Phylogenetic analysis and possible function of bro

Journal of General Virology (2003), 84, 2531–2544
DOI 10.1099/vir.0.19256-0
Phylogenetic analysis and possible function of brolike genes, a multigene family widespread among
large double-stranded DNA viruses of
invertebrates and bacteria
Dennis K. Bideshi,1,2 Sylvaine Renault,3 Karine Stasiak,1,3 Brian A. Federici1
and Yves Bigot1,3
1
Department of Entomology and Interdepartmental Graduate Program in Genetics, University of
California, Riverside, CA 92521, USA
Correspondence
Yves Bigot
(at Université François Rabelais)
2
[email protected]
3
Received 26 March 2003
Accepted 1 May 2003
Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene
family, typically with multiple copies per genome, known to occur among certain insect dsDNA
viruses and bacteriophages. Little is known about the evolutionary history and function of the
proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur
among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in
prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal
region, consisting of two subdomains, is the most conserved region and contains a DNA-binding
motif that has been characterized previously. Phylogenetic analysis indicated that these proteins
are distributed among eight groups, Groups 1–7 consisting of invertebrate virus proteins and
Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified
in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in
prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic
relationship between bro genes suggests that they have resulted from recombination of viral
genomes that allowed the duplication and loss of genes, but also the acquisition of genes by
horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in
different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these
proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro
gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for
replication, but may enhance replication during the occlusion phase of reproduction.
California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504-3297, USA
Laboratoire d’Etude des Parasites Génétiques, FRE CNRS 2535, Université François Rabelais,
UFR des Sciences et Techniques, Parc de Grandmont, 37200 Tours, France
INTRODUCTION
A common feature of many eukaryotic dsDNA viruses is the
presence of multigene families (MGFs) composed of related
repeated open reading frames (ORFs) dispersed along the
genome. MGFs are abundant in viruses of the family
Phycodnaviridae (Van Etten & Meints, 1999), Asfarviridae
(Almendral et al., 1990; De la Vega et al., 1990; Gonzalez
et al., 1990; Vydelingum et al., 1993; Rodriguez et al., 1994;
Yozawa et al., 1994; Yanez et al., 1995; Pires et al., 1997),
Herpesviridae (Gompels et al., 1995), Chordopoxviridae
(Goebel et al., 1990; Massung et al., 1994) and vertebrate
The sequences reported here have been deposited in the DDBJ/
EMBL/GenBank sequence database under accession nos AJ292546–
AJ292551.
0001-9256 G 2003 SGM
Iridoviridae (Schnitzler et al., 1987), where they occur in the
terminal regions of the genomes.
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
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2531
D. K. Bideshi and others
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 100–150
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 a–e) in
BmNPV are expressed early, about 2–4 h after initiation of
2532
virus replication, and that transcription initiates 50–
70 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 BROd 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.
METHODS
Virus strains. The ascoviruses used, SfAV1a and HvAV3c, were
isolated initially from the noctuid hosts Spodoptera frugiperda and
Heliothis virescens, respectively (Federici et al., 1990). DpAV4 was
isolated from its wasp host, Diadromus pulchellus (family Ichneumonidae), and from pupae of its lepidopteran host, Acrolepiopsis assectella (Family Yponeumonidae; Bigot et al., 1997). The iridovirus (IIV;
iridovirus type 31, so-called IV31) was isolated from the terrestrial
isopod Armadillidium vulgare, and has been described previously
(Federici, 1980). Propagation and preparation of viral DNA were
performed as described previously (Bigot et al., 1997; Federici et al.,
1990).
Genomic DNA libraries. Fifty mg 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
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Journal of General Virology 84
bro and bro-like genes
were blunted with SI nuclease and T4 DNA polymerase (New
England Biolabs) and EcoRI linkers were ligated at both ends.
