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Journal of Virology, September 2006, p. 8439-8449, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00464-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Genome of Invertebrate Iridescent Virus Type 3 (Mosquito Iridescent Virus)

Gustavo Delhon,1,3,9* Edan R. Tulman,1,4,5 Claudio L. Afonso,1,6 Zhiqiang Lu,1 James J. Becnel,2 Bettina A. Moser,2,7,8 Gerald F. Kutish,1,5,6 and Daniel L. Rock1,9

Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York 11944,1 CMAVE, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, Florida 32608,2 Area of Virology, School of Veterinary Sciences, University of Buenos Aires, 1427 Buenos Aires, Argentina,3 Department of Pathobiology and Veterinary Science,4 Center of Excellence for Vaccine Research, University of Connecticut, Storrs, Connecticut 06269,5 Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia 30605,6 U.S. Army Veterinary Laboratory Europe, APO AE 09180,7 Microbiology Department, Veterinary Laboratory Europe, Geb. 3810, Zi. 122B, 66849 Landstuhl-Kirchberg, Germany,8 Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 618029

Received 6 March 2006/ Accepted 1 June 2006


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ABSTRACT
 
Iridoviruses (IVs) are classified into five genera: Iridovirus and Chloriridovirus, whose members infect invertebrates, and Ranavirus, Lymphocystivirus, and Megalocytivirus, whose members infect vertebrates. Until now, Chloriridovirus was the only IV genus for which a representative and complete genomic sequence was not available. Here, we report the genome sequence and comparative analysis of a field isolate of Invertebrate iridescent virus type 3 (IIV-3), also known as mosquito iridescent virus, currently the sole member of the genus Chloriridovirus. Approximately 20% of the 190-kbp IIV-3 genome was repetitive DNA, with DNA repeats localized in 15 apparently noncoding regions. Of the 126 predicted IIV-3 genes, 27 had homologues in all currently sequenced IVs, suggesting a genetic core for the family Iridoviridae. Fifty-two IIV-3 genes, including those encoding DNA topoisomerase II, NAD-dependent DNA ligase, SF1 helicase, IAP, and BRO protein, are present in IIV-6 (Chilo iridescent virus, prototype species of the genus Iridovirus) but not in vertebrate IVs, likely reflecting distinct evolutionary histories for vertebrate and invertebrate IVs and potentially indicative of genes that function in aspects of virus-invertebrate host interactions. Thirty-three IIV-3 genes lack homologues in other IVs. Most of these encode proteins of unknown function but also encode IIV3-053L, a protein with similarity to DNA-dependent RNA polymerase subunit 7; IIV3-044L, a putative serine/threonine protein kinase; and IIV3-080R, a protein with similarity to poxvirus MutT-like proteins. The absence of genes present in other IVs, including IIV-6; the lack of obvious colinearity with any sequenced IV; the low levels of amino acid identity of predicted proteins to IV homologues; and phylogenetic analyses of conserved proteins indicate that IIV-3 is distantly related to other IV genera.


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INTRODUCTION
 
Iridoviruses (IVs) (family Iridoviridae) are large, icosahedral, double-stranded DNA (dsDNA) viruses that cause subclinical to lethal infections in invertebrates and poikilothermic vertebrates. IV particles are 120 to 300 nm in diameter and consist of a central core of nucleic acid and proteins, an intermediate lipid membrane, a T = 147 capsid comprised of copies of the major capsid protein (MCP), and, in virions that bud from the plasma membrane, an external envelope (90, 95). IV genomic DNA is a linear molecule which is circularly permuted and terminally redundant (30). Although IV replication includes nuclear and cytoplasmic stages, virion assembly occurs exclusively in the cytoplasm (28). In contrast to most dsDNA insect viruses, insect IVs are not occluded in a protein matrix.

Based on particle size, host range, DNA cross-hybridization, the presence of a methyl transferase, and the MCP gene sequence, IVs are classified into five genera, with viruses of two genera (Iridovirus and Chloriridovirus) infecting invertebrates, mostly insects (invertebrate iridescent viruses [IIVs]), and viruses of the other three (Ranavirus, Lymphocystivirus, and Megalocytivirus) infecting cold-blooded vertebrates (vertebrate iridoviruses [VIVs]) (47, 84, 91). Complete genome sequences have been determined for viruses representing four of the five genera, including lymphocystis disease virus 1 (LCDV-1) and LCDV-China (LCDV-C) of the genus Lymphocystivirus; tiger frog virus (TFV), frog virus 3 (FV-3), Ambystoma tigrinum virus (ATV), and Singapore grouper iridovirus (SGIV) of the genus Ranavirus; rock bream iridovirus (RBIV) and infectious spleen and kidney necrosis virus (ISKNV) of the genus Megalocytivirus; and IIV-6 (or Chilo iridescent virus) of the genus Iridovirus (22, 34, 35, 42, 43, 69, 73, 76, 98). Also available are the genome sequences of grouper iridovirus (GIV) and orange-spotted GIV (OSGIV), viruses currently lacking taxonomic classification (53, 78). IV genomes range in size from 105 to 212 kbp and contain 96 to 234 largely nonoverlapping open reading frames (ORFs), a G+C content ranging from 27 to 55%, and complex repeat sequences mostly located between coding regions. Genomes exhibit little to no colinearity among genera (see Table 1).


