehringer Mannheim). Hybridization was performed at 65 °C in 0·1 % SDS, 0·5 M Na2HPO4/NaH2PO4 buffer, pH 7, and post-hybridization washes were performed at 65 °C using 0·5x SSC (high stringency) or 2x SSC (low stringency).
Disruption of the AcMNPV orf2 (bro) in E. coli BJ5183.
The recombinant AcMNPV bacmid AcBacP+1 and methods used for baculovirus gene disruption by homologous recombination in E. coli BJ5183 (recBC sbsBC; Hanahan 1983; Chartier et al., 1996
) have been described previously (Bideshi & Federici, 2000
). The AcMNPV orf2 (Ayres et al., 1994
) was obtained as a 2·2 kbp fragment by PCR with Taq DNA polymerase (Promega), using primers ORF2a (5'-AAGCGAGGATCTACAACGTT-3') and ORF2b (5'-TAAAATGTTTCCCGCGCGTT-3') and AcBacP+1 as the DNA source. The PCR product was cloned in pGEM-T Easy to generate pGEMT-orf2. The restriction sites used for orf2 disruption were SwaI and BstEII located at, respectively, positions +23 and +677 relative to the translation initiation codon of orf2. pGEM-Bro/tet, which retained the BRO-N domain coding sequence, was generated by inserting the 1·6 kbp SspIMscI fragment with the tetracycline resistance gene (tetr) from pBR322 (Biolabs) into the blunted BstEII site in pGEM-T-orf2. pGEM-Bro/chl was constructed by inserting the blunted 1·2 kb BspHIXmnI fragment with the chloramphenicol resistance marker (chlr) from pBCSK(-) (Stratagene) in the blunted BstEII and SwaI sites in pGEM-T-orf2. The 3·9 kb and 2·8 kb fragments in, respectively, pGEM-Bro/tet and pGEM-Bro/chl were obtained by PCR with the ORF2a and ORF2b primers and used to disrupt the orf2 in AcBacP+1 harboured in E. coli BJ5183. E. coli BJ5183 strains with recombinant bacmids AcP+4M:T12 and AcBacP+1:brochlABD were recovered on LB agar containing, respectively, tetracycline (15 µg ml-1) and kanamycin (45 µg ml-1) or chloramphenicol (15 µg ml-1) and kanamycin (45 µg ml-1). Disruption of orf2 was confirmed by PCR using the ORF2a and ORF2b primers and by Southern blot hybridization with the orf2 probe.
In vitro and in vivo replication of recombinant AcMNPV bacmids.
DNAs from AcBacP+1 (polh+, kanr, chls, tets, orf2+), AcP+4M:T12 (polh+, kanr, chls, orf2 disrupted with tetr) and AcBacP+1:brochlABD (polh+, kanr, tets, orf2 disrupted with chlr) were purified using the Nucleobond AX kit (Clontech). Insect cells were grown in TC-100 medium (Gardiner & Stockdale, 1975) with 10 % foetal bovine serum (TC-100/FBS). Cells of Trichoplusia ni (BTI-TN5-B1-4; Invitrogen) or S. frugiperda (SF21; Pharmingen) were transfected in triplicate with approximately 1 µg viral DNA, or mock-transfected using the Cellfectin liposome reagent (Gibco BRL). Transfected cell cultures of AcBacP+1, AcP+4M:T12 and AcBacP+1:brochlABD were incubated at 28 °C for 7 days after which the percentage of cells containing polyhedra was assessed. A total of 300 cells was counted in each of the transfected cultures. Two ml of each transfected culture medium was collected by centrifugation at 1000 r.p.m. for 5 min, diluted 1 : 100 in TC-100/FBS and used to infect T. ni and S. frugiperda cells. After incubation for 4 days at 28 °C, budded virions were collected from the culture medium and viral DNAs were isolated. The presence of recombinant bacmids was confirmed by PCR using the ORF2a and ORF2b primers and by Southern blot hybridization using the orf2 probe. Larvae of T. ni were grown on a semi-defined medium (Shorey & Hale, 1965
). For insect inoculation, 100 µl culture medium from BTI-TN5-B1-4 cells infected with AcBacP+1, AcP+4M:T12, AcBacP+1:brochlABD, or mock-infected cells were mixed with 100 µl Grace's insect cell culture medium (Gibco BRL). Two µl of the mixture was injected into 10 early fourth instar T. ni using the Microapplicator model M microinjector (Instrumentation Specialities Company). To determine whether the virus was infectious by feeding, BTI-TN5-BI-4 cells containing polyhedra were suspended in 1 ml Grace's medium. Twenty µl of this suspension was added to 0·5 mg of growth medium, which was then fed to 10 early fourth instars of T. ni.