Fragments of approximately 0?85–1?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 uC
for 30 s, annealing at 50 uC for 15 s and polymerization at 70 uC 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 (1–250 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, chl r and tet r; see below)
were prepared using a Dig DNA labelling and detection kit
(Boehringer Mannheim). Hybridization was performed at 65 uC in
0?1 % SDS, 0?5 M Na2HPO4/NaH2PO4 buffer, pH 7, and posthybridization washes were performed at 65 uC using 0?56 SSC
(high stringency) or 26 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
(59-AAGCGAGGATCTACAACGTT-39) and ORF2b (59-TAAAATGTTTCCCGCGCGTT-39) 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 SspI–MscI fragment with the tetracycline resistance gene
(tetr) from pBR322 (Biolabs) into the blunted BstEII site in pGEMT-orf2. pGEM-Bro/chl was constructed by inserting the blunted
1?2 kb BspHI–XmnI fragment with the chloramphenicol resistance
marker (chlr) from pBCSK(2) (Stratagene) in the blunted BstEII
and SwaI sites in pGEM-T-orf2. The 3?9 kb and 2?8 kb fragments
http://vir.sgmjournals.org
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 mg ml21) and kanamycin (45 mg ml21) or chloramphenicol
(15 mg ml21) and kanamycin (45 mg ml21). 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, chl s, tet s, 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 mg 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 uC 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 uC, 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 ml 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 ml Grace’s insect cell culture medium (Gibco BRL). Two ml 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 ml of this suspension was added to
0?5 mg of growth medium, which was then fed to 10 early fourth
instars of T. ni.
RESULTS
Cloning of bro-l genes from the genomes of
ascoviruses and the crustacean iridovirus IIV
Based on BLAST searches with sequences generated from
our ascovirus and iridovirus genomic libraries and from
DpAV4 sequences reported previously (Bigot et al., 1997,
2000; Stasiak et al., 2000), several bro and bro-l sequences
were identified, with the quality of the observed similarities
based on smallest sum probabilities ranging from 1.e2200
to 1.e210 (30–45 % similar to the most closely related BRO
proteins). Eleven bro-l ORFs were identified in SfAV1a
(SfAV1a-bro-l1 to -l11), three in DpAV4 (DpAV4-bro-l1
to -l3), two in HvAV3c (HvAV3c-bro-l1 and -l2) and one
in iridovirus IIV (IIV-bro-l) (Table 1).
The bro-l genes present in the HvAV3c and IIV genomes
were confirmed by Southern blot hybridization. Several
HvAV3c fragments hybridized with the HvAV3c-bro-l1
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2533
D. K. Bideshi and others
Table 1. bro and bro-like genes found in invertebrate and bacterial viruses
Name of bro or
bro-like genes
Ac-bro-B40781
Ac-bro-B38477
Ag-bro-a
Ag-bro-b
AmEPV-bro-la
AmEPV-bro-lb
AmEPV-bro-lc
AmEPV-bro-ld
AmEPV-bro-le
Bm-bro-a
Bm-bro-b
Bm-bro-c
Bm-bro-d
Bm-bro-e
CIV-bro-la
CIV-bro-lb
CIV-bro-lc
Cn-bro-a
Cn-bro-b
Cn-bro-c
Cn-bro-d
Cn-bro-e
Cn-bro-f
DpAV4-bro-l1
DpAV4-bro-l2
DpAV4-bro-l3
Ha-bro-a
Ha-bro-b
Ha-bro-c
Ha-bro-d
Ha-bro-e
Ha-bro-f
HaEPV-bro-l
HvAV3c-bro-l1
HvAV3c-bro-l2
Hz-bro-a
Hz-bro-b
Hz-bro-c
Hz-bro-d
Hz-bro-e
IIV-bro-l
Ld-bro-a
Ld-bro-b
Ld-bro-c
Ld-bro-d
Ld-bro-e
Ld-bro-f
Ld-bro-g
Ld-bro-h
Ld-bro-i
Ld-bro-j
Ld-bro-k
Ld-bro-l
2534
Virus or
transposons
Virus or transposon
family
Host name
Gene
accession no.
Protein
accession no.
