Genomic Sequence of Spodoptera frugiperda Ascovirus 1a, an Enveloped, Double-Stranded DNA Insect Virus That Manipulates Apoptosis for Viral Reproduction

Ascoviruses (family Ascoviridae) are double-stranded DNA viruseswith circular genomes that attack lepidopterans, where theyproduce large, enveloped virions, 150 by 400 nm, and cause achronic, fatal disease with a cytopathology resembling thatof apoptosis. After infection, host cell DNA is degraded, thenucleus fragments, and the cell then cleaves into large virion-containingvesicles. These vesicles and virions circulate in the hemolymph,where they are acquired by parasitic wasps during ovipositionand subsequently transmitted to new hosts. To develop a betterunderstanding of ascovirus biology, we sequenced the genomeof the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a).The genome consisted of 156,922 bp, with a G+C ratio of 49.2%,and contained 123 putative open reading frames coding for avariety of enzymes and virion structural proteins, of whichtentative functions were assigned to 44. Among the most interestingenzymes, due to their potential role in apoptosis and viralvesicle formation, were a caspase, a cathepsin B, several kinases,E3 ubiquitin ligases, and especially several enzymes involvedin lipid metabolism, including a fatty acid elongase, a sphingomyelinase,a phosphate acyltransferase, and a patatin-like phospholipase.Comparison of SfAV-1a proteins with those of other viruses showedthat 10% were orthologs of Chilo iridescent virus proteins,the highest correspondence with any virus, providing furtherevidence that ascoviruses evolved from a lepidopteran iridovirus.The SfAV-1a genome sequence will facilitate the determinationof how ascoviruses manipulate apoptosis to generate the novelvirion-containing vesicles characteristic of these viruses andenable study of their origin and evolution.

INTRODUCTION

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Introduction

The family Ascoviridae was erected recently to accommodate severalnew species of large double-stranded DNA (dsDNA) viruses withcircular genomes that attack insects of the order Lepidopteraat the larval and pupal stages, causing a chronic, fatal disease(38). Viruses of this family are characterized by large, envelopedvirions with a distinctive reticulate surface pattern. Dependingon the species, virions are either bacilliform or allantoid(sausage shaped), contain an internal lipid membrane surroundingthe DNA/protein core, and are composed of at least 12 structuralproteins, ranging in mass from 10 to 200 kDa (40).

These structural characteristics of the virions are sufficientto distinguish ascoviruses from all other large dsDNA viruses.However, the most novel feature of ascoviruses is not theirvirion structure, but rather their unusual cellular pathologyand transmission. Unlike for all other viruses, a variety ofevidence suggests that ascoviruses induce apoptosis as partof a mechanism that enhances their reproduction and transmission.A typical pattern of cytopathology, as exemplified by Spodopterafrugiperda ascovirus 1a (SfAV-1a), the type species, beginswith nuclear hypertrophy and cleavage of host DNA, followedby lysis of the nucleus and fragmentation of the nuclear membrane.Recent studies demonstrating that SfAV-1a synthesizes an executionercaspase provide evidence that this virus plays a direct rolein initiating apoptosis (16). After nuclear lysis, cellularhypertrophy ensues and what appear to be destined to becomeapoptotic bodies begin to form at the cell periphery by membraneinvagination. However, rather than degenerate, the developingapoptotic bodies are rescued by the virus and go on to formlarge virion-containing vesicles, also referred to as viralvesicles, that typically range from 5 to 10 µm in diameter.These disseminate to the hemolymph, where they circulate forweeks, until the infected larva or pupa dies (39).

Ascoviruses are very poorly infectious per os, an unusual traitfor insect viruses other than iridoviruses (family Iridoviridae).Thus, circulation of viral vesicles in the hemolymph apparentlyevolved to facilitate transmission. Several field and laboratorystudies have shown that endoparasitic wasps acquire ascovirusvirions and viral vesicles on their ovipositor while probinginfected larvae or pupae during egg-laying. Contamination ofthe ovipositor results in very high levels of infection, typicallygreater than 80%, in larvae subsequently probed by these wasps.That this mechanism of transmission operates under field conditionsis supported by studies showing that ascovirus isolates canbe obtained by allowing field-collected wasps to probe laboratory-rearedlarvae (46, 103).

Whereas field studies of ascoviruses indicate that they occurcommonly, particularly in populations of lepidopteran speciesbelonging to the family Noctuidae and especially when parasiticwasp populations are high, only five ascovirus species are recognizedcurrently (37). In addition to SfAV-1a, these are Trichoplusiani ascovirus 2a (TnAV-2a), Heliothis virescens ascovirus 3a(HvAV-3a), Diadromus pulchellus ascovirus 4a (DpAV-4a), andSpodoptera exigua ascovirus 5a (SeAV5a). Of these, with theexception of DpAV-4a, which attacks the leak moth, Acrolepiopsisassectella (family Hyponomentoidae), at the pupal stage, allknown ascovirus species attack noctuids. Some ascoviruses, suchas TnAV-2a and HvAV-3a, have broad host spectra, being ableto replicate in species of several noctuid genera. In contrast,SfAV-1a has a narrow host range, apparently capable of replicatingonly in Spodoptera species. The most unusual ascovirus is DpAV-4a,which replicates in its wasp vector and is transmitted verticallyto its lepidopteran host, in which it replicates much more extensively(17, 18).