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RESULTS |
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Comparison of the SfAV1a-bro-l1 to -l11, or HvAV3c-bro-l1 and -l2 nucleotide sequences showed that they were about 65 % similar to each other and shared no significant homology with the 3·1 kbp and 1·1 kbp repeated sequences (accession nos AJ279828 and AJ279829) described previously for SfAV1a and HvAV3c (Bigot et al., 2000). In contrast, the three bro-l sequences from DpAV4a were 8089 % identical to each other and were located within three different 980 bp repeated sequences (accession nos X85006, nt 4101400; AJ27918, nt 6801675; AJ279813, nt 52906250, respectively; Bigot et al., 1997
, 2000
). In DpAV4a, these bro-l genes included at least 80 % of the 980 bp repeated sequences and analysis of their sequence revealed that they were non-functional bro-l genes since the ORF was interrupted by frame shifts or stop codons. We therefore classified the bro-l genes that could be aligned as being either fossil or active, depending on whether the ORFs were interrupted by frame shifts or stop codons, or not.
When SfAV1a-bro-l2 was used to probe BamHIHindIII fragments of three SfAV1 variants (SfAV1a, -1b and -1c; Stasiak et al., 2000), the probe hybridized to three fragments ranging in size from 15 to 20 kbp in SfAV1a (Fig. 1c
, lane1). However, only one fragment of about 1·5 kbp in SfAV1b (Fig. 1c
, lane 2) and in SfAV1c (Fig. 1c
, lane 3) hybridized with the probe. Similar polymorphisms were observed in 12 different HvAV3 isolates (data not shown).
Ubiquity of bro and bro-l genes in large dsDNA viruses, bacterial phages and transposons
Using sequences reported in this study and by Iyer et al. (2002), 128 BRO and BRO-l proteins were identified by BLAST searches. These sequences were restricted to invertebrate viruses and prokaryotic genetic elements (Tables 1 and 2
). Ninety-six were encoded by ascoviruses, baculoviruses, insect iridoviruses and entomopoxviruses, one by the phycodnavirus ESV (ORF 117) and 31 were found in bacteriophage or prophages integrated in bacterial genomes. In agreement with Iyer et al. (2002)
, sequences with significant homology to bro-l genes were not identified in eukaryotic genomes, including those of Caenorhabditis elegans, Drosophila melanogaster and Anopheles gambiae for which complete genome sequences are known, or in vertebrate viruses or their mobile genetic elements.
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Sequence relationships between BRO and BRO-l proteins and their coding genomes
The high level of polymorphism and repetition of the bro MGF in invertebrate viruses makes it difficult to determine orthological relationships between bro genes and their respective genomes. None the less, we attempted to assess whether bro genes have co-evolved with their respective viruses or within viral families by inferring phylogenetic relationships using complete N-terminal domain (BRO-N) sequences (Iyer et al., 2002) from 114 BRO and BRO-l proteins. Three analytic methods parsimony, neighbour-joining and unweighted pair-group with arithmetic means each with 1000 bootstrap replicates, were used for analyses. The topologies of the trees generated by these methods were similar, but bootstrap values obtained by parsimony were more significant and were used to construct the tree shown in Fig. 2
. Eight groups (Groups 18) composed of 103 of the 114 sequences used were clearly defined in the tree. Other notable characteristics of the tree were that the bootstrap values at the intergroup nodes were lower than 50 %, whereas the values at the terminal nodes in each group were more robust, typically >50 %, and that Group 2, 3, 5 and 6 contained BRO-N domains encoded by viruses belonging to different virus families. In view of data previously published on baculovirus evolution (Bulach et al., 1999
), the relationships between N-terminal domains in groups 1, 2 and 5 indicate that numerous horizontal transfers have also occurred between the genomes of these viruses. Finally, Group 8 was composed of closely related bro-l genes from bacteriophages and bacterial transposons. Surprisingly, this group also contained phycodnavirus ESV-bro-l, suggesting that this eukaryotic sequence has a prokaryotic origin.