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  		TABLE 1. Characteristics of IV genomes

The type species and currently sole member of the genus Chloriridovirus is Invertebrate iridescent virus type 3 (IIV-3), also known as mosquito iridescent virus (MIV). IIV-3 is characterized by its restricted host range (mosquitoes [Diptera]) and relatively large particle size (180 nm) (17, 90). In contrast, IIVs of the Iridovirus genus have been isolated from diverse hosts of the orders Diptera, Lepidoptera, Hemiptera, and Coleoptera and are approximately 120 nm in diameter (89). Iridovirus species include IIV-1, the first reported IV; IIV-6, the type species of the genus; and several tentative species which remain to be fully characterized (25, 89, 94). Notably, IIV-6 can also infect mosquitoes but causes only sublethal (covert) infections and reduced fitness in Aedes aegypti relative to noninfected conspecifics (55).

IIV-3 was originally isolated from larvae of the salt marsh mosquito Ochlerotatus (formerly Aedes) taeniorhynchus (Wiedemann) (19), with successful transmission to an additional mosquito host, Ochlerotatus sollicitans (93). IIV-3 has been isolated from several other mosquito species, including Aedes vexans, Psorophora ferox, Culiseta annulata, and Culex territans, which are important pests of both humans and domestic animals (14, 17, 93). Early mosquito larval stages are most susceptible to IIV-3 infection, but clinical disease (yellow-green iridescence beneath the epidermis) and death rates are highest in the fourth instar (93). IIV-3 infection of O. taeniorhynchus results in virus replication in the fat body and to a lesser extent in the dermis, imaginal disks, trachea, gonads, and hemocytes (33). Oral and transovarian transmission of IIV-3 have been documented for larval mosquitoes (19, 51, 52, 93). Two IIV-3 strains have been described, a field isolate referred to as regular strain (RMIV) and a laboratory isolate referred to as turquoise strain (TMIV) (93); these cause orange and blue-green iridescence, respectively, in infected larvae.

Despite the role of mosquitoes as significant vectors of human disease, chloriridoviruses are the least studied members of the family Iridoviridae. To date, IIV-3 genomic data are limited to the sequence of the DNA polymerase gene of RMIV (71, 83) and estimations that the TMIV genome contains about twice as much repetitive DNA as that of RMIV (82). Here, we report the complete genomic sequence, with analysis, of IIV-3, RMIV strain. These data, combined with previous work on viruses belonging or related to the other four IV genera, provide a first comparative overview of IV genomics.


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MATERIALS AND METHODS
 
IIV-3 DNA isolation, cloning, and sequencing. The RMIV strain was originally obtained from field-collected Ochlerotatus taeniorhynchus (Diptera: Culicidae) larvae and maintained by horizontal transmission in the laboratory. RMIV-infected O. taeniorhynchus larvae were ground with a Tekmar Tissuemizer in deionized water and filtered through 400 mesh to remove large insect parts. The filtrate was layered on a continuous HS-40 Ludox gradient and spun for 30 min at 16,000 x g. The band containing RMIV was collected, resuspended in deionized water, and spun for 30 min at 16,000 x g, and genomic DNA was extracted from the pellet as previously described (87). Genomic DNA was incompletely digested with TacI endonuclease (New England Biolabs, Beverly, Mass.), and fragments larger than 1.0 kbp were cloned into the dephosphorylated EcoRI site of pUC19 plasmid vector and grown in Escherichia coli DH10B cells (Gibco-BRL, Gaithersburg, Md.). Plasmids were purified by alkaline lysis according to the manufacturer's instructions (Eppendorf-5 Prime, Boulder, Colo.). DNA templates were sequenced from both ends with M13 forward and reverse primers using dideoxy chain terminator sequencing chemistries (65) and the Applied Biosystems PRISM 3700 automated DNA sequencer (Applied Biosystems, Foster City, Calif.). Chromatogram traces were basecalled with Phred (23) and linearly assembled with Phrap (http://www.phrap.org), with the quality files and default settings used to produce a consensus sequence which was manually edited with Consed (31). An identical sequence was assembled by using the Cap3 assembler with quality files and clone length constraints (38). While the IIV-3 genome assembled essentially in a circular fashion consistent with circular permutation, specifically, randomly located but overlapping termini were observed in independent linear assemblies. For clarity, the IIV-3 genome is presented here in a linear fashion, with overlapping sequence removed from the right terminus of one assembly and the first base of the left terminus arbitrarily numbered base one. The final DNA consensus sequence represented on average 10-fold redundancy at each base position (5,483 reactions) and had a Consed estimated error rate of 0.04/10 kbp.