To begin to develop a better understanding of the genetic basisof ascovirus pathogenesis, host spectra, and evolution, we sequencedthe SfAV-1a genome, which we report here. Consistent with itsunusual pathology, we show that this genome contains many genesunique among viruses.

MATERIALS AND METHODS

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Materials and Methods

Genomic DNA library. Propagation of SfAV-1a, referred to hereafter as SfAV, and preparationof viral DNA were performed as described previously (17, 40),using a clone of this species originally propagated in S. frugiperda(40). Briefly, 50 µg of SfAV DNA was sheared by sonication(20 W for 2.5 min with 1-s pulses) to generate fragments rangingin size from 0.5 to 3 kbp. DNA fragments were blunted with S1nuclease and T4 DNA polymerase (New England Biolabs), and EcoRIlinkers were ligated at both ends. Fragments of approximately0.85 to 1.1 kbp were purified from agarose gel by using a QIAquickgel extraction kit (QIAGEN) and ligated to the EcoRI site inpUC18.

DNA sequencing. Plasmids were isolated and purified using standard protocols(8). Nucleotide sequences were determined by dideoxynucleotidesequencing (84) using a Sequitherm long-read cycle sequencingkit with universal and reverse IRD800 fluorescently labeledprimers (Epicentre Technologies). DNAs were amplified by PCR(25 cycles of denaturation at 94°C for 30 s, annealing at50°C for 15 s, and polymerization at 70°C for 1 min).Nucleotide sequences for both strands were generated using amodel 4200 DNA long-read sequencer (Li-cor). Each nucleotidewas sequenced an average of eight times to verify accuracy.

Nucleotide sequence accession number. The nucleotide sequence of the SfAV genome has been depositedin the GenBank/EMBL/DDBJ databases with the accession number(AM 398843).

RESULTS AND DISCUSSION

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Results and Discussion

General characteristics and coding capacity of the SfAV genome. Of the five recognized species of ascoviruses, none has beensequenced. Thus, we determined the complete genome sequenceof SfAV, the ascovirus type species, by sequencing 1,600 overlappingclones, each approximately 1 kilobase pair (kbp) in length,from a shotgun library in pUC18. The SfAV genome consisted ofa circular dsDNA molecule of 156,922 bp, with a G+C contentof 49.26%.

Other than ascoviruses, several types of large dsDNA virusesare known to attack insects. The most common of these are thebaculoviruses (family Baculoviridae), iridoviruses (family Iridoviridae),and entomopoxviruses (family Poxviridae). Of these, based onmolecular phylogenetic data, the ascoviruses are most closelyrelated to the iridoviruses and appear to have evolved froma lepidopteran iridovirus (98, 99). Interestingly, however,the genome size and G+C content of SfAV differed significantlyfrom those of the Chilo iridescent virus (CIV), the only lepidopteraniridovirus sequenced to date. The CIV genome is 212 kbp, witha G+C content of 28.63%. Of nine other iridoviruses for whichthe genome sequence is available, eight of these are smaller(98 to 140 kbp) than SfAV, whereas the Lymphocystivirus iridovirus(lymphocystis disease virus isolated in China) has a largergenome (186 kbp). The G+C contents of these viruses differ widely,ranging from 27.25 to 54.8% (72). Comparison to other dsDNAviruses of insects shows that the genome size of SfAV is largerthan those of most baculoviruses. These range in size from approximately81 kbp to 178 kbp, with G+C contents ranging from 32.4% to 50.9%(61). With respect to insect poxviruses, these have larger genomesand lower G+C ratios than SfAV. The grasshopper entomopoxvirusof Melanoplus sanguinipes, for example, is 236 kbp, with a G+Cratio of 18.3%, and the lepidopteran entomopoxvirus from Amsactamoorei is 232 kbp, with a G+C content of 17.8% (3, 12). Whereasascoviruses contain some genes related to those of baculovirusesand nudiviruses, molecular phylogenetic analyses of replicationenzymes and structural proteins (for example, DNA polymeraseand major capsid protein genes, respectively) indicate thatthey are much more closely related to the large dsDNA virusesthat initiate replication in the cytoplasm, viruses such asChilo IV and the phycodnaviruses (97, 98).

In regard to other general characteristics of the SfAV genome,two large repeat sequences of 2.9 kbp, each with inverted terminalrepeats (ITRs), were found head-to-head between the $~$ 80-kbp and94-kbp positions (Fig. 1). A fragment of the 5'- and 3'-truncated2.9-kbp repeated sequence is also present in the region spanningthe $~$ 147.5-kbp to 149.5-kbp positions. These ITRs may be delineatingregions in the SfAV genome where reciprocal changes of configurations(linear versus circular) might occur during the replication.This possibility is based on prior observations that these ITRscan differ in size, being smaller in two variants, SfAV-1b andSfAV-1c (19), which suggested that only a portion of these largerepeats may be essential for this virus.

