Virology 286, 182–196 (2001) doi:10.1006/viro.2001.0963, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Analysis of the First Complete DNA Sequence of an Invertebrate Iridovirus: Coding Strategy of the Genome of Chilo Iridescent Virus 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 Iridoviridae 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 polymerase, DNA polymerase, protein kinase, thymidine and thymidylate kinase, thymidylate synthase, 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 fish (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 viruses. © 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, Chloriridovirus, Lymphocystivirus, and stranded DNA molecule (212 kb), which was found to be Ranavirus (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 enzymes involved