AcMNPV
"
AgMNPV
"
AmEPV
"
"
"
"
BmMNPV
"
"
"
"
CIV (IV6)
"
"
CuniNPV
"
"
"
"
"
DpAV4
"
"
HaNPV
"
"
"
"
"
HaEPV
HvAV3c
"
HzMNPV
"
"
"
"
IIV (IV31)
LdMNPV
"
"
"
"
"
"
"
LsMNPV
"
"
"
Baculoviridae
"
Baculoviridae
"
Poxviridae
"
"
"
"
Baculoviridae
"
"
"
"
Iridoviridae
"
"
Baculoviridae
"
"
"
"
"
Ascoviridae
"
"
Baculoviridae
"
"
"
"
"
Poxviridae
Ascoviridae
"
Baculoviridae
"
"
"
"
Iridoviridae
Baculoviridae
"
"
"
"
"
"
"
Baculoviridae
"
"
"
Autographa californica
"
Anticarsia gemmatalis
"
Amsacta moorei
"
"
"
"
Bombyx mori
"
"
"
"
Chilo suppressalis
"
"
Culex nigripalpus
"
"
"
"
"
Diadromus pulchellus
"
"
Helicoverpa armigera
"
"
"
"
"
Heliothis armigera
Heliothis virescens
"
Helicoverpa zea
"
"
"
"
Armadillidium vulgare
Lymantria dispar
"
"
"
"
"
"
"
Lymantria dispar
"
"
"
L22858
"
Y17753
"
AF250284
"
"
"
"
L33180
"
"
"
"
AF003534
"
"
AF403738
"
"
"
"
"
X85006
AJ279818
AJ279813
AF303045
"
"
"
"
"
AF022176
AJ292549
AJ292550
"
"
"
"
AF275264
AJ292551
AF081810
"
"
"
"
"
"
"
AF081810
"
"
"
B40781
B38477
–
–
–
–
–
–
O92398
O92457
O92458
O92507
O92508
–
–
Q91FW9
Q91FN5
Q919R4
Q919G9
Q919G8
Q919R0
Q919I2
Q919R1
–
–
–
Q8QMF6
Q99GY7
Q99GY8
Q8QMF5
Q91BV2
Q91BA1
–
–
–
Q8V5T5
Q8V5T7
Q9E231
Q8V5T6
–
–
Q9YMU2
Q9YMU1
Q9YMQ6
Q9YMQ5
Q9YMQ4
Q9YMQ3
Q9YMQ2
Q9YML5
Q9YML4
Q9YML3
Q9YML2
Q9YMI2
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Journal of General Virology 84
bro and bro-like genes
Table 1. (cont.)
Name of bro or
bro-like genes
Virus or
transposons
Virus or transposon
family
Host name
Gene
accession no.
Protein
accession no.
Ld-bro-m
Ld-bro-n
Ld-bro-o
Ld-bro-p
Ls-bro
Mc-bro-c
Mc-bro-d
Mc-bro-e
Mc-bro-f
Mc-bro-g
MsEPV bro-l1
MsEPV bro-l2
MsEPV bro-l3
MsEPV bro-l4
MsEPV bro-l5
MsEPV bro-l6
MsEPV bro-l7
Op-bro
PuGV-bro
Se-bro
Sl-bro-a
Sl-bro-b
Sl-bro-c
SfAV1a-bro-l1
SfAV1a-bro-l2
SfAV1a-bro-l3
SfAV1a-bro-l4
SfAV1a-bro-l5
SfAV1a-bro-l6
SfAV1a-bro-l7
SfAV1a-bro-l8
SfAV1a-bro-l9
SfAV1a-bro-l10
SfAV1a-bro-l11
TnGV-bro-1
TnGV-bro-2
TnGV-bro-3
XcGV bro-a
XcGV bro-c
XcGV bro-e
XcGV bro-f
XcGV bro-g
XcGV bro-j
ESV-bro-l
"
"
"
"
LsMNPV
MacoNPV
"
"
"
"
MsEPV
"
"
"
"
"
"
OpMNPV
PuGV
SeMNPV
SlMNPV
"
"
SfAV1a
"
"
"
"
"
"
"
"
"
"
TnGV
"
"
XcGV
‘‘
"
"
"
"
ESV
"
"
"
"
Baculoviridae
Baculoviridae
"
"
"
"
Poxviridae
"
"
"
"
"
"
Baculoviridae
Baculoviridae
Baculoviridae
Baculoviridae
"
"
Ascoviridae
"
"
"
"
"
"
"
"
"
"
Baculoviridae
"
"
Baculoviridae
"
"
"
"
"
Phycodnaviridae
"
"
"
"
Leucania separata
Mamestra configurata
"
"
"
"
Melanoplus sanguinipes
"
"
"
"
"
"
Orgyia pseudotsugata
Pseudaletia unipuncta
Spodoptera exigua
Spodoptera littura
"
"
Spodoptera frugiperda
"
"
"
"
"
"
"
"
"
"
Trichoplusia ni
"
"
Xestia c-nigrum
"
"
"
"
"
Ectocarpus siliculosus
"
"
"
"
AB009612
AF467808
"
"
"
"
AF063866
"
"
"
"
"
"
U75930
D14871
AF169823
AF143953
AF325155
"
AJ292546
AJ292547
AJ292547
