Virology 286, 182–196 (2001) doi:10.1006/viro.2001.0963, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Analysis of the First Complete DNA Sequence of an Invertebrate Iridovirus: Coding Strategy of the of Chilo Iridescent

Nurith J. Jakob, Kristin Mu¨ller, Udo Bahr, and Gholamreza Darai1

Institut fu¨r Medizinische Virologie, Universita¨t Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Federal Republic of Germany Received February 27, 2001; returned to author for revision March 20, 2001; accepted April 18, 2001

Chilo iridescent virus (CIV), the type species of the genus Iridovirus, a member of the family, is highly pathogenic for a variety of insect larvae. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity and strategy of the CIV genome was elucidated by the analysis of the complete DNA nucleotide sequence of the viral genome (212,482 bp) using cycle sequencing by primer walking technology. Both DNA strands were sequenced independently and the average redundancy for each nucleotide was found to be 1.85. The base composition of the viral genomic DNA sequence was found to be 71.37% AϩT and 28.63% GϩC. The CIV genome contains 468 open reading frames (ORFs). The size of the individual viral gene products ranges between 40 and 2432 amino acids. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs were nonoverlapping. The comparison of the deduced amino acid sequences to entries in protein data banks led to the identification of several genes with significant homologies, such as the two major subunits of the DNA-dependent RNA , DNA polymerase, protein , thymidine and , , ribonucleoside- diphosphate reductase, major capsid protein, and others. The highest homologies were detected between putative viral gene products of CIV and lymphocystis disease virus of (LCDV). Although many CIV putative gene products showed significant homologies to the corresponding viral proteins of LCDV, no colinearity was detected when the coding strategies of the CIV and LCDV-1 were compared to each other. An intriguing result was the detection of a viral peptide of 53 amino acid residues (ORF 160L) showing high homology (identity/similarity: 60.0%/30.0%) to sillucin, an antibiotic peptide encoded by Rhizomucor pusillus. Iridovirus homologs of cellular genes possess particular implications for the molecular evolution of large DNA . © 2001 Academic Press Key Words: cytoplasmic DNA viruses; Iridoviridae; Chilo iridescent virus; insect iridescent virus type 6; DNA nucleotide and amino acid sequence alignment; computer analysis; protein alignment.

INTRODUCTION considered a potential agent for biological control, since these viruses cause lethal infections in important pest Iridoviruses are large icosahedral cytoplasmic de- insect species (Kleespies et al., 1999; Hernandez et al., oxyriboviruses. A major characteristic of iridoviruses is their large icosahedral capsid and their replication and 2000). An advantage of CIV is that it can be easily grown assembly in cytoplasm. Members of the Iridoviridae fam- and propagated in cultured Choristoneura fumiferana ily have been isolated from poikilothermic animals, in- (CF-124) cells (Cerutti et al., 1981). The icosahedral virus cluding various insects and some cold-blooded verte- particle of CIV comprises a capsid, intermediate lipid brates. Iridoviruses are subdivided into four genera in- layer, and viral genome with a single linear double- cluding Iridovirus, , , and stranded DNA molecule (212 kb), which was found to be (van Regenmortel et al., 2000). The type spe- circularly permuted and terminally redundant (Delius et cies for the genus Iridovirus is Chilo iridescent virus al., 1984; Schnitzler et al., 1987; Soltau et al., 1987; (CIV); an alternative name is insect iridescent virus type Fischer et al., 1990). This genomic feature is common to 6. CIV was isolated from the stem-boring lepidopteran other iridoviruses too, such as frog virus 3 (FV3, type Chilo suppressalis (rice stem borer; Fukaya and Nasu, species of the genus Ranavirus; Goorha and Murti, 1982), 1966). As to biological relevance CIV is of particular lymphocystis disease virus (LCDV, type species of the economical and ecological importance, since it infects a genus Lymphocystivirus; Darai et al., 1983, 1985; Schnit- number of herbivore insects that cause immense dam- zler et al., 1990), and insect iridescent virus type 9 (Ward ages in agriculture, e.g., rice farming and stone fruits and Kalmakoff, 1987). Further biological and genomic (Smith, 1976). Consequently this insect iridovirus can be properties of CIV such as the virion structure, viral pro- teins, enzymatic activities (Cerutti and Devauchelle, 1980, Cerutti et al., 1981; Darai, 1982, 1985; and 1990), the 1 To whom correspondence and reprint requests should be ad- positions of at least six origins of replication, and repet- dressed. Fax: ϩ49-6221-56-4104. E-mail: [email protected]. itive DNA elements (Fischer et al., 1988a,b; Handermann