Sequence analysis. DNA composition, structure, repeats, and restriction enzyme patterns were analyzed as previously described (1). ORFs longer than 30 codons with a methionine start codon were evaluated for coding potential by the Hexamer (ftp.sanger.ac.uk/pub/rd), Glimmer (64), and Framefinder (http://www.ebi.ac.uk/~guy) computer programs. Minor ORFs contained within larger ORFs were excluded. Here, 126 ORFs are annotated as potential genes and numbered from left to right. Given the predicted nature of all IIV-3 genes and gene products, ORF names are used throughout this work to indicate both the predicted gene and its putative protein product. DNA and protein comparisons with entries in genetic databases (PROSITE, Pfam, Prodom, Sbase, Blocks, and GenBank) were performed with the BLAST (4), PsiBlast (5), FASTA, TFASTA (63), and HMMER (70) programs. The GCG (20), MEMSAT (44), Psort (61), and SAPS (13) programs were used for general analysis, transmembrane prediction, and physical characterization of predicted proteins. DNA repeats were analyzed with Lalign (http://workbench.sdsc.edu) and Dot Plot (GCG package). Sequence quality, sequence depth, and the lack of obvious polymorphism observed in IIV-3 repeat regions were consistent with that of the rest of the IIV-3 genome assembly. Multiple alignments were performed with Clustal, Dialign-T, and Kalign (49, 72, 75). Phylogenetic analysis was performed with the PHYLO_WIN, TREE-PUZZLE, IQPNNI, PHYLIP, PHYML, and MRBAYES software packages (16, 24, 26, 32, 39, 66), and evolutionary models were selected with ModelGenerator (http://bioinf.nuim.ie/software/modelgenerator). Additional analyses were conducted on alignments where poorly aligned regions were removed with Gblocks (16).

Nucleotide sequence accession number. The IIV-3 genome sequence has been deposited in the GenBank database under accession no. DQ643392.


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RESULTS AND DISCUSSION
 
Genome organization. IIV-3 DNA was assembled in a contiguous sequence of 190,132 bp, a figure that differs significantly from previous size estimates based on restriction endonuclease analysis (135,000 bp) and sucrose gradient centrifugation (383,000 bp) (82, 88). IIV-3 nucleotide composition averaged 48% G+C, which is lower than previously reported (54%) (82) and closer to the ranavirus and megalocytivirus genomes than to IIV-6, the other fully sequenced IIV (Table 1). The IIV-3 base composition was not uniformly distributed throughout the genome, with lower G+C values (42%) found in regions containing repeated sequences.

Based on coding potential analysis and similarity to known proteins, 126 IIV-3 ORFs were annotated here (Fig. 1; Table 2). These ORFs, which encode proteins of 60 to 1,377 amino acids (aa), represent a coding density of 68%, one of the lowest among fully sequenced IVs (Table 1). IIV-3 contains 33 unique genes, 27 homologues of genes present in all sequenced IVs, and 52 genes present in IIV-6 but not in VIVs. Consistent with the lack of gene colinearity observed between IIV-6 and VIV and even between LCDV-1 and LCDV-C, viruses likely belonging to the same genus, Lymphocystivirus, the IIV-3 genome exhibited no obvious colinearity with any other completely sequenced IV genome.


Figure 1
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  		FIG. 1. Linear map of the IIV-3 genome. ORFs are numbered from left to right. ORFs transcribed to the right or to the left are located above and below the horizontal line, respectively. Red boxes represent genes encoded by all currently sequenced IV genomes. Blue, yellow, and white boxes are genes unique for IIVs, IIV-3, and selected IVs, respectively. Repetitive DNA sequences are shown as numbered black boxes.