AJ312700
AJ312700
AJ312701
AJ312702
AJ312702
AJ312703
AJ312699
AJ292548
D58376
D58375
D58377
AF162221
"
"
‘‘
‘‘
"
AF204951
Q9YMH8
Q9YMH5
Q9YMH4
Q9YMG7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Q9J8C2
Q9JAD6
Q91BA1
Q91BA9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Q9PYY3
Q9PYT4
Q9PYR5
Q9PYR4
Q9PYN7
–
–
probe (Fig. 1a). An HpaII restriction sites was present at
position 196 in the sequence of the probe used, but DraI and
EcoRV sites were absent. The two hybridizing fragments
observed in the DraI and EcoRV DNA digests indicated that
another sequence homologous to the HvAV3c-bro-l1 was
present in the HvAV3c genome (Fig. 1a, lanes 1 and 2). Due
to its nucleic acid sequence similarity to HvAV3c-bro-l1
http://vir.sgmjournals.org
(less than 65 %), probing with HvAV3c-bro-l2 did not reveal
the same bands (data not shown). The results suggested
that at least three bro-l genes were present in this genome.
Similar studies were carried out with iridovirus IIV. Two to
three fragments were detected in IIV DNA digested with
restriction enzymes with sites absent in the nucleotide
sequence of the probe (Fig. 1b, lanes 1 and 2). This indicated
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2535
D. K. Bideshi and others
(a)
1 2
3
kbp
21.7
5.5
4.9
3.5
(b)
1
2
3 kbp
21.7
(c)
1
2
3
kbp
21.7
5.5
4.9
5.5
4.9
3.5
3.5
2.0
2.0
1.5
0.9
2.0
1.5
1.5
Fig. 1. Identification of bro genes in ascovirus and iridovirus
genomes by Southern hybridization. (a) Hybridization of bro-l1
cloned from HvAV3c to HvAV3c genomic DNA digested with
DraI (lane 1), EcoRV (lane 2) and HpaII (lane 3). (b)
Hybridization of bro-l1 cloned from the IIV (type 31) genome to
IIV DNA digested with ClaI (lane 1), EcoRI (lane 2) and EcoRV
(lane 5). (c) Hybridization of SfAV1a-bro-l2 to the genome of
three different variants of SfAV1, SfAV1a (lane 1), SfAV1b
(lane 2) and SfAV1c (lane 3), each digested with BamHI/
HindIII. The four SfAV1a fragments that hybridized with bro-l2
are indicated by arrows in the left margin. Molecular masses
are indicated in the right margins.
that at least three related copies of bro-l genes were present
in the IIV genome.
Eleven genes with their 59 and 39 flanking regions encoding
BRO-l proteins ranging from 70 to 370 amino acids were
found in the 500 SfAV1a genomic sequences. Five of them,
SfAV1a-bro-l5, -l7, -l8, -l9 and -l10, presented cardinal
features of functional genes. One, SfAV1a-bro-l2, lacked a
start codon and SfAV1a-bro-l3 and -l4 lacked sequences
encoding approximately 150 N-terminal residues. SfAV1abro-l1, -l6 and -l11 corresponded to partial bro-l gene
sequences. SfAV1a-bro-l2, -l3 and -l4 were apparently nonfunctional based on their small size and coding capacity,
suggesting the presence of functional and non-functional
bro-l genes in the SfAV1a genome.