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FIG. 1. Schematic illustration of the organization of the Spodoptera frugiperda ascovirus 1a genome. Predicted ORFs are indicated by their locations, orientations, and putative sizes. White arrows represent ORFs in the forward strand, whereas gray arrows identify those in the complement strand. The relative positions of the 2.9-kbp fragments, organized as inverted repeats at one locus spanning the $~$ 80-kbp to 94-kbp positions and as a 5'- and 3'-truncated repeat at a second locus spanning the $~$ 147.5-kbp to 149.5-kbp positions, are also shown.

For the purpose of orientating the open reading frames (ORFs)contained in the SfAV genome, the A in the translation startcodon (ATG) of the DNA polymerase gene was designated the firstnucleotide position. Computer-assisted analyses of the SfAVgenome revealed 123 ORFs, which were predicted to code for proteinscontaining 65 to 1,157 amino acid residues containing conserveddomains and motifs (Fig. 1 and Table 1) . Of these, only three(44, 53, and 98) completely overlapped with ORFs in the complementarystrand (Fig. 1). In addition, 67 other putative ORFs, typicallysmall, encoding peptides of 49 to 202 amino acids in length,were found. However, these showed no significant level of homologywith other proteins in database searches and lacked conserveddomains and motifs (Table 2).

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TABLE 1. Properties of ORFs within the SfAV-1a genome^a

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TABLE 2. Properties of small ORFs within the SfAV-1a genome^a

The average protein encoded by these ORFs contained 225 residues.Of these, 70 (57%) were encoded by ORFs present in the forwardorientation and 53 (43%) in the reverse. Using a 30-bp windowto scan the genome for variations in base pair ratios, we determinedthat the G+C contents of the 2.9-kbp repeats were lower ( $~$ 43%)than those for the rest of the genome. Similarly, when the averageORF of 675 bp (3 x 225 residues) was used as a scanning windowto investigate variations in base ratios along the SfAV genometo suppress local nonsignificant differences due to coding capacities,we observed that G+C peaks lower than 46.7% occurred regularlyalong the sequence (Fig. 2, vertical black arrows on the horizontalaxis). All these peaks were located in intergenic regions. Otherintergenic regions did not contain lower G+C peaks, indicatingthat two types of putative intergenic regions were present inSfAV: G+C-rich and G+C-poor regions. Data published previouslyon transcription of CIV indicate that genes are transcribedtemporally as mono- or polycistronic mRNA (31), suggesting thatthe G+C-poor regions delineate regions in which transcriptionis arrested at the termination of mRNA synthesis. In agreementwith previous ascovirus sequence analyses (97), we observedthat all G+C-poor regions contained short inverted repeats thatmight function as transcription termination signals.

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FIG. 2. Variations in G+C content (vertical axis) along the Spodoptera frugiperda ascovirus 1a genome (horizontal axis). The graphic representation was calculated using the plot option in Vector NTI (Invitrogen) and a window of 675 nucleotides. G+C-poor intergenic regions are identified by vertical black arrows. Large light gray arrows and a gray box identify the 2.9-kbp repeats, organized as inverted repeats at one locus (spanning the $~$ 80-kbp to 94-kbp positions) and as a 5'- and 3'-truncated repeat at the second locus (spanning the $~$ 147.5-kbp to 149.5-kbp positions). The line located at a G+C content of 46.7% corresponds to the threshold used to differentiate intergenic regions from those putatively transcribed as mono- and polycistronic mRNA. G+C peaks lower than 46.7% along the SfAV-1a genome sequence are indicated by arrows.

Similarity of SfAV proteins to those of iridoviruses and baculoviruses. Comparison of SfAV genes with those of other large dsDNA virusesthat attack insects provided further evidence that this ascovirusshares a common evolutionary lineage with iridoviruses (98,99). Thirty-five SfAV ORFs (28%) were identified, for whichthe closest orthologs were those of CIV (Table 1). In addition,22 (18%) orthologs of baculovirus genes were found in SfAV (Table1). At least 20 (16%) of the remaining putative proteins sharedsignificant levels of similarity with proteins encoded by eukaryoticgenomes (Table 1).