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  		TABLE 2. IIV-3 ORFs

Repeated sequences. The IIV-3 genome contained highly repetitive DNA sequences in 15 distinct regions (designated R1 to R15 from left to right) that were 0.8 to 4.6 kbp in length, irregularly distributed, and apparently noncoding (Fig. 1). IIV-3 DNA repeats were complex, mostly direct and imperfect, and they could be divided into two groups based on sequence similarity, with group I repeats located at R2, R4, R6 to R8, R11 to R13, and R15 and group II repeats located at R1, R3, R5, R9, R10, and R14. Group I repeats were comprised of a 100-nucleotide (nt) sequence present in 2 to 10 copies which had 83 to 100% nucleotide identity. Group II repeats were also comprised of a sequence of approximately 100 nt and were present in two to six copies which had 80 to 99% nucleotide identity. Group I and group II repeats had only 62 to 68% nucleotide identity. Repeated sequences in each group contained invariant motifs (e.g., TAAATTTC, AATC, and GCAT in group I repeats; GAGTT, ATGCGT, and GAAATTT in group II repeats) flanked by less-conserved sequences.

DNA repeats are present in the intergenic regions of all fully sequenced IVs; however, their extent, arrangement, localization, and repeated sequence motifs differ between genera (22, 34, 35, 42, 43, 53, 69, 73, 76, 78, 98). In contrast to previous reports, IIV-3 repeated sequences were extensive (20% of the genome), resembling IIV-9 and IIV-16, in which 25% and 39% of the genomes, respectively, are made up of repetitive DNA (10, 58, 89). Although late transcription has been detected from IIV-9 repeated DNA regions, the role of IV DNA repeats is unknown (59). Repeated sequences are known to be involved in genome replication and gene transcription in other DNA viruses. For example, in nucleopolyhedroviruses (NPVs), repetitive DNA sequences function as enhancers of transcription and as origins of genomic DNA replication (3, 74).

Notable IIV-3 genes. (i) Viral transcription and DNA replication. The IIV-3 genome contained several genes with predicted roles in viral transcription and RNA processing, including five RNA polymerase II subunit (Rpb) homologues, and in viral DNA replication, metabolism, and maintenance (Table 2).

IIV3-053L is a novel IV protein with limited similarity to Rpb7, a DNA-dependent RNA polymerase II subunit highly conserved among eukaryotes and having roles in DNA repair, transcription, and RNAi-directed chromatin silencing (18, 21, 56) (Fig. 2). Cellular Rpb7 forms a complex with Rpb4 near the transcript exit groove and the C-terminal domain (CTD) linker region of RNA polymerase II, interacts with various transcription factors and CTD phosphatase FCP1, and is essential for cell viability in yeast (18). IIV3-053L was most similar to Encephalitozoon cuniculi Rpb7 (25% amino acid identity over 142 amino acids) (Fig. 2), particularly in the N-terminal half of the protein and including G64 in the predicted tip loop involved in binding of Rpb7 to the RNA polymerase II core (6). Less conserved was the C-terminal half of IIV3-053L, which lacked 20 aa and a predicted three-stranded antiparallel ß-sheet relative to E. cuniculi Rpb7 (6, 77). IIV3-053L lacked recognizable Rpb7 Pfam signatures, suggesting that it, like homologues in Giardia, Methanosarcina, and mimivirus, is a highly divergent Rpb7 whose function in transcription remains to be determined.


Figure 2
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  		FIG. 2. Multiple amino acid sequence alignment of IIV3-053L with selected Rpb7 proteins. Asterisks, colons, and single dots denote identical, conserved, and semiconserved residues, respectively (74). Rpb7 protein sequence names correspond to the following accession numbers: mosquito (Anopheles gambiae), XM_320916; rat, NM_053948; Saccharomyces cerevisiae, NC_001136; E. cuniculi, NC_003234; mimivirus (Acanthamoeba polyphaga mimivirus), NC_006450.

IIV3-092R had limited similarity with predicted Rpb5-like proteins encoded by African swine fever virus (ASFV), mimivirus, and several IVs. Differences between IIV3-092R and other viral Rpb5-like proteins were greatest in the N-terminal two-thirds of the protein, a region in cellular Rpb5 proposed to interact with transcriptional regulators (85).

IIV3-104L was similar to the FCP1 phosphatase catalytic domain (FCPH), with orthologues in all fully sequenced IVs. FCP1 is responsible for dephosphorylating Rpb1 CTD and, together with specific kinases, driving the transition of RNA polymerase II from the initiation to elongation modes. Like SCP1, a recently described FCP1-like phosphatase with similar roles in transcription regulation, IIV3-104L and IV homologues lack most sequences N-terminal of the FCPH domain, including BRCT and TFIIF binding domains (96). Given the lack of recognizable CTD heptapeptide repeats in IV Rpb1 homologues, IV IIV3-104L-like proteins may conceivably target host Rpb1, a protein which is involved in the nuclear phase of IV transcription (29). Whether IIV3-104L-like proteins represent the IV virion-associated protein (VATT) proposed to modify host RNA polymerase II and to initiate iridoviral immediate-early transcription remains to be determined (92).