Comparison of the SfAV1a-bro-l1 to -l11, or HvAV3c-brol1 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
80–89 % identical to each other and were located within
three different 980 bp repeated sequences (accession nos
2536
X85006, nt 410–1400; AJ27918, nt 680–1675; AJ279813,
nt 5290–6250, 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 BamHI–HindIII
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.
Additionally, five bro-l sequences, not previously described,
were identified in bacterial class II transposons. In Pseudomonas alcaligenes, a bro-l gene was located in Tn5563, a
transposon related to ISXc5 and ISXc4 (IS3 family; Mahillon
& Chandler, 1998), upstream from the transposase A ORF
in a different coding frame (accession no. U88088, nt
30716–31717). In Pseudomonas putida, four bro-l genes
were present in Tn5542, a transposable element related to
ISPs1 (IS5 family, IS427 group; Mahillon & Chandler, 1998).
One of these was found upstream from the transposase A
gene and was fused in the same coding frame (accession
no. AF148496, nt 4724–5110) and the other three were
interrupted by several frame shifts and stop codons. We
also identified sequences in O. pseudotsugata MNPV,
Pseudaletia unipuncta GV, T. ni GV and X. c-nigrum GV
(XcGV-bro-j) that contained bro-l sequences interrupted by
several stop codons and frame shifts. Two bro-l sequences
with regions encoding a truncated N-terminal domain
were present in Leucania separata MNPV (Table 1) and in
Epiphyas postvittana MNPV (Hyink et al., 2002).
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Journal of General Virology 84
bro and bro-like genes
Table 2. bro-like genes found in genomes of bacteriophage and in bacterial transposons
Name of
bro-like gene
Virus or
transposons
Virus or
transposon family
A2-bro-l
BIL309-bro-l
BK5T-bro-l
CP-933N-bro-l
LLH-bro-l
Mx8-bro-l
N15-bro-l
P27-bro-l
phiPV83-bro-l
phi11-bro-l
r1t-bro-l
Hi-bro-l1
TnpPa-bro-l
Ll-bro-l
TnpPp-bro-l
Bm-bro-l1
Bm-bro-l2
Cl-bro-l
Li-bro-l
Ng-bro-l
Se-bro-l1
Se-bro-l2
Sp-bro-l1
Sp-bro-l2
Sp-bro-l3
Sc-bro-l
Xf-bro-l-1
Xf-bro-l-2
Xf-bro-l-3
Xf-bro-l-4
Yp-bro-l
A2
BIL309
BKT5
CP-933N
LLH
Mx8
N15
P27
PhiPV83
Phi11
r1t
Unnamed
ISP1
Unnamed
Tn5542
Unnamed
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
Bacteriophage
"
"
"
"
"
"
"
"
"
"
"
IS5
Bacteriophage
IS5
Prophage
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
Host name
Gene accession no.
Protein accession no.
Lactobacillus casei
Lactococcus lactis
"
Lactobacillus delbrueckii
Myxococcus sp.
Escherichia coli
"
Staphylococcus aureus
"
Lactococcus lactis
Haemophilus influenzae
Lactococcus lactis
Pseudomonas alcaligenes
Pseudomonas putita
Brucella melitensis
"
Clostridium perfringens
Listeria inoculata
Neisseria gonorrhoeae
Staphylococcus epidermidis
"
Streptococcus pyogenes
"
"
Streptomyces coelicolor
Xylella fastidiosa
"
"
"
Yersinia pestis
Y12813
A323670
L44593
AE005325
L42315
AF396866
U63086
AJ298298
AB044554
AF424781
U38906
U32821
A47218
U88088
AF148496
AE009530
AE009601
AP003189
AL596163
AJ004687
AF269322
AF269376
AE10005
AE00979
Ae006544
AF096822
AE003912
AE004059
AE003992
AE003985
AJ414151
–
Q9AZQ4
–
Q8X797
–
Q94MQ6
–
Q8W644
–
–
–
P44189
–
–
–
Q8YHA3
Q8YF64
Q8XLD2
Q92FM4
–
–
–
–
–
–
–
Q9FHP4
Q9PCU2
Q9PAJ2
Q9PAK9
Q8ZEN5
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, neighbourjoining 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 1–8) 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 %,
http://vir.sgmjournals.org
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.