0042-6822/01 $35.00 Copyright © 2001 by Academic Press 182 All rights of reproduction in any form reserved. ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 183 et al., 1992; Sonntag and Darai, 1992; Stohwasser et al., nucleotide from each strand was determined with an 1993; Schnitzler et al., 1994a,b; Tidona and Darai, 1997) average redundancy of 1.85. The base composition of the have been reported. Furthermore some important viral viral genomic DNA sequence was found to be 71.37% genes have been identified and analyzed, including the AϩT and 28.63% GϩC, which is in agreement with val- major capsid protein, an evolutionarily highly conserved ues described previously (Delius et al., 1984). The com- viral protein, the largest subunit of the DNA-dependent plete DNA sequence was deposited with GenBank (Ac- RNA polymerase, the ATPase, and the DNA polymerase cession No. AF303741). The analysis of the viral DNA (Stohwasser et al., 1993; Schnitzler et al., 1994a,b; revealed the presence of numerous short direct, in- Sonntag et al., 1994a,b; Bahr et al., 1997; Tidona et al., verted, and palindromic repetitive DNA sequences. In 1998; Mu¨ller et al., 1999). addition three clusters of large repetitive DNA elements Recently we determined the complete DNA sequence were detected within the DNA sequences of the EcoRI of LCDV-1 (Tidona and Darai, 1997), which is the caus- CIV DNA fragments H and C (261R; 396L, 443R). The ative agent of lymphocystis disease in fish and belongs positions of two repetitive DNA elements are in agree- to the genus Lymphocystivirus within the Iridoviridae ment with the data reported previously (Fischer et al., family. This was the first complete DNA sequence of an 1988a,b, 1990). iridovirus genome. Several viral gene products with sig- nificant homologies to entries in protein data banks were Coding capacity of the CIV genome identified. However, a handicap in this analysis was the Computer-assisted analysis of the DNA sequence of lack of sequence information of other iridoviruses. The the CIV genome revealed the presence of 468 potential goal of the present study was the analysis of the coding open reading frames (ORFs) with coding capacities for capacity and strategy of the genome of this ecological polypeptides ranging from 40 to 2432 amino acids. The important insect iridovirus—Chilo iridescent virus—by analysis of the coding capacity of the CIV genome re- elucidation of the primary structure of the viral genome. vealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were nonoverlapping. The properties of 215 po- RESULTS AND DISCUSSION tential viral ORFs (Ͼ150 nucleotides), including 8 over- lapping ORFs, that were detected within the DNA nucle- Integrity of the viral genome organization otide sequence of the upper (R) and lower (L) DNA The organization of the viral genome and the integrity strands of CIV genome are summarized in Table 1. The of the genomic library of CIV (Schnitzler et al., 1987; classical or slightly modified canonical promoters and Soltau et al., 1987) were determined by PCR analysis and termination signals were found upstream and down- determination of the DNA nucleotide sequences of the stream of the start and termination codons of the majority terminal regions of the cloned EcoRI CIV DNA fragments of the individual viral ORFs. However, due to the high AT (Bahr et al., 1997). Based on the data obtained from the content of the viral DNA molecule the identification of the analysis of the DNA nucleotide sequence of the individ- AT-rich transcriptional signals of individual viral ORFs is ual EcoRI DNA fragments further DNA oligonucleotide difficult. primers were constructed and used for amplification of the corresponding region of the viral genome by PCR. Relatedness of CIV gene products to other proteins These studies revealed that each PCR product pos- In analogy to other large DNA viruses of eukaryotes it sesses the corresponding EcoRI restriction site flanked was found that iridoviruses encode a number of cellular by adjacent EcoRI DNA fragments as determined by protein homologs. The majority of these proteins repre- physical mapping of the viral genome (Schnitzler et al., sent orthologues of cellular involved in tran- 1987; Soltau et al., 1987; Fischer et al., 1990; Bahr et al., scription, replication, and nucleotide metabolism. Impli- 1997). The results of this study confirmed the integrity of cations of iridovirus homologs of cellular genes during the viral genome arrangement. the molecular evolution of large DNA viruses are of particular importance (Tidona and Darai, 2000). The iden- Determination of the viral genomic DNA sequence tified putative gene products of CIV showed significant The complete DNA nucleotide sequence of the CIV homology to proteins in databases, including enzymes DNA molecule (212,482 bp) was determined using a and structural proteins involved in virus replication, tran- primer walking strategy. More than 1200 DNA oligonu- scription, protein modification, and virus–host interaction cleotide primers were designed, synthesized, and used (Table 1 and Fig. 1). Furthermore several conserved in sequencing reactions to obtain contiguous DNA nu- protein domains and signatures were detected in puta- cleotide sequences from both viral DNA strands. The tive viral gene products as shown in Table 1. average read length obtained from individual sequenc- Enzymes that are involved in DNA replication are evo- ing reactions ranged from 550 to 950 nucleotides. Both lutionarily important gene products that are present in DNA strands were sequenced independently and each prokaryotes and eukaryotes as well as in large DNA TABLE 1 Properties of Selected Open Reading Frames Detected within the CIV Genome