IIV3-080R and IIV3-111R contained domains characteristic of MutT-like proteins, including the signature motif [G-X5-E (D in IIV3-080R)-X4-5-C/T-L/A-X-RE-F/L-X-EE-X-G/T] at positions 136 to 157 and 76 to 98, respectively. The MutT (or nudix) domain is found in certain phosphohydrolases which are believed to eliminate toxic nucleotide derivatives and to regulate the levels of signaling nucleotides in both eukaryotes and prokaryotes (57). While IIV3-111R had additional similarity with IIV-6 and LCDV MutT-like proteins, IIV3-080R is a novel IV ORF more similar to poxvirus MutT proteins (28% amino acid identity to variola minor virus F10R over 169 aa) (Table 2). A poxvirus MutT-like protein, vaccinia virus (VV) D10R, is essential for virus infectivity and appears to function as a repressor of host and viral transcription and translation (67). Whether MutT proteins play a similar role in IV remains to be determined.

IIV3-060L had similarity with viral homologues of proliferating cell nuclear antigen (PCNA). While most similar to the Paramecium bursaria chlorella virus (PBCV-1) PCNA-like protein A193L (25% amino acid identity, over 239 aa), IIV3-060L was less similar to PCNA-like proteins encoded by all currently sequenced IVs (17 to 21% amino acid identity) and by NPVs and mimivirus. Cellular PCNA, an acidic protein (pI 4.5) with critical roles in DNA replication and repair, assembles as a ring-shaped homotrimer around dsDNA and is highly conserved among eukaryotes. Nine R and K residues within each monomer confer a net positive electrostatic potential to the central channel, a feature thought to be important for PCNA sliding clamp function (48). In contrast, IV PCNA-like proteins are highly divergent between genera, have predicted pI values ranging from 6.4 (IIV-6) to 9.2 (IIV-3) and, with the exception of conserved residues K37, R102, K168, and K231 in IIV3-060L, contain a pattern and distribution of positive charges distinct from that of cellular PCNA. VV G8R, one of the most divergent viral PCNA-like proteins, is an essential protein that affects late transcription rather than viral DNA replication (46). While the function of IV PCNA-like proteins is unknown, they also may have a novel role during viral replication.

(ii) IIV-3 genes lacking VIV homologues. IIV-3 shared with IIV-6 52 genes absent in VIVs (Fig. 1). These potentially IIV-specific genes encoded several proteins with predicted functions involving DNA replication and maintenance, including DNA topoisomerase IIA (IIV3-086L), Pif1-like SFI helicase (IIV3-106R), NAD-dependent DNA ligase (IIV3-052L), HMGB-like protein (IIV3-068R), and Swib/mdm2 homology domain-containing protein (IIV3-070L), and several predicted to function in protein modification, including OTU-like cysteine protease (IIV3-084L), type 2 metalloprotease (IIV3-095L), and RING finger proteins (IIV3-021L and IIV3-027R). Additionally, a protein containing the BRO motif found in proteins encoded by a range of other insect viruses (IIV3-019R) was identified.

IIV-specific proteins predicted to manipulate DNA contained novel features relative to cellular or viral homologues. IIV3-086L, similar to topoisomerase IIA encoded by other viruses, lacked the C-terminal domain important for nuclear targeting and for interaction with other proteins (50). Similarly, IIV3-106R contained all seven motifs (I, Ia, and II to VI) characteristic of cellular Pif1-like helicases but lacked most of the N-terminal sequences implicated in Pif1/chromatin factor interaction (reviewed in reference 8). The IIV3-052L NAD-dependent DNA ligase contained a putative C-terminal BRCT domain (position 648 to 720) absent in entomopoxviral homologues, suggesting that IIV DNA ligases, similar to cellular homologues, might direct multimeric complex assemblies through this domain (27).

IIV3-068R contained A and B boxes (positions 70 to 138 and 143 to 185, respectively) similar to those found in non-histone chromatin proteins of the high mobility group B (HMGB), proteins that bind and distort DNA and interact with a number of transcription factors and DNA repair and recombination proteins (reviewed in reference 2). IIV HMGB-like proteins lack the acidic tail found in cellular HMGBs. Interestingly, a cellular HMGB mutant lacking the acidic tail exhibits 100-fold-higher DNA binding affinity than the wild-type protein, leading to a block in nucleosome sliding (11). Iridovirus HMGB may conceivable play a structural role in IIV genome conformation.