Proteins with conserved domains and motifs and putative functions in SfAV. Compared to other groups of large DNA viruses, ascoviruses havereceived relatively little study, and thus, the functions ofonly a few SfAV proteins were known (98). Based on sequenceanalyses, using the numerical designations herein, these includethe major capsid protein (ORF 41), DNA polymerase (ORF 1), thymidinekinase (ORF 40), and ATPase III (ORF 110). Additionally, werecently reported a functional executioner caspase (ORF 73)(16). Based on computer-assisted analyses of the SfAV genomefor identification of conserved structural and functional domainsand motifs of the various ORFs, as well as their predicted subcellularlocations, we were able to assign putative functions to a largenumber of these proteins (Table 1). Most of the SfAV proteinsfor which functional assignments could be made were enzymesinvolved in virus replication, transcription, protein modification,nucleotide metabolism, and virus-host interactions. Additionally,and of potential significance to the unique rescue of apoptoticbodies characteristic of SfAV, we identified several enzymesinvolved in lipid metabolism not known to occur as a group inother viruses. Below, we describe the apparent functions ofthese proteins and their likely roles in SfAV replication andpathobiology.

Proteins involved in DNA replication, recombination, and repair. SfAV encodes a ${delta}$ DNA polymerase (ORF 1) of the B family, which,like the principal replication enzymes in prokaryotes and eukaryotes,contains a nucleotide-polymerizing domain (II-VI-III-I-V) fusedto an N-terminal 3' to 5' exonuclease domain (67). No homologueof the proliferating cell nuclear antigen (PCNA)-like clamp,a ring-shaped protein known as a processing factor of DNA polymerasedelta, was identified in SfAV. A PCNA-like DNA clamp factoris encoded by other viruses, including vertebrate poxvirusesand baculoviruses (56, 81), but as with SfAV, it is not knownto occur in entomopoxviruses and iridioviruses. The absenceof a PCNA-like clamp protein in SfAV suggests that a processivityfactor for DNA replication is provided by the host cell, asis the case for the AcMNPV baculovirus, where the viral PCNAgene is not required for viral replication (81).

SfAV also encodes a homologue (ORF 99, 95 kDa) of the poxvirusD5 family of proteins, which belongs to helicase superfamilyIII within the AAA+ ATPase class. Helicase superfamily III includesprimary replicative helicases encoded by several DNA and RNAviruses (44, 57). The vaccinia virus D5 protein (90 kDa), anucleic acid-independent nucleoside triphosphatase, is requiredfor viral DNA replication (35). Recently, it has been shown(57) that the poxvirus D5 protein contains a primase domain,which is also present in SfAV ORF 99. The presence and conservationof poxvirus D5 homologues in SfAV and iridoviruses (72) suggestthat ORF 99 is essential for viral DNA replication.

Other proteins encoded by SfAV with known or presumed functionsin viral DNA replication, recombination, and repair that aretypical of large dsDNA viruses include a thymidine kinase (ORF40), known to be involved in the synthesis of deoxyribonucleotidesin cells with suboptimal pools of nucleotides (34, 41); a topoisomeraseI (ORF 86), which alters DNA topology by transiently breaking,passing, and rejoining single DNA strands (22, 90); and a DNAligase (ORF 32) that seals nicked duplex DNA substrates (95).Other SfAV enzymes that likely play roles in DNA metabolisminclude an ATPase (ORF 110), ssDNA and dsDNA nucleases (ORF37 and ORF 75), a helicase (ORF 95), serine/threonine kinases(ORF 64, ORF 82, and ORF 104), and a tyrosine kinase (ORF 90)(68, 74) as well as members of the Rec/Sbc/Rad superfamilies.These include RecD exonuclease V (ORF 95), SbcC-like and SbcD-likeexonucleases (ORF 59 and ORF 103, respectively), and a Rad50-likenuclease (ORF 101) (2, 26, 35, 80, 94, 104).

SfAV also encodes a putative FEN-1/FLAP-like nuclease (ORF 66).FEN-1/FLAP nucleases share structural and functional similaritieswith eubacterial DNA polymerases, Rad2, and the Xeroderma pigmentosumG (XPG N and I regions) repair endonuclease and also possessRNase H activity (102). Homologues of ORF 66 are also presentin iridovirus, poxvirus, Emiliania huxleyi virus, and mimivirus,all of which are among the large nuclear and cytoplasmic DNAviruses, the so-called NCLDV family (56). A FEN-1/FLAP homologueis known to function in RNA primer removal during T4 phage DNAreplication, and in poxvirus, a homologue (G5R) apparently functionsin the early stage of viral morphogenesis (30, 56). Interestingly,it has been shown that the homologous enzyme in herpesvirusesinhibits cellular gene expression in infected cells, destabilizingpreexisting host mRNAs and thereby ensuring rapid turnover ofviral mRNA. Thus, ORF 66 could have several functions in SfAVvirogenesis.

Genes involved in transcription and RNA metabolism. SfAV gene products that apparently participate in mRNA biogenesisincluded the DNA-dependent RNA polymerase subunit C (ORF 8)and RNA polymerase II subunits 1 and 2 (ORF 67 and ORF 52, respectively).In addition, three transcription factor-like proteins, TFIIF(ORF 109), VLTF2 (virus-like transcription factor 2, ORF 113),and Yabby-like TF (ORF 91), were encoded by SfAV. The Yabby-likeproteins in plants constitute a group of putative transcriptionfactors with a Cys-containing zinc finger. These proteins alsocontain a DNA-binding domain with high similarity to the HMG(high-mobility-group) family of transcription factors (73).As Yabby proteins participate in abaxial-adaxial identity determinationin lateral organs in Arabidopsis thaliana, and HMG proteinsare known to modulate gene expression by interacting with nucleosomes,transcription factors, nucleosome-remodelling machines, andhistone H1 (14), SfAV ORF 91 could have similar functions. Althougha homologue of ORF 91 is present in CIV (T03180), the functionof this protein is unknown. In our database searches, no homologueswere identified in other virus families.