Note. ORFs (coding for putative polypeptides & gt; 50 amino acid residues) are listed from nucleotide position 1 to 212,482. ORFs marked with an asterisk (*) are overlapping. ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 185

TABLE 1—Continued 186 JAKOB ET AL.

TABLE 1—Continued ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 187

TABLE 1—Continued 188 JAKOB ET AL.

TABLE 1—Continued ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 189

TABLE 1—Continued

viruses, like iridoviruses, poxviruses, and herpesviruses. Sonntag and Darai, 1995). CIV encodes putative proteins This is true for cytoplasmic DNA viruses such as iridovi- for viral transcription such as the largest subunit of the ruses as they have no direct access to cellular DNA DNA-dependent RNA polymerase (176R; Schnitzler et al., replication enzymes that are almost exclusively located 1994b; Sonntag and Darai, 1995) and five small subunits in the nucleus of the cell (Carroll and Moss, 1997; Moss of the DNA-dependent RNA polymerase (107L, 343L, et al., 1996). Consequently these viruses depend on their 349L, 428L, and 454R). The CIV putative gene product of own replication machinery, representing a minimal solu- ORF 343L showed homology to an evolutionarily impor- tion for efficient replication of large DNA molecules. tant DNA-directed RNA polymerase subunit AЉ (Koonin et Proteins involved in DNA replication, modification, and al., 2001). The phylogenetic tree derived from the com- processing like the DNA polymerase (037L), two exo- parison of the amino acid sequence of the 343L gene nucleases (012L and 244L), a topoisomerase II (045L), product and the proteins of DNA-directed RNA polymer- and an endonuclease homolog (369L) were identified ase subunit AЉ of different species is shown in Fig. 2A (Mu¨ller et al., 1999). and the corresponding alignment of the two conserved DNA-dependent RNA polymerase subunits are re- domains of this viral protein is given in Fig. 2B. With the quired for replication of cytoplasmic DNA viruses like exception of a CIV putative gene product, 142R, that is a vaccina virus and iridoviruses (Jones et al., 1987; homolog of the double-stranded RNA-specific ribonucle- 190 JAKOB ET AL.