Several IIV-specific proteins shared similarity or functional motifs with proteins involved in modification of other viral or host proteins. Similar to cellular and viral members of the OTU (for Drosophila "ovarian tumor gene") cysteine protease superfamily, IIV3-084L contained in its C terminus the four conserved motifs of an OTU homology domain (position 596 to 719), including residues in motif I (C601) and motif IV (H718) thought to be important for cleavage (54, 68). IIV3-084L also contained a highly positively charged region (position 137 to 218) and a putative bipartite NLS (position 410), features shared by IIV-1 (Tipula iridescent virus) late protein L96 (35% amino acid identity) and IIV-6 232R (22% amino acid identity) (37). The classification of OTU proteins as cysteine proteases was originally based solely on similarity to the arterivirus NSP2 cysteine protease; however, a number of deubiquitinating enzymes exhibiting proteolytically active OTU domains, including a potent downregulator of NF-{kappa}B, were recently described (7, 12, 54, 86). IIV3-084L may similarly affect ubiquitin-mediated protein degradation to counteract antiviral cellular responses.

IIV3-095L, a protein with homologues in viruses infecting insects, including IIV-6, granuloviruses, and entomopoxviruses, was similar to mammalian type 2 matrix metalloproteinases (MMp). IIV3-095L contained an N-terminal signal peptide, the MMp prodomain consensus sequence (or cysteine switch) (PRCXXPD [position 117 to 123]), the catalytic domain signature (HEXXHXXGXXH [position 275 to 285]), a conserved Met at position 293 (reviewed in reference 62), and RRRR and RTRR motifs similar to the equivalently located RRKR furin cleavage target motif of mammalian MMp, indicating that IIV3-095L is likely activated by furin-mediated proteolysis of the prodomain (81). IIV3-095L lacked predicted transmembrane domains and the hemopexin-like domain which affects substrate recognition in many mammalian MMps. Relative to IIV3-095L, the MMp homologue in IIV-6 (165R) lacks 100 N-terminal amino acids, including the signal peptide, the cysteine switch signature, and the potential furin cleavage site, suggesting differences in compartmentalization and activation mechanisms between IIV MMps. IIV3-095L could conceivably function as is the case for entomopathogenic bacterial proteins, targeting receptors, antimicrobial peptides, or other factors involved in insect responses to pathogens, thus facilitating virus replication. Alternatively, viral MMp could act on the extracellular matrix (e.g., peritrophic matrix of lepidopterous larvae), facilitating virus spread within the host (80). Notably, an MMp-like prodomain is also present in IIV3-114L; however, lack of the prodomain Cys residue critical for MMp function and other MMp features make IIV3-114L an unlikely MMp.

IIV3-021L and IIV3-027R contained C-terminal C3HC4-type RING finger motifs which are characteristic of a diverse array of single-subunit E3 ubiquitin ligases and critical for E3 recruitment of ubiquitin-conjugating enzymes to specific substrates (36). Given the importance of the RING domain in cellular E3 ubiquitin ligase function, its presence in IIV3-021L and IIV3-027R suggests that they too may affect protein ubiquitination to mediate specific virus-host interactions. Notably, the IIV3-021L RING motif (CX2CX4RX5PCXHX3CX2CX9CPXC) exhibits particular similarity to the RING motif present in the E3-like baculovirus inhibitor of apoptosis protein 3 (IAP-3) and is preceded by a single, truncated domain (CXCX10EX5H [position 126 to 145]) similar to the baculovirus IAP repeat (BIR) domain involved in caspase inhibition by viral and mammalian IAPs (60). These features suggest that IIV3-021L may also participate in caspase inhibition to affect apoptosis in the host cell.

IIV3-019R contained an N-terminal baculovirus repeated ORF (BRO)-family homology domain (Bro-N domain, position 24 to 132), 62% identical to the IIV-6 201R Bro-N domain, and included a single-stranded DNA binding motif at position 24 to 57. The C terminus of IIV3-019R, however, is most similar to the C-terminal domains of proteins lacking Bro-N domains (entomopoxvirus AMV207 and AMV209; 27% amino acid identity), consistent with the extensive domain shuffling found in BRO family proteins (40). BRO family proteins are encoded, often in multiple copies, by many invertebrate dsDNA viruses and by bacteriophages. While three BRO genes have been identified in IIV-6, IIV-3, like IIV-31, contains only one (9, 41). Bombyx mori NPV BRO proteins (BRO-a to BRO-e) are expressed early during infection, with BRO-a and BRO-c proteins interacting with chromatin (45, 97). The requirement of BRO proteins for virus replication seems to vary with the specific virus-host system. Disruption of the single BRO gene in the Autographa californica multicapsid NPV did not affect virus replication or virus pathogenicity in instar larvae (9). Conversely, inability to recover B. mori NPV BRO-d deletion mutants suggested a function essential for virus replication (45).