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Conservation of the BRO-N and BRO-C domains among invertebrate BRO proteins
Consensus sequences for invertebrate BRO and BRO-l proteins were assembled for each of the groups and then aligned with each other (Fig. 3). Analysis of the alignment confirmed that BRO-N (aa 1150) was the most conserved. This analysis, however, also revealed that this feature varied from that proposed previously. Indeed, only the second half of the motif proposed by Zemskov et al. (2000)
as being the origin of the structure responsible for the non-specific DNA binding activity [K/R]-X25-[K/R]-X412-[F/Y]-X214-[F/Y]-X613-[F/Y]-X119-[K/R]-X326-[F/Y/W]-X612-[K/R] was found to be significantly conserved. Moreover, conserved residues at positions 49, 5254, 6061 and 6465 (Fig. 3
) suggested that the DNA binding domain of BRO and BRO-l proteins might be different from that proposed. HCA analyses confirmed this and also revealed that the N-terminal domain contains two subdomains separated by a hinge, which varied in size from approximately 1 to 40 amino acids (Fig. 3
, aa 7895; unpublished data). As a result, our data and those of Iyer et al. (2002)
suggested that the folding of the N-terminal domain of the BRO proteins might correspond to a structure yet to be determined through crystallographic studies.
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BRO protein encoded by AcMNPV orf2 is not required for replication
Previous studies of bro genes in BmNPV suggested that BRO-a, BRO-c and BRO-d play an important role in the biology of this virus, since mutants with single or double deletions in these genes could not be recovered from BmN-4 cells (Kang et al., 1999). However, it is not known whether disruption of these genes is deleterious to BmNPV, or whether bro-a and bro-c compensate for their deletion. Because bro-d and the unique bro gene (orf2) in the AcMNPV share a high level of homology, we disrupted orf2 to determine its effect on AcMNPV replication in insect cells and larvae.
PCR, Southern blot and sequence analyses confirmed that the unique bro orf2 was disrupted in the two mutant AcMNPV. Specifically, using the ORF2a and ORF2b primers and viral DNAs prepared from budded virions, PCR products of 2·2, 3·9 and 2·8 kb were obtained from, respectively, the intact orf2 in the control AcBacP+1 (Fig. 4a, b, lanes 1 and 3), orf2 disrupted with tetr (orf2-tetr) in AcP+4M:T12 (Fig. 4a
, lanes 2 and 4) and orf2 disrupted with chlr (orf2-chlr) in AcBacP+1:brochlABD (Fig. 4b
, lanes 2 and 4). All of the PCR fragments hybridized with the orf2 probe (Fig. 4a, b
, lanes 3 and 4).
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DISCUSSION |
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Although the list of sequences used in the present study was more extensive than previously reported, our sequence analyses are in general agreement with Iyer et al. (2002) that BRO proteins encoded by invertebrate viruses contain a conserved N-terminal DNA binding domain (BRO-N) associated with a highly variable C-terminal domain (BRO-C). In BRO-l proteins of prokaryotic origin, BRO-N appears to be more homogeneous than those encoded by invertebrate viruses and is linked to highly variable BRO-C domains from at least seven different origins (Iyer et al., 2002
).
Interestingly, bro gene homologues appear to be absent from vertebrate genomes, vertebrate viruses and transposons of invertebrates including C. elegans, An. gambiae and D. melanogaster for which complete genome sequences are known, and from prokaryotic genomes with the exception of those found in prophages. Thus, it is tempting to propose that bro genes, like genes that encode capsid proteins, are native components of invertebrate dsDNA viruses. In this regard, contrary to the proposal that many virus-encoded proteins, such as those involved in DNA metabolism or in anti-apoptotic pathways (Domingo et al., 1999; Huang et al., 2000
), have been pirated from host chromosomes, the unique characteristics of BRO proteins, particularly the N-terminal domain, and the apparent absence of these proteins in eukaryotes suggest that the evolution of bro genes has not been mediated by genetic exchanges between invertebrate viruses and their hosts.