to the of different species and particu- larly to those of LCDV (136R) and channel catfish virus (Davison, 1992). The alignment of the amino acid resi- dues of the thymidylate of CIV (251L) and variola-, vaccinia, and African swine fever virus (Koonin and Senkevich, 1992; Aguado et al., 1992; Smith et al., 1989; Yanez et al., 1993) is shown in Fig. 3A and the phylogenetic tree derived from the comparison of the amino acid sequence of the 251L protein and the corre- sponding protein of other species is shown in Fig. 3B. The data indicate that the CIV gene product of 251L shows closer phylogenetic relatedness to eukaryotic thy- midylate kinases. Several putative gene products of CIV contain highly conserved domains and motifs of enzymes involved in protein processing and modification such as two cysteine proteinases (224L, 361L) and three protein kinases (123R, 380R, and 389L) which showed significant homology to members of the serine/threonine and ty- rosine families (Traktman et al., 1989). The identification of two high-mobility-group (195L and 401R) homologs encoded by CIV is of particular interest, since these viral gene products may be involved in the process of DNA binding and packaging (Koonin, 1993). Baculoviruses encode small zinc finger proteins that are able to block apoptosis induced by viral infection (Crook et al., 1993). As shown in Table 1 and Fig. 1 CIV putative proteins 157L, 193R, and 332L show significant homology to the apoptosis inhibitors (iap) of baculovi- ruses (Crook et al., 1993). Although the function of the CIV proteins remains to be elucidated it can be assumed FIG. 1. Graphic representation of the homologies of CIV gene prod- that this class of small zinc finger proteins may play an ucts that are grouped according to their possible function or related- important role in blocking apoptosis during infection by ness to other species. Single groups are marked by distinct colors. different insect viruses. Another baculovirus protein that shows homology to a putative CIV protein (172L) is the ase (Srivastava and Srivastava, 1996), there is no further global transactivator of Autographa californica nuclear significant evidence for the presence of enzymes in- polyhedrosis virus. Global transactivators are a class of volved in the processing of viral RNA. non-DNA-binding proteins that have been implicated in The detection of a putative gene product of CIV (414L) the regulation of homeotic genes (Tamkun et al., 1992). that contains the bacterial mutator protein signature (Miller and Michaels, 1996) is of particular interest, since Relatedness of CIV gene products to LCDV-1 other large cytoplasmic deoxyriboviruses such as LCDV (022R; Tidona and Darai, 1997), African swine fever virus High homologies were detected between putative (g5R), and vaccinia virus (D9 and D10) encode function- gene products of CIV and LCDV (Tidona and Darai, 1997) ally similar proteins (Koonin, 1993). The alignment of the as shown in Fig. 1. This includes the DNA polymerase amino acid residues of the conserved domain of the (037L), the viral ATPase (075L), the large chain of the bacterial mutator mutT sequence of the corresponding ribonucleoside-diphosphate reductase (085L and 376L), viruses is illustrated in Fig. 2C. the ribonuclease (142R), and a putative protein (143R) CIV codes for a number of proteins that interfere with showing similarities to the putative thymidine kinase of the nucleic acid metabolism of the host cell, i.e., homol- LCDV. An alignment of the protein sequences of the ogous proteins: two subunits of ribonucleoside-diphos- ATPase from CIV, LCDV, and frog virus 3 is shown in Fig. phate reductase (085L and 376L), thymidylate synthase 4. In addition the largest subunit of the DNA-dependent (225R) (Mu¨ller et al., 1998), thymidylate kinase (251L), RNA polymerase (176R), two cysteine proteinases (224L nucleoside-triphosphatase (022L), and thioredoxin and 361L), a protein showing homology to a putative (453L). The CIV putative protein 143R shows similarities LCDV DNA puff II/9-2 (DNA-binding) protein precursor ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 191