SfAV ORF 66, which has a Fen-1/FLAP-like nuclease domain (notedabove), also contains a motif within the XPG N-I regions thatcorresponds to sequences found in various virion host shutoff(VHS) proteins encoded by herpesviruses, vericella-zoster virus,and pseudorabies virus. VHS proteins inhibit cellular gene expressionin infected cells and destabilize preexisting host mRNAs, therebyensuring rapid turnover of virus-specific mRNAs (69, 93).

In addition, SfAV contains two genes (ORF 22 and ORF 23) thatencode proteins with similarities to prokaryotic RNase III.ORF 22 contains a well-conserved RNase III catalytic domainthat is required for cleavage of dsRNA templates. In prokaryotesand eukaryotes, RNase III participates in a variety of functions,including the processing of cellular and virus-encoded precursorRNAs into mRNAs, rRNAs, and tRNAs, and also is involved in thedegradation of specific mRNAs (33, 79). The RNase III catalyticdomain is also present in Dicer, also a member of the RNaseIII protein superfamily, which cleaves precursor dsRNA intosmall temporal RNAs and short interfering RNA (siRNA) of $~$ 22nucleotides (5, 79). siRNAs participate in gene-specific inactivation,whereas small temporal RNAs function in the control of developmentalprocesses (79).

Database searches indicated that SfAV RNase III homologues arenot encoded by dsDNA viruses other than ascoviruses and theiridescent virus Paramecium bursaria chlorella virus 1 (familyPhycodnaviridae). Zhang et al. (111) have shown that the Parameciumbursaria chlorella virus 1 RNase III is an active enzyme, butas this protein lacks additional N-terminal domains (ATPase/helicase,DUF283, and PAZ) present in Dicer proteins (79, 110), it isunlikely that the viral RNase III functions in gene-specificsilencing during virogenesis. Interestingly, however, it hasbeen shown that an RNase III encoded by the sweet potato chloroticstunt virus, an RNA virus (family Closteroviridae), enhancessuppression of host-induced RNA silencing by a sweet potatochlorotic stunt virus-encoded p22 protein (66). Thus, it ispossible that the RNase III encoded by these dsDNA viruses couldfunction in a similar way to evade an antivirogenic responseelicited by host-directed siRNA.

Cellular homologues involved in protein modification, processing, and apoptosis. (i) Protein kinases and oxidoreductases. SfAV codes for several enzymes that apparently modify proteinsby phosphorylation/dephosphorylation and oxidation/reductionprocesses. SfAV protein kinases potentially involved in bothupregulating and downregulating diverse cellular regulatoryprocesses and pathways, including transcription, translation,and cell division, include a tyrosine protein kinase (ORF 90),two serine/threonine kinases (ORF 64 and ORF 104), and a thymidinekinase (ORF 40).

ORFs related to the vaccinia virus thiol oxidoreductase (E10R)and thioredoxin (G4L) are also present in iridoviruses, poxviruses,and other large nuclear and cytoplasmic viruses (45, 72, 87,88). Thiol-oxidase-like proteins (ORF 61 and ORF 116) are alsoencoded by SfAV. These enzymes are members of the Evr1/Alr (Evr1,essential for respiration and vegetative growth; Alr, augmenteror liver regeneration) family of flavin adenine dinucleotide-containingsulfhydryl oxidases that use oxygen as an electron acceptor.Evr1/Alr proteins contain a conserved domain of about 100 aminoacid residues with an invariant C-X-X-C active-site motif thatis implicated in redox function (86, 87, 88). Representativesof the Evr1/Alr proteins include the Saccharomyces cerevisiaeEvr1 protein, required for mitochondrial development, and mammalianhematopoietin (ALR). The G4L has thiol transferase activityand is required for formation of disulfide bonds (108). Recently,Senkevich et al. (87, 88) have shown that E10R and G4L functionin the formation of disulfide bonds between conserved cysteineresidues of viral structural membrane-bound proteins and arerequired for virion morphogenesis. Thus, the SfAV ORF 61 andORF 116 proteins may play a similar role in proteins bound tothe virion membrane.