These results reflect the evolutionary relationships of BRO
and BRO-l N-terminal domains. In an attempt to verify
whether they also reflect the evolutionary history of
C-terminal domains and therefore that of complete BRO
and BRO-l proteins, psi-BLAST searches were carried out
with each of the C-terminal domains and the BLOSUM45
as a starting matrix. At equilibrium, the first scores for all
searches indicated that each C-terminal domain was always
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2537
D. K. Bideshi and others
Fig. 2. Consensus unrooted tree illustrating the phylogenetic relationships of BRO and BRO-l proteins. The tree is based on
amino acid sequences from aa 1 to 160, as shown in Fig. 3. Due to the large number of protein sequences used in this
analysis, bootstrap values at nodes (1000 replicates) are represented by red, violet, blue and green spots that are scaled in
the left margin. All the references for each protein are given in the Tables 1 and 2. Depending on their origin, the names of
different BRO and BRO-l proteins are identified with different colours as follows: NPVs (dark green), GVs (green), Culex
nigripalpus NPV (light blue), entomopoxviruses (red), iridoviruses (dark blue), ascoviruses (pink), bacteriophages (orange),
bacterial transposon (brown) and the phycodnavirus ESV (black). The different groups of BRO and BRO-l proteins are
identified by grey ellipses and numbered from 1 to 8. The putative BRO and BRO-l proteins that are encoded by truncated
genes or a pseudogenes are typed within boxes filled in yellow.
more related to the C-terminal domains of BRO proteins
with N-terminal domains present in the same group. This,
therefore, indicated that N- and C-terminal domains of the
BRO proteins belonging to each of the seven invertebrate
groups have co-evolved together. However, our results
also indicated that the 11 unclassified proteins (Ag-bro-b,
2538
CIV-bro-l1, CIV-bro-l2, Ha-bro-a, Hz-bro-b, Ld-bro-m,
Mc-bro-c, Mc-bro-g, Mc-bro-f, Se-bro and SfAV1-bro-l5)
were probably chimeric proteins resulting from fusion of
the N-terminal BRO domain from one group with the
C-terminal domain of a different group. In agreement with
the results of Iyer et al. (2002), we observed that the
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Journal of General Virology 84
bro and bro-like genes
Fig. 3. Alignment of the consensus sequences deduced from amino acid sequence analyses of each of the eight groups
(G1–G8) of BRO and BRO-l proteins identified in dsDNA genomes of invertebrate viruses and bacteriophages. In each
conserved motif, empty columns indicate short sequences of the complete alignment that corresponded to insertions or
deletions that were deleted. Conserved positions between the eight consensus sequences are indicated in white, with
identical positions occurring in at least 50 % of the positions in black boxes and those that are similar are in grey boxes. The
residues in clear grey boxes correspond to positions that are identical or similar in more than 75 % of the BRO sequences
used to define the consensus of each group. The matrix of substitutions used was that described by Nevill-Manning et al.
(1998). Only one substitution group was added: R=K=H. The residues proposed by Zemskov et al. (2000) as
corresponding to the conserved motif responsible of the non-specific DNA binding activity in BRO proteins are indicated
below the alignment at aa 9–62. The two subdomains contained in the conserved N-terminal region of the BRO and BRO-l
proteins are located by boxes filled in light grey, whereas the hinge region is located by grey hatching.
C-terminal domain of the BRO-l protein from bacteriophages was the most diverse from the others. This indicated
that the N- and C-terminal domains of these proteins
http://vir.sgmjournals.org
were probably more mobile and thus subject to greater
chimerization than those of their homologues contained
in the genomes of the invertebrate DNA viruses.