(iii) IIV-3 genes encoding conserved IV domains. IIV3-016R, IIV3-033L, IIV3-056L, and IIV3-107R are proteins of unknown function similar to proteins encoded by all currently sequenced IVs. IIV3-016R contained in the central region of the protein a conserved 90-aa domain which included the motif YXCX8-9GX3NX11-12PCCY (position 502 to 533), also found in entomopoxvirus proteins (MSV063 and AMV105) which have similarity with VV A7L early transcription factor subunit. IIV3-033L contained a putative N-terminal transmembrane domain and a 100-aa domain also present in proteins of unknown function encoded by ascovirus (accession no. CAC19143) and Symbiobacterium thermofilum, an uncultivable bacterium that depends on bacterial commensalism for growth (79). IIV3-056L and IIV3-107R, predicted to contain an N-terminal transmembrane domain, shared with IV homologues a 50-aa domain which included the motifs F/Y-X4-V/I-R-G-X11-A-X2-h-h-X13-14-G-X-P-X-P (position 144 to 187, where "h" is hydrophobic) and R-X5-D-P/F-IRGD-L/V-X-I-X-P-X5-F-X5-P-X3-L-X2-G (position 116 to 151), respectively. While conserved, the functional relevance of these protein domains is unknown.

Comparison between IIV-3 and other IV genomes. IIV-3 overall resembled other IVs in genome size, DNA composition, and gene complement. Data also indicated that among completely sequenced IVs, IIV-3 most closely resembled IIV-6, the only other sequenced IIV. Despite these similarities, IIV-3 contained novel genomic features that indicate its distant relationship to other IV genera and confirm its unique position within the family Iridoviridae.

IIV-3 contained homologues of 27 genes present in all currently sequenced IVs (Fig. 1, red boxes), indicating that these genes play critical and likely essential roles in aspects of IV biology. Approximately half of the conserved genes are involved in viral transcription/DNA replication (IIV3-009R, IIV3-029R, IIV3-048L, IIV3-055R, IIV3-060L, IIV3-076L, IIV3-087L, IIV3-090L, IIV3-101R, IIV3-104L, IIV3-120R, and IIV3-121R). Others encode homologues of protein kinase, MCP, FV3 IE ICP46-like protein, ATPase, and Erv1/Alr-like protein (IIV3-010L, IIV3-014L, IIV3-039R, IIV3-088R, and IIV3-096R, respectively). The functions of nine IIV-3 genes conserved among IVs, including two genes encoding putative transmembrane proteins (IIV3-006R and IIV3-107R), are unknown.

Fifty-two IIV-3 genes have IIV-6 but not VIV counterparts (Fig. 1, blue boxes), suggesting that these genes (IIV-3/IIV-6 genes) function in infection of invertebrate hosts. Although approximately a third of the IIV-3/IIV-6 genes were clustered in three regions in the IIV-3 genome (positions 29876 to 35147, 99860 to 106597, and 143564 to 157359), even these regions lacked discernible colinearity. Only 12 of 52 IIV-3/IIV-6 genes have a predicted function/activity, with 7 genes likely involved in DNA replication/maintenance or gene expression (IIV3-026R, IIV3-052L, IIV3-059L, IIV3-068R, IIV3-070L, IIV3-078R, IIV3-086L, and IIV3-111R), 4 genes encoding protein modification enzymes (IIV3-020R, IIV3-067L, IIV3-095L, and IIV3-098L), and 1 gene encoding an apoptosis regulator (IIV3-021L). Of the IIV-3/IIV-6 genes with unknown function, six encode predicted transmembrane proteins (IIV3-025R, IIV3-037L, IIV3-066L, IIV3-073R, IIV3-085L, and IIV3-112R), three encode Zn finger proteins (IIV3-012R, IIV3-027R and IIV3-034R), and one encodes a Bro-like protein (IIV3-019R). An additional similarity between IIV-3 and IIV-6 was the lack of specific genes present in VIVs, including those encoding DNA methyltransferase and proteins proposed to play roles in evading host responses (e.g., ß-OH steroid oxidoreductase, eIF-2{alpha}, caspase recruitment domain-containing protein). Notably, the only homologue of IIV3-015R is a gene similarly located adjacent to the MCP gene in IIV-22, an IIV infecting the dipteran Simulium variegatum (15).