FIG. 2. The phylogenetic tree (A) based on the alignment of the DNA-directed RNA polymerase subunit AЉ amino acid sequences of 11 species including CIV (RPA2_CIV) (ORF 343L) with Halobacterium halobium (RPA2_HALHA), Halococcus morrhuae (RPA2_HALMO), Archaeoglobus fulgidus (RPA2_ARCFU), Thermoplasma acidophilium (RPA2_THEAC), Methanobacterium thermoautotrophicum (RPA2_METTW), Methanococcus vannielii (RPA2_METVA), Pyrococcus abyssi (RPA2_PYCAB), Thermococcus celer (RPA2_THECE), Sulfolobus acidocalcarius (RPA2_SULAC), and Aeropyrum pernix (RPA2_AERPN). The distances are proportional to the relative sequence deviations between individual amino acid sequences. The phylogenetic analyses were carried out with the CLUSTAL, PROTDIST, and KITSCH programs (Bairoch, 1991). (B) The alignment of the two conserved domains of the amino acid sequences of the DNA-directed RNA polymerase subunit AЉ of 11 species including CIV. (C) The alignment of the amino acid residues of the conserved domains of the bacterial mutT protein of Chilo iridescent virus (414L), lymphocystis disease virus (022R, L 63545), African swine fever virus (g5R, CAA50807), and vaccinia virus (D9, P04311 and D10, P04312). A gray shaded box in the corresponding region of the amino acid sequence indicates the position of the mutT domain signature. Bold letters and asterisks denote identical amino acids. One dot indicates the positions of weakly conserved amino acids. Dots indicate the positions of well-conserved amino acids.

(287R), and a structure-specific endonuclease (369L) FV3 (Beckman et al., 1988). Further homologies were were identified. detected between CIV and LCDV gene products, includ- CIV putative gene product 380R shows significant ho- ing CIV putative gene products 414L, 428L, and 453L, mology to three protein kinases of LCDV (010L, 080R, and which are homologous to the bacterial mutator mutT 088R) which showed significant homology to members of protein; the DNA-directed RNA polymerase II, subunit 2; the serine/threonine and tyrosine protein kinase families, and a thioredoxin, respectively. This indicates that these and it has been reported previously that they showed conserved gene products seem to be iridovirus-specific internal amino acid sequence homologies, when their proteins that are common to vertebrate and insect iri- amino acid sequences were compared to one another doviruses. (Tidona and Darai, 1997). CIV 393L shows homology to In conclusion, the comparative analysis of CIV and LCDV 047L and the immediate-early protein ICP-46 of LCDV revealed that 183 ORFs of CIV putative FIG. 3. (A) Alignment of the amino acid sequences of the thymidylate kinases of CIV (KTHY_CIV), variola virus (KTHY_VARV), vaccinia virus (KTHY_VACCV), and African swine fever virus (KTHY_ASFB7). Bold letters and asterisks denote identical amino acids. One dot indicates the positions of weakly conserved amino acids. Two dots indicate the positions of well-conserved amino acids. Dashes indicate artificial gaps that have been introduced to achieve maximal amino acid matching. The position of the ATP/GTP (P-loop) of the protein is indicated by a box with a gray shadow in the corresponding region of the amino acid sequence. (B) Phylogenetic tree based on the amino acid sequence alignment of the thymidylate kinase of 30 species, including CIV (KTHY_CIV), Archaeoglobus fulgidus (KTHY_ARCFU), Thermotega maritima (KTHY_THEMA), Sulfolobus solfutarius (KTHY_SULSO), Ureaplasma urealyticum (KTHY_UREA), Aeropyrum pernix (KTHY_AERPE), variola virus (KTHY_VARV), vaccinia virus (KTHY_VACCV), Mycobacterium tuberculosis (KTHY_MYCTU), African swine fever virus (KTHY_ASFB7), Caenohabditis elegans (KTHY_CAEEL), Mus musculus (KTHY_MOUSE), Homosapiens sapiens (KTHY_HUMAN), Schizosaccharomyces pombe (KTHY_SCHPO), Saccharomyces cervisiae (KTHY_YEAST), Chlamydia trachomatis (KTHY_CHLTR), Chlamydia pneumonia (KTHY_CHLPN), Neisseria meningitis (KTHY_NEIME?), Deinococcus radiodurans (KTHY_DEIRA), Haemophilus influenzae (KTHY_HAEIN), Yersinia pestis (KTHY_YERPE), Escherichia coli (KTHY_ECOLI), Bacillus subtilis (KTHY_BACSU), Methanococcus jannischii (KTHY_METJA), Aquifex aeolicus (KTHY_AQUAE), Rickettsia prowazekii (KTHY_RICPR), Methanobacterium thermoautotrophicum (KTHY_METTW), Mycoplasma pneumoniae (KTHY_MYCPN), Mycoplasma genitalium (KTHY_MYCGE), and Helicobacter pylori (KTHY_HELPJ). The distances are proportional to the relative sequence deviations between individual amino acid sequences. The phylogenetic analyses were carried out with the CLUSTAL, PROTDIST, and KITSCH programs (Bairoch, 1991).