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2539
D. K. Bideshi and others
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 1–150) 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]-X2–5-[K/R]-X4–12-[F/Y]-X2–14[F/Y]-X6–13-[F/Y]-X1–19-[K/R]-X3–26-[F/Y/W]-X6–12-[K/R] –
was found to be significantly conserved. Moreover,
conserved residues at positions 49, 52–54, 60–61 and 64–65
(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 78–95; 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.
In contrast to BRO-N, alignments of the consensus sequences showed that the BRO-C domain was less conserved
among BRO proteins in Groups 1, 2, 3, 5 and 7 (Fig. 3);
the C-terminal sequences of the Group 4 proteins were
unrelated to BRO-C.
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).
All of the bacmid strains replicated in BTI-TN5-B1-4 and
2540
(a)
kbp
(b)
1
2
3
4
2
4
kbp 1
2
3
4
3
2
Fig. 4. Gel analysis illustrating disruption of the AcMNPV bro
orf2 gene. (a) PCR products from budded virions obtained
using ORF2a and ORF2b primers with DNA from AcBacP+1
containing the intact orf2 (lanes 1 and 3) and AcP+4M:T12
containing orf2 disrupted with the tetracycline resistance gene
(lanes 2 and 4). (b) PCR products obtained from AcBacP+1
(lanes 1 and 3) and AcBacP+1:brochlABD (lanes 2 and 4).
Results of hybridization with the orf2 probe are shown in lane
3 and 4 (a, b).
SF21 cells, and budded virions produced in these cells were
lethal for T. ni and Spodoptera exigua fourth instar larvae.
None of the larvae injected with budded virions survived.
However, significant differences were observed in the polyhedra production in cells transfected or infected with the
different bacmid strains, at 7 days post-infection (Fig. 5).
For example, 5 days after transfection, approximately
61 % and 46 % of BTI-TN5-B1-4 cells transfected with
AcBacP+1 and AcP+4M:T12, respectively, contained
numerous polyhedra in the nuclei (more than 100 per
nuclei), whereas the percentage value was markedly reduced
to 11 % for AcBacP+1:brochlABD (Fig. 5). Furthermore,
relatively few polyhedra, generally between 5 and 25 and
occasionally only one, were observed in nuclei of cells
infected with AcBacP+1:brochlABD.
DISCUSSION
Previous studies have identified and partially characterized
the bro MGF in baculoviruses, poxviruses and bacteriophages (Afonso et al., 1999; Hayakawa et al., 1999; Kang
et al., 1999; Kuzio et al., 1999; Bawden et al., 2000; Iyer et al.,
2002). In this study, we have shown that the bro and bro-l
MGF is present in two other invertebrate virus families,
Ascoviridae and Iridoviridae, and in at least two bacterial
class II transposons.
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 (BROC). 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).
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Journal of General Virology 84
bro and bro-like genes
Fig. 5. Polyhedra observed in BTI-TN5-B1-4 cells at 7 days post-transfection. Cells were mock transfected (A), or transfected with 1 mg bacmid DNA (A), or transfected with AcBacP+1 (polh+, kanr, chls, tets, orf2+) (B), AcP+4M:T12 (polh+,
kanr, chls, orf2 disrupted with tet r) (C) or AcBacP+1:brochlABD (polh+, kanr, tets, orf2 disrupted with chl r) (D). For each of
the three virus mutants, three assays were performed. In B, C and D, white arrows indicate the polyhedra in the infected cells.
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
http://vir.sgmjournals.org
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
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2541
D. K. Bideshi and others
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 2–4 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 virus–
host 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 virus–host
relationship. They may provide a general mechanism to
maintain the ‘virulence’ of these viruses within specific
2542
hosts, resulting from host resistance to viruses and bacteriophages with a large dsDNA genome that is common to many
invertebrates and bacteria.
ACKNOWLEDGEMENTS
We thank Dr M. V. Demattei and J. J. Johnson for their assistance
throughout our investigations. This work was supported by grants
from the CNRS (UPRES-A 6035), NATO and the Ministère de
l’Education Nationale, de la Recherche et de la Technologie to Y. Bigot
and from US National Science Foundation Grant INT-9726818 to
B. A. Federici.
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