(ii) IAPs. SfAV encodes four inhibitor of apoptosis protein (IAP)-likeproteins (ORF 15, ORF 16, ORF 25, and ORF 74). The SfAV-1a IAP-likehomologues ORF 16, ORF 25, and ORF 74 are most closely relatedto cellular proteins from Danio rerio (zebrafish), Xenopus laevis(African clawed frog), and rodent, respectively, whereas ORF15 is most closely related to a baculovirus IAP. Nevertheless,based on phylogenetic studies, baculovirus IAPs are thoughtto have been derived from lepidopteran hosts (54). The presenceof at least four IAP-like proteins in the SfAV genome is notunusual. In baculovirus genomes, from one to four IAP-like proteinshave been reported (55), and in the invertebrate CIV genome,two copies are present (58). IAPs are metalloproteins that containone or more zinc-binding motifs referred to as baculovirus IAPrepeats and a RING (really interesting new gene) domain (105).First described in baculoviruses (24, 27), cellular homologuesof IAPs have been reported in organisms ranging from yeaststo humans and, most recently, plants (51). Many IAPs are knownto function by directly inhibiting caspases, thereby haltingthe execution phase of programmed cell death (105) and maximizingefficiency of viral replication (24). Thus, SfAV IAPs couldfunction in the transient regulation of the apoptotic response.One possibility is that shortly after infection, one or moreof the SfAV IAPs act in a cascade in which apoptosis is initiallyinhibited to enable the ascovirus to gain access to the nucleusand establish infection, after which the caspase gene is expressedto trigger cell death and initiate the development of apoptoticbodies. Other possible roles for SfAV IAPs include posttranslationalmodification and degradation of proteins via ubiquitination(see below).

(iii) E3 ubiquitin ligases and IAPs. The ubiquitin-proteasome system is a well-conserved system inmetazoans that is used for posttranslational modification anddegradation of proteins. Three essential components are requiredin a cascade that targets proteins for ubiquitination: E1 ubiquitin-activatingenzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase(77). E1 transfers ubiquitin, a protein of 76 amino acid residues,to E2 ubiquitin, which, together with E3, ligates ubiquitinto targets doomed for degradation by proteasomes. E3 ubiquitinligase contains a zinc-binding motif called a RING domain thatis essential for ubiquitination (6). SfAV ORFs 16, 81, and 97encode proteins with conserved RING domains that are characteristicof the E3 ubiquitin ligase. Members of the nucleo-cytoplasmiclarge DNA viruses are also known to encode ubiquitin homologues(56).

The Op-IAP3 baculovirus IAP (inhibitor of apoptosis protein;see above), which also contains a RING (C3H4) domain (105),functions as an E3 ubiquitin protein ligase, as shown recently(75). As noted above, the SfAV genome encodes four IAPs (ORF15, ORF 16, ORF 25, and ORF 74), which therefore could potentiallyfunction in the ubiquitin pathway. Interestingly, a breast-and ovarian-specific tumor suppressor-like protein (BRCA-1)is also encoded by SfAV ORF 97. BRCA-1, together with anotherprotein (BARD1), has been shown to function as an E3 ubiquitinligase (96). Ubiquitin posttranslational protein modificationis involved in numerous cellular processes, including signaltransduction, the cell cycle, and programmed cell death, inthe last of which the E3 ubiquitin ligase targets include theproapoptotic proteins, including Hid, Reaper, Grim, and caspases(75). Alternatively, more-recent data suggest that an E3 ubiquitinligase encoded by the white spot syndrome virus functions asan inhibitor of apoptosis to ensure efficient virus propagation(48). Thus, it is possible that the SfAV E3-ubiquitin ligasesand the IAP-like and BRCA-1-like proteins all play similar rolesduring SfAV replication.

Proapoptotic proteins. (i) Executioner caspase. The cytopathology induced by most ascoviruses is novel amongviruses in that virogenesis involves a modified form of celldeath in which apoptotic bodies, as they develop, are convertedinto virion-containing vesicles (36, 39). This suggested thatSfAV encoded proteins unique to this virus type that induceapoptosis and rescue the developing apoptotic bodies. As notedabove, we demonstrated recently that SfAV encodes a functionalexecutioner caspase (ORF 73) that apparently plays a directrole in initiating apoptosis (16). As executioner caspases alsoare known to be involved in the maturation of viral proteins(13), the SfAV caspase may also be involved in virion maturation.

(ii) Cathepsin B (SfAV ORF 114). Although caspases are the key enzymes that regulate programmedcell death, over the past few years it has been shown that cathepsins,particularly cathepsins B, D, and L, also play crucial rolesin this process (23). Cathepsins are cysteine proteases thatbelong to the C1 family of enzymes, the so-called papain family.These enzymes are synthesized as zymogens and are glycosylatedposttranslationally, after which they are compartmentalizedin lysosomes, a process mediated by cellular mannose-6-phosphatereceptors. Activated cathepsins released from lysosomes throughvarious stimuli, including sphingomyelin diphosphodiesterase(SMase) activity (see below), direct many cellular pathways,such as protein degradation, proenzyme activation, antigen processing,metabolism, and apoptosis. With regard to proapoptotic mechanisms,it is interesting to note that SfAV encodes a protein (ORF 114)that contains a well-conserved peptidase domain found in cathepsinB. Recent evidence suggests that cathepsin B could regulateapoptosis by attacking the mitochondria directly causing therelease of cytochrome c or by activating proapoptotic proteinsof the Bcl2 superfamily, such as Bid and Bax (23), that activateinitiator caspases, which in turn activate executioner caspasesand possibly the SfAV caspase (ORF 73). Cathepsin homologuesare also known in baculoviruses and the CIV (58, 92). The functionof the cathepsin B homologue in CIV is unknown. However, thebaculovirus cathepsin L homologue is thought to be linked todegradation and liquefaction of host tissues during virus pathogenesis(47, 92).