192 ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 193

FIG. 4. Alignment of the amino acid sequences of the ATPase of frog virus 3 (M80551), lymphocystis disease virus (054R), and CIV (075L). Bold letters and asterisks denote identical amino acids. One dot indicates the positions of weakly conserved amino acids. Two dots indicate the positions of well-conserved amino acids. Dashes indicate artificial gaps that have been introduced to achieve maximal amino acid matching. The position of the ATP/GTP binding site of the protein is indicated by a box with gray shadow. gene products possess LCDV homologous proteins, in- Asp. According to our knowledge CIV-VAB is the first cluding 125 small polypeptides (40 to 60 amino acid discovered viral peptide that shows high homology to a residues). Twenty-four of 125 small viral ORFs were known antibiotic peptide. The transcriptional activity of found to be nonoverlapping. However, no colinearity was the CIV-VAB gene was determined using RT-PCR. Under detected when the coding strategies of the CIV and the conditions described under Materials and Methods LCDV-1 were compared to each other. and using two DNA oligonucleotide primers that corre- spond to the 5Ј and 3Ј termini of the CIV-VAB gene (160L), CIV encodes for an antibiotic peptide a DNA fragment of 176 bp was amplified from RNA of An intriguing result of the analysis of the relatedness CIV-infected cells (Fig. 5B, lane 2). The subsequent de- of the individual CIV putative gene products was the termination of the DNA nucleotide sequence of the RT- detection of a viral peptide of 53 residues (ORF 160L) PCR product revealed 100% identity to the DNA nucleo- showing high homology to sillucin, an antibiotic peptide tide sequence of the CIV ORF 160L (data not shown). against gram-positive bacteria encoded by Rhizomucor This indicates that the CIV-VAB gene is transcribed in pusillus (Bradley and Somkuti, 1979). The sequence of infected cells. The fate and functional role of this viral this CIV peptide, termed CIV-VAB, is shown in Fig. 5. peptide during viral replication in vivo is still unknown Sillucin is 30 amino acids in size, containing 8 Cys and needs further investigation. However, it is of partic- residues. CIV-VAB contains an N-terminal secretory sig- ular interest to determine whether the presence of the nal (aa 1–23) and a potential secreted peptide (aa 24– VAB gene in the genome of CIV is a unique event specific 53). It is remarkable that 18 residues of the C-terminal to Chilo iridescent virus or it is a common feature of the part of the CIV-VAB (aa 24–53), including 7 Cys, are members of the iridovirus genera. identical (60%) compared to sillucin. The percentage of A major dilemma in this analysis was the lack of the well-conserved amino acids of the CIV-VAB was sequence information of other iridoviruses. Accordingly found to be 30%. As shown in Fig. 5A the 16th amino acid the determination of the genomic DNA sequence of other residue of the sillucin (indicated by X) is unknown (Brad- iridoviruses is of particular importance since it makes it ley and Somkuti, 1979). This fact and the lack of genetic possible to identify conserved iridovirus-specific pro- information corresponding to the N-terminal amino acid teins and similarities between the coding strategies of composition of sillucin are due to the method used to different iridovirus species. The elucidation of the coding identify the amino acid composition of sillucin, which strategy of the complete genome of Chilo iridescent was elucidated by the Edman procedure. Therefore the virus, the type species of the Iridoviridae, is a milestone amino acid position 16 of sillucin can be either Asn or and straightforward prerequisite for the genetics and 194 JAKOB ET AL.

