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Identification and Characterization of a DNA Photolyase-Containing Baculovirus from Chrysodeixis Chalcites $

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Identification and Characterization of a DNA Photolyase-Containing Baculovirus from Chrysodeixis Chalcites $ View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Virology 330 (2004) 460–470 www.elsevier.com/locate/yviro Identification and characterization of a DNA photolyase-containing baculovirus from Chrysodeixis chalcites $ Monique M. van Oersa,*, Elisabeth A. Hernioub, Magda Usmanya, Gerben J. Messelinkc, Just M. Vlaka aLaboratory of Virology, Wageningen University, 6709 PD Wageningen, The Netherlands bDepartment of Biological Sciences, Imperial College London, Ascot Berkshire SL5 7PY, United Kingdom cApplied Plant Research, Business Unit Glasshouse Horticulture, 2670 AA Naaldwijk, The Netherlands Received 27 August 2004; returned to author for revision 20 September 2004; accepted 22 September 2004 Abstract A hitherto unknown single nucleocapsid nucleopolyhedrovirus (SNPV) with a unique property was isolated from larvae of the looper Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). Polyhedrin, lef-8, and pif-2 gene sequences were obtained by PCR with degenerate primers and used for phylogenetic analysis. ChchNPV belonged to class II NPVs and its polyhedrin sequence was most similar to that of class II NPVs of other members of the subfamily Plusiinae. Further genetic characterization involved the random cloning of HindIII fragments into a plasmid vector and analysis by end-in sequencing. A gene so far unique to baculoviruses was identified, which encodes a putative DNA repair enzyme: cyclobutane pyrimidine dimer (CPD) DNA photolyase (dpl). The transcriptional activity of this gene was demonstrated in both ChchNPV-infected C. chalcites larvae and infected Trichoplusia ni High Five cells by RT-PCR and 5Vand 3VRACE analysis. The possible role of this gene in the biology of the virus is discussed. D 2004 Elsevier Inc. All rights reserved. Keywords: Baculovirus; Chrysodeixis chalcites; CPD DNA photolyase; Phylogeny Introduction (IJkel et al., 2000; Pearson et al., 2000). NPV virions occluded in polyhedra may contain single nucleocapsids The Baculoviridae form a family of large circular (SNPVs) or multiple nucleocapsids (MNPVs). dsDNA viruses, pathogenic for insects. The baculoviruses To date, the genome sequences of over 23 baculoviruses are divided into the genera Nucleopolyhedrovirus (NPV) have been determined from lepidopteran, dipteran, and and Granulovirus (GV) (Van Regenmortel et al., 2000). hymenopteran hosts. A few hundred baculoviruses are Phylogenetic analyses have shown a further division of awaiting characterization and may reveal unique features. NPVs into group I and group II NPVs (Bulach et al., 1999; Sixty-two genes are shared among all lepidopteran baculo- Herniou et al., 2001, 2003). One of the major differences viruses sequenced so far (Li et al., 2002), 29 of which have between the group I and group II NPVs is the absence of homologues in all baculoviruses and are seen as baculovirus the baculovirus envelope protein GP64 from group II NPVs core genes. The combined sequences of conserved baculo- virus genes may be used to obtain trees representing the evolution of the baculovirus genomes (Herniou et al., 2001, $ GenBank accession numbers: AY456389, AY456390, AY545786, 2003). AY545787. Among those conserved genes, the lef-8 gene encoding a * Corresponding author. Laboratory of Virology, Wageningen Univer- sity, Binnenhaven 11, 6709 PD Wageningen, The Netherlands. Fax: +31 viral RNA polymerase subunit (Acharya and Gopinathan, 317 484820. 2002; Guarino et al., 1998; Passarelli et al., 1994) and the E-mail address: [email protected] (M.M. van Oers). per os infectivity factor pif-2 (Ac22 in AcMNPV) (Pijlman 0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2004.09.032 M.M. van Oers et al. / Virology 330 (2004) 460–470 461 et al., 2003)provedtobetwoofthemostreliable Infection in cell culture baculovirus markers for phylogenetic analyses at the virus family level (Herniou et al., 2004). These analyses have In order to facilitate the study of the cytopathology and further shown that lepidopteran baculoviruses form a replication of ChchNPV, we tested several insect cell lines monophyletic group. for their susceptibility to the virus. The best results were In addition to the set of conserved genes, baculovirus obtained for Trichoplusia ni High Five cells. Hemolymph genomes contain genes for so-called auxiliary functions. isolated from infected larvae 3 days postinfection at 27 8C These genes can be shared by a group of related induced cytopathic effects in T. ni High Five insect cells, baculoviruses or be present in a limited number of related such as enlargement of the nucleus and rounding up of the or unrelated