Enzymes involved in lipid metabolism. A unique feature of SfAV and other ascoviruses, such as TnAV-2aand HvAV-3a, is that after nuclear lysis and host DNA degradation,cell cleavage similar to apoptosis occurs. However, insteadof forming typical apoptotic bodies, the cell cleaves into numerousvirion-containing vesicles, each of which occludes numerousvirions in various stages of morphogenesis along with a virogenicstroma, apparently still active. A characteristic of this processis that de novo membrane synthesis occurs as these virion-containingvesicles form. Most host cell DNA is degraded by the time theviral vesicles form, suggesting that the elaboration of membranesthat delimit these vesicles may be a product of lipid-metabolizingenzymes encoded by SfAV. Consequently, our search of the SfAVgenome revealed genes encoding the following enzymes, knownto play a role in lipid metabolism. Whereas some of these occurindividually in other viral genomes, they do not occur togetherin any other viral genome.

(i) SfAV ORF 87, encoding fatty acid elongase (ELO). The SfAV ORF 87 protein belongs to the GSN1/SUR4 enzyme family(11), which catalyzes the biosynthesis of long-chain (26 carbons)fatty acid precursors for ceramide and sphingoid lipogenesis.In yeast, allelic variants of ELO2 and ELO3 (VBM1 and VBM2,respectively), also involved in long-chain fatty acid elongationand sphingoid synthesis, appear to play an important role inprotein sorting and trafficking of secretory vesicles (59).A gene homologue (AJ58127) is present in the fowlpox virus.

(ii) SfAV ORF 59, encoding sphingomyelin phosphodiesterase. SfAV ORF 59, which also contains an SbcD DNA repair exonucleasedomain (see above), has a well-conserved overlapping calcineurin-likephosphoesterase domain found in a wide variety of phosphoesterases,including protein phosphoserine phosphatases, nucleotidases,2'-3' cyclic AMP phosphodiesterase, and SMases (4). SMases cleavesphingomyelin to release phosphocholine and ceramide. A genehomologue (Q91FS8) is present in CIV.

(iii) SfAV ORF 112, encoding phosphate acyltransferase. A conserved domain in SfAV ORF 112 suggests that this proteinbelongs to the phosphate acyltransferase (PlsC) family of proteins.Members of this family, which include 1-acylglycerol-3-phosphateacyltransferase, function in de novo biosynthesis of cell membranephospholipids and have glycerophosphate, 1-acylglycerolphosphate,or 2-acylglycerolphosphoethanolamine acyltransferase activities(1, 29). No viral homologues of this enzyme were identifiedin database searches.

(iv) SfAV ORF 93, encoding a patatin-like phospholipase/alpha/beta hydrolase. The SfAV ORF 93 protein contains a FabD/lysophospholipase-likedomain related to the patatin-like phospholipases (53, 83).A homologue (Q91F63) is present in CIV. The patatin domain overlapswith a highly conserved domain found in members of the alpha/betahydrolase fold superfamily of enzymes. These enzymes have diversecatalytic functions, including lipolytic, thioesterase, andpeptidase activities. Patatin, a storage protein in potato tubers,also has an intrinsic lipid acylhydrolase activity that catalyzesthe cleavage of fatty acids from membrane lipids. The structuralcharacteristics of the active site in patatin, which containsa Ser-Asp dyad, are similar to those of cytosolic phospholipaseA₂ (PLA₂) (53). The PLA₂ family of enzymes plays a central rolein several cellular processes, such as phospholipid digestionand metabolism, host defense, and signal transduction (52, 112),and is also implicated in the induction of apoptosis via ceramidebiogenesis (7, 60) and nuclear shrinkage in caspase-independentcell death (89). Interestingly, parvovirus capsid proteins havean intrinsic PLA₂-like activity required for endocytic entryof the virus (21, 101), a possible function of ORF 93. In addition,patatin was shown to inhibit growth of the corn root worm (Diabroticasp.) (100). Ascovirus-infected larvae exhibit decreased feedingand growth, and though unlikely, this patatin-like protein maybe partly responsible for this characteristic.

(v) SfAV ORF 13, encoding carboxylesterase/JHE. The SfAV ORF 13 protein contains a well-conserved carboxylesterasedomain found in the alpha/beta hydrolase lipase family and isalso related to the juvenile hormone esterase (JHE) of Apismellifera. Carboxylesterases are lipases that catalyze the hydrolysisof a wide variety of ester- and amide-containing molecules,including short- and long-chain acylglycerols, long-chain acylcarnitine,and long-chain acyl-coenzyme A esters (85). JHE degrades thejuvenile hormone, which plays diverse roles in the insect lifecycle, including development, morphogenesis, reproduction, diapause,and metabolism (43, 62). No other viral homologues were identifiedin database searches.