FIG. 5. (A) Alignment of the gene product of CIV ORF 160L (CIV-VAB) showing high homology to the amino acids of sillucin, an antimicrobial peptide of Rhizomucor pusillus (Bradley and Somkuti, 1979). Asterisks denote identical amino acids. Dots indicate the positions of well-conserved amino acids. Dashes indicate artificial gaps that have been introduced to achieve maximal amino acid matching. (B) Polyacrylamide gel electrophoresis of the RT-PCR products using total RNA obtained from CIV-infected cells and the oligonucleotide primers CIV-AB-FT and CIV-AB-RT (for details see Materials and Methods). Lane 1 served as negative control in which the was performed without reverse transcription. Lane 2 harbors the product of the RT-PCR (176 bp). Lane 3 served as negative control in which prior to cDNA synthesis the total RNA of CIV-infected cells was treated with DNase and RNase. Lane M contains the molecular weight marker (100 bp ladder; Pharmacia Biotech, Germany).

of these viruses. The determination of isolation, incubation, and electrophoresis were carried the CIV primary structure opens new paths to gain in- out as described previously (Schnitzler et al., 1987). sight into the functions of the different CIV gene products and will be of great help to understand the ecology and Polymerase chain reaction pathogenicity of iridoviruses. Oligonucleotide primers were synthesized with an Oligo 1000 M DNA synthesizer (Beckman Instruments MATERIALS AND METHODS GmbH, Munich, Germany). The PCR was performed us- ␮ Virus and cells ing 0.5 fmol of the individual template DNA in 100- l volumes containing 80 mM Tris–HCl, pH 8.9, 20 mM CIV was propagated in Cho. fumiferana CF-124 cell (NH4)2SO4,5mMMgCl2, 12.5 nmol of each dNTP, 5 pmol cultures as described previously (Fukaya and Nasu, of each primer, and 2.5 units of Taq DNA polymerase 1966; Delius et al., 1984). (Applied Biosystems GmbH, Weiterstadt, Germany). Thirty-five cycles were run in an automated temperature Viral DNA isolates and genomic library cycling reactor (GeneE; Techne, Cambridge, UK) under The recombinant plasmids harboring specific DNA cycling conditions of 96°C for 30 s, 55°C for 1 min, and sequences of the CIV genome used in this study were 72°C for 2 min per cycle. For RT-PCR total cellular RNA obtained from a defined gene library, which was estab- was extracted from infected cells. CF-124 cells were lished and described elsewhere (Schnitzler et al., 1987; infected with CIV at the m.o.i. of 10 PFU/cell. The total Soltau et al., 1987). CIV DNA used for polymerase chain cellular RNA was extracted 48 h after infection using the reactions were obtained from original virus stocks guanidinium/cesium chloride method as described pre- (Schnitzler et al., 1987). viously (Glisin et al., 1974; Ullrich et al., 1977). Two oligo- nucleotide primers were synthesized corresponding to DNA nucleotide positions 65228–65195 [5Ј-ccccccAT- Enzymes and DNA isolation GTCGATATTACTTAAAATTTTATTTAATTG-3Ј; CIV-AB-FT Restriction endonucleases were purchased from (38-mer)] and 65070–65035 [5Ј-CACACTAGTTACGCTA- Roche Diagnostics GmbH (Mannheim, Germany). DNA TAATTTATAAGTACAATTccctaggg-3Ј; CIV-AB-RT (42-mer)] ANALYSIS OF THE GENOME OF Chilo IRIDESCENT VIRUS 195 of the viral genome that correspond to DNA nucleotide iridescent virus between the genome coordinates 0.101 and 0.391: positions 1 to 33 and 129 to 162 (including TAA stop Similarities in coding strategy between insect and vertebrate iridovi- codon) of the CIV-VAB gene (160L). The reverse transcrip- ruses. Virus Genes 17, 59–66. Bairoch, A. (1991). PROSITE: A dictionary of sites and patterns in tion step was performed using the commercially avail- proteins. Nucleic Acids Res. 19, 2241–2245. able RNA LA PCR Kit Ver. 1.1 (Takara Shuzo Co., Japan). Beckman, W., Tham, T. N., Aubertin, A. M., and Willis, D. B. (1988). Structure and regulation of the immediate early frog virus 3 gene that DNA sequencing encodes ICR489, an animal DNA virus, is circularly permuted and terminally redundant. J. Virol. 