virus species (i.e., helicase-2). These auxiliary cells. At 3 days postinfection, a small percentage of the cells genes seem to reflect gene acquisitions that provided was lost due to what appeared to be apoptotic processes additional fitness to their bearer during baculovirus evolu- based on cell morphology: Blebbing was observed in a tion (Herniou et al., 2003). The collection of auxiliary genes small portion of the cells resulting in the formation of is likely to give each baculovirus specific characteristics, vesicles. In the nucleus of a low percentage (b10%) of the which may determine its virulence, host range, tissue cells, a small number of polyhedra were visible after 1 specificity, and other biological features. Newly discovered week. The infection process developed much slower than baculoviruses are likely to harbor novel genes belonging to for a fully permissive replication of, for instance, Auto- this category. grapha californica MNPV (Wickham et al., 1992) in these The moth Chrysodeixis chalcites is a polyphageous cells, suggesting that this cell system may be semi- looper and a major pest in Dutch greenhouses in sweet permissive for ChchNPV, but may still be adequate for pepper and tomato. A new single nucleocapsid nucleopo- transcription studies. lyhedrovirus was isolated (tentatively named ChchNPV) from laboratory cultures of this moth, originating from a Phylogenetic analyses greenhouse in Naaldwijk, the Netherlands. In this paper, we characterized this virus by restriction enzyme analysis Partial sequences of the pif-2 and lef-8 genes (GenBank and end-in sequencing of a HindIII restriction fragment accession numbers AY545786 and AY545787, respectively) library. Its evolutionary relationship with other baculovi- were obtained using degenerate PCR primers (Table 1). A ruses was explored using polh, lef-8, and pif-2 sequences combined phylogenetic analysis of these sequences showed obtained by PCR. A unique new baculovirus gene, a clear, highly supported division between the GVs and the homologous to class II DNA photolyases (dpl), was NPVs. Among NPVs, viruses of the group I NPVs form a discovered, putatively encoding a light-inducible DNA highly supported clade, to which ChchNPV does not belong repair enzyme. We analyzed the transcription of this gene (Fig. 1A). The combined lef-8 + pif-2 tree provides poor in ChchNPV-infected insect cells and in vivo in C. resolution with low bootstrap support (b50%) for the basal chalcites larvae. Its possible role in baculovirus ecology relationships of the NPVs. The phylogenetic position of is discussed. ChchNPV is at the base of the tree among other NPVs for which the phylogenetic relationships are unresolved. These baculoviruses have in previous analyses been indicated as Results group II NPVs (Bulach et al., 1999; Zanotto et al., 1993). From this phylogenetic analysis, it can be concluded that the Virus morphology lef-8 and pif-2 sequences from ChchNPV are not closely related to other group II NPVs, at least not to those for C. chalcites larvae regularly showed mortality, which which lef-8 and pif-2 sequence data are available. was accompanied by liquefaction of the cadavers, in Since polyhedrin sequences are available for many more laboratory rearings. Light microscopy preparations of NPVs, the ChchNPV polyhedrin gene (polh) was analyzed. these larvae showed large numbers of polyhedron- Therefore, the sequence of a 1447-bp DNA fragment shaped particles, which also appeared to be alkali (GenBank accession number AY456390), including the sensitive, a characteristic of baculovirus polyhedra. total polyhedrin coding region, was determined. PCR Electron microscopy on these particles confirmed the amplification products were obtained with polyhedrin presence of viral occlusion bodies (polyhedra) typical of degenerate primers (designed by de Moraes and Maruniak, nucleopolyhedroviruses (NPVs). Ultrathin sections (not 1997) and sequenced. The sequence was extended by shown) revealed that the virions in these polyhedra genome walking using ChchNPV polyhedrin-specific pri- contained single nucleocapsids, characteristic of SNPVs. mers for sequencing of the viral DNA. The polyhedrin gene The isolated virus was tentatively named C. chalcites contains an open reading frame of 738 bp, encoding a nucleopolyhedrovirus, ChchNPV, according to the cur- protein with a predicted molecular mass of 28.8 kDa. The rent approved ICTV nomenclature (Van Regenmortel et ChchNPV polh gene harbors an ATAAG baculovirus late al., 2000). promoter element located at À47 to À43 relative to the 462 M.M. van Oers et al. / Virology 330 (2004) 460–470 Table 1 Oligonucleotides Name Sequence Amino acid motif a Reference Polyhedrin F1 TAYGTGTAYGAYAACAAG YVYDNK (de Moraes and Maruniak, 1997) Polyhedrin R1 TTGTARAAGTTYTTCCAG WENFYK (de Moraes and Maruniak, 1997) Polyhedrin
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