Previous data (Lopez-Ferber et al., 2001) and ours indicate that both intra- and interspecific polymorphisms in bro and bro-l genes are a general feature of insect dsDNA viruses. One of the major factors that could explain the high level of polymorphism and redundancy of the bro MGF in viral genomes is the process of gene duplication and differentiation. However, it is interesting to note that the relatedness of many bro genes and therefore their evolutionary origin and differentiation was not related to the virus genomes in which they occurred. Thus, the main process that is responsible for the plasticity of the bro and bro-l gene is very probably a result of the recombination events that occur within viral genomes and between different viruses that infect the same invertebrate hosts. Regardless of the mechanisms that maintain bro diversity, our analysis revealed that the differentiation of the bro MGF was largely independent of the evolutionary history of invertebrate viruses.
The presence of non-functional or fossil bro genes in several invertebrate viruses and bacteriophages is somewhat unexpected, since it would be presumed that the lack of selective pressure would lead to elimination of these sequences during viral genome evolution. However, it is possible that these fossil sequences are maintained in viral populations by processes such as intertypic recombination between different viruses or by horizontal transfer of bro sequences, as noted above.
The high degree of variation among BRO proteins encoded by different viruses, and even among BRO proteins encoded by the same virus, for example those of LdMNPV and BmNPV, suggests that these proteins constitute a multifunctional or diversified protein family, the function of which could be to initiate and terminate transcription, translation or replication at different stages during virus pathogenesis. However, early expression of the five BmNPV bro genes by 24 h post-infection (Kang et al., 1999; Suzuki et al., 2001
) and the DNA binding activity of the BRO protein (Zemskov et al., 2000
) in B. mori cells infected with BmNPV suggest that their functions might be limited to early events, including infection and replication, in virus pathogenesis.
The BmNPV BRO-a, BRO-c and BRO-d proteins appear to play important roles in the biology of this virus, potentially being involved in transcription of virus genes and BmNPV replication (Kang et al., 1999; Zemskov et al., 2000
). Here we have shown that disruption of the BRO-c domain (recombinant AcP+M4:BT12) in the unique AcMNPV BRO (orf2), a homologue of BmNPV BRO-d, had little detrimental effect on replication and pathogenesis of this virus in cells of S. frugiperda and T. ni, or in T. ni and S. exigua larvae infected with budded virus or per os with polyhedra. Instead, we found that disruption of BRO-N in orf2 in the recombinant AcBacP+1:brochlABD affected the terminal stage of AcMNPV replication by markedly reducing the number of polyhedra produced in infected nuclei. In this regard, orf2 could function, directly or indirectly, in maximizing polyhedra formation in infected larvae, thereby maintaining high numbers of infective virions in the field following larval death. Further studies are required to determine the exact role orf2 plays in AcMNPV replication.
The function of orf2 homologues may not be essential for other baculoviruses that are closely related to AcMNPV. For example, the A. falcifera MNPV and R. ou MNPV, variants of the AcMNPV, which replicate efficiently in the same lepidopteran hosts, apparently lack orf2 (Federici & Hice, 1997; Harrison & Bonning, 1999
). This indicates that the role of BRO proteins might be host dependent. The absence of bro genes in P. xylostella GV (Hashimoto et al., 2000
) provides additional support that BRO function may not be essential for all baculoviruses. Whether there are bro homologues present in insect hosts that compensate for deficiency in BRO function in these baculoviruses is not known.
In conclusion, the structural features of active bro genes and BRO proteins in different viral species, which include putative motifs for temporal bro expression and BRO function (Kang et al., 1999; Zemskov et al., 2000
; Iyer et al., 2002
), suggest that BRO proteins mediate specific virushost interactions during virus pathogenesis. Taking into account the plasticity, variability and putative host-specific function of BRO proteins, it is tempting to propose that bro and bro-like genes have the features of a genetic system resulting from the evolution of the virushost relationship. They may provide a general mechanism to maintain the virulence of these viruses within specific hosts, resulting from host resistance to viruses and bacteriophages with a large dsDNA genome that is common to many invertebrates and bacteria.
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ACKNOWLEDGEMENTS |
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Received 26 March 2003;
accepted 1 May 2003.
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