62, 1271–1277. The DNA templates and PCR products were prepared Bradley, W. A., and Somkuti, G. A. (1979). The primary structure of as described previously (Tidona and Darai, 1997). The sillucin and antimicrobial peptide from Mucor pusillus. FEBS Lett. 97, PCR products and the cloned fragments of the viral 81–83. genomic DNA were automatically sequenced (Smith et Carroll, M. W., and Moss, B. (1997). Poxviruses as expression vectors. al., 1986) with a 373A Extended DNA sequencer using Curr. Opin. Biotechnol. 8, 573–577. Cerutti, M., and Devauchelle, G. (1980). Inhibition of macromolecular the DyeDeoxy terminator–Taq cycle sequencing tech- synthesis in cells infected with an invertebrate virus (iridovirus type nique (Applied Biosystems GmbH). Sequencing reac- 6 or CIV). Arch. Virol. 63, 297–303. tions were performed as described previously (Tidona Cerutti, M., Gue´rillon, J., Arella, M., and Devauchelle, G. (1981). [Repli- and Darai, 1997). The nucleotide sequences obtained cation of type 6 iridovirus in various cell lines.] C. R. Seances Acad. from individual sequencing reactions were assembled Sci. III 292, 797–802. Cerutti, M., and Devauchelle, G. (1982). Isolation and reconstitution of using the Sequence Navigator software (Version 1.0, Ap- chilo iridescent virus membrane. Arch. Virol. 74, 145–155. plied Biosystems GmbH). Cerutti, M., and Devauchelle, G. (1985). Characterization and localiza- tion of CIV polypeptides. Virology 145, 123–131. Computer-assisted analysis Cerutti, M., and Devauchelle, G. (1990). 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M. Medicine (Bethesda, MD). Amino acid sequences were (1985). Molecular cloning and physical mapping of the genome of scanned for known active site motifs and fish lymphocystis disease virus. Virology 146, 292–301. signatures (PROSITE 13.0; Bairoch, 1991), N-terminal se- Davison, A. J. (1992). Channel catfish virus: A new type of herpesvirus. cretory signal sequences (PSIGNAL; Von Heijne, 1986), Virology 186, 9–14. ␣ Delius, H., Darai, G., and Flu¨gel, R. M. (1984). DNA analysis of insect and transmembrane -helices (HELIXMEM; Eisenberg et iridescent virus 6: Evidence for circular permutation and terminal al., 1984). The search for homologous proteins was car- redundancy. J. Virol. 49, 609–614. ried out using the FASTA and FSTPSCAN (Lipman and Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R. (1984). Analysis Pearson, 1985) programs. Protein alignments were gen- of membrane and surface protein sequences with the hydrophobic erated using the CLUSTAL program (Higgins and Sharp, moment plot. J. Mol. Biol. 179, 125–142. Fischer, M., Schnitzler, P., Delius, H., and Darai, G. (1988a). Identifica- 1988). Significant homology between two proteins was tion and characterization of the repetitive DNA element in the ge- defined as amino acid identity of over 20.0% or amino nome of insect iridescent virus type 6. Virology 167, 485–496. acid identity of over 15.0% when conserved protein do- Fischer, M., Schnitzler, P., Scholz, J., Ro¨sen-Wolff, A., Delius, H., and mains and signatures were present in both proteins. Darai, G. (1988b). DNA nucleotide sequence analysis of the PvuII DNA fragment L of the genome of insect iridescent virus type 6 ACKNOWLEDGMENTS reveals a complex cluster of multiple tandem, overlapping, and interdigitated repetitive DNA elements. Virology 167, 497–506. 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