While certain features of the IIV-3 genome were most similar to IIV-6, others made clear the unique nature of IIV-3 and its distant relationship to IIV-6. These included differences in the nature and extent of repetitive DNA, genome composition, gene colinearity, and gene complement. Thirty-three predicted IIV-3 proteins are not encoded by other IVs (Fig. 1, yellow boxes) and, with the exception of IIV3-044L (protein kinase), IIV3-053L (Rpb7), and IIV3-080R (MutT-like protein), they lacked similarity to any other protein. VIV gene homologues present in either IIV-3 or IIV-6, but not both, include IIV3-007R, which encodes a homologue of the FV-3 IE 31-kDa protein absent in IIV-6, and thymidylate synthase and dUTPase genes, present in IIV-6 and many VIVs but absent in IIV-3. While predicted IIV-3 proteins were most similar to IIV-6 homologues in pairwise comparisons, amino acid identities ranged only from 22 to 53%. Finally, phylogenetic analyses clearly separated IIV-3 from IIV-6 (Fig. 3). Analysis of a large, concatenated protein data set indicated that IIV-3 and IIV-6, while grouping together within the Iridoviridae, have genetic distances comparable to those between other IV genera, consistent with their classification into separate genera (Fig. 3A). Analysis of available IIV MCP data also indicated a clear separation between IIV-3 and IIV-6 and indicated that IIV-3, while unique, was more similar to a group of viruses which included IIV-22, consistent with IIV-3/IIV-22 colinearity at the IIV3-015/016 locus (Fig. 3B). Notably, several other members of this group (IIV-1, IIV-9, IIV-16) are currently classified as members of the genus Iridovirus. Given that here IIV-1, IIV-9, and IIV-16 were as phylogenetically distinct from IIV-6 (genus Iridovirus) as was IIV-3 (genus Chloriridovirus), reconsideration of current IIV taxonomy may be in order (Fig. 3B).


Figure 3
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  		FIG. 3. Phylogenetic analysis of IIV-3 proteins (A) and IIV MCP (B). (A) Eleven conserved IIV-3 proteins (IIV3-009R, IIV3-014L, IIV3-048L, IIV3-055R, IIV3-076L, IIV3-087L, IIV3-088R, IIV3-090L, IIV3-101R, IIV3-120R, and IIV3-121R) were concatenated and aligned with similar data sets from other iridoviruses by using Kalign. The unrooted tree for 9,190 aligned characters was generated by the maximum likelihood tree search strategy, including the WAG model for correction for multiple substitutions, the four-category discrete gamma model for correction for among-site rate variation, 100 bootstrap replicates, and default settings, as implemented in Phyml. Bootstrap values greater than 70 are indicated at the appropriate nodes, and dots indicate values of 100. Sequences from the following viruses and accession numbers were compared: IIV-3, DQ643392; IIV-6, AF303741; LCDV-1, L63545; LCDV-C, AY380826; GIV, AY666015; SGIV, AY521625; FV-3, AY548484; ATV, AY150217; TFV, AF389451; ISKNV, AF371960; RBIV (strain KOR-TY1), AY532606; OSGIV, AY894343. (B) IIV3-014L was aligned with available IIV MCP sequences and LCDV MCP as an outgroup with Clustal, and the tree was generated as described above. Sequences from the following viruses and accession numbers were compared: IIV-24, AF042340; IIV-30, AF042336; IIV-29, AF042339; AgIV, AF042343; IIV-2, AF042335; IIV-9, AF025774; IIV-23, AF042342; IIV-22, M32799; IIV-1, M33542; IIV-16, AF025775; IIV-3, DQ643392; IIV-31, AF042337; PjIV, AF042338; IIV-6, AF303741; LCDV, AY849392. Scales indicate estimated distances. Similar topologies were obtained with maximum likelihood as implemented in MRBAYES and/or TREE-PUZZLE and with neighbor-joining and maximum-parsimony algorithms as implemented in PHYLO_WIN.

Conclusions. The genome sequence of IIV-3, sole member of the genus Chloriridovirus, has been determined. IIV-3 contains a number of novel genes which are likely important for infection of the natural mosquito host. Comparison between IIV-3, IIV-6, and available non-IIV-6 IIV gene sequences suggests that diversity within IIV may be greater than previously recognized. Genes common to all four IV genera, which include those encoding enzymes involved in RNA transcription, DNA replication, and protein processing, likely define the genetic core of the family Iridoviridae.


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ACKNOWLEDGMENTS
 
We thank A. Lakowitz and A. Zsak for providing excellent technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Ave., Urbana, IL 61802. Phone: (217) 244-3989. Fax: (217) 244-7421. E-mail: gadelhon{at}uiuc.edu. Back


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Journal of Virology, September 2006, p. 8439-8449, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00464-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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