Lipid metabolism and SfAV-induced apoptosis. The presence of a large number of genes coding for enzymes involvedin lipid metabolism is unusual for known prokaryotic and eukaryoticviruses. Collectively, these enzymes could play important rolesin the reorganization of existing membranes and de novo membranesynthesis during SfAV maturation (38) and in initiating andmediating the apoptotic response through sphingoid signaling.A number of studies have linked oxidative stress induced byreactive oxygen species with ceramide generation during theapoptotic program (4, 91), and it is well established that ceramideis an endogenous regulator of apoptosis (20, 65). In this regard,it is interesting to note that lysosomal permeabilization andcathepsin B release (see above) can be initiated by a numberof extralysosomal and intralysosomal stimuli that include oxidativestress induced by reactive oxygen species and, in particular,by sphingoid derivatives, especially ceramides, produced bysphingomyelinase and ceramidase enzymatic activities (4, 23).Recently, it has also been shown that apotosis induced by endoplasmicreticulum stress in insulin-secreting cells is amplified byoverexpression of phospholipase A₂ (iPLA₂B) (82). Based on theseobservations, it is tempting to speculate that the putativeSfAV cathepsin B, SMase, patatin-like PLA₂, and SfAV caspasefunction in concert to provide a pathway that leads to the rapidonset of the apoptotic response observed in SfAV-infected cellsand larvae (16, 36).

The SfAV viral lipases, particularly the patatin-like phospholipaseand coesterase/JHE alpha/beta hydrolases, could also functionin viral membrane biogenesis and egress of virus from the cell.In this regard, Baek et al. (9) have shown that the major envelopeprotein (p37) encoded by the vaccinia virus is a lipase thatparticipates in the de novo synthesis of the double membranesof this virus and is also involved in viral entry and exit frominfected cells.

Enzymes with other functions. (i) SfAV ORF 107, encoding PutA. The SfAV ORF 107 protein contains a domain conserved in thePutA family of NAD-dependent aldehyde dehydrogenases. Membersof the PutA family in prokaryotes and eukaryotes have two activeenzymes, proline oxidase and delta 1-pyrroline-5-carboxylatedehydrogenase. In eukaryotes, proline oxidase is located onthe inner mitochondrial membrane, is involved in the proline/pyrroline-5-carboxylatecycle, and catalyzes the formation of reactive oxygen species.Oxidative stress induced by reactive oxygen species and theaccumulation of pyrroline-5-carboxylate have been shown to induceand/or contribute to the apoptotic response (32, 70, 71).

(ii) SfAV ORF 14, encoding matrixin. The SfAV ORF 14 protein contains a zinc-binding motif (HEXGHXXGXXH)in the catalytic domain conserved in the matrixin zinc-dependentfamily of metalloproteases. These proteins, which include collagenasesand gelatinases that cleave denatured collagens, are peptidasesthat participate in the degradation of the extracellular matrix(106). A possible role for this protein could be the degradationof the extracellular matrix surrounding adipocytes, a cellulartarget of SfAV (39, 46).

(iii) Baculovirus repeated open reading frame genes (bro). The bro genes, first identified in baculoviruses, constitutea multigene family typically with many copies per genome andare also known to occur among other large insect dsDNA virusesand bacteriophages (15). These sequences are absent in vertebrategenomes and vertebrate viruses. Although a few baculoviruseslack bro homologues, others are known to contain from 1 to 16copies that are either active or inactive genes (15). SfAV containsseven bro-like gene homologues that encode from 97 to 363 aminoacid residues (ORFs 30, 31, 69, 70, 72, 79, and 80). We observedthat three of these appeared to be alleles, suggesting thatvariations occurred by mutation and recombination in this ascovirus.Little is known about the function(s) of BRO proteins in viralbiology, but at least one baculovirus BRO protein has been shownto bind nucleic acid, associate with nucleosomes, and interactwith host cell laminin (63, 109). In another study (15), inactivationof a single-copy bro gene in a baculovirus showed that it wasnot essential for virus replication but may enhance replicationduring the late phase of the viral life cycle.

(iv) SfAV ORF 46, encoding a protein with multifunctional domains. The putative protein encoded by ORF 46 contains several well-conservedoverlapping domains (Smc, Hec1, SbcC, filament, and Reo_Sigma1).The Smc family consists of chromosome segregation ATPases thatfunction during the cell division cycle (78). The Hec1 proteinsare known to interact with Smc proteins (64). The SbcC ATPasesare involved in DNA replication, recombination, and repair (2,26, 35, 80, 94, 104). The Reo_Sigma1 family is composed of glycoproteinsthat share homology with the reoviral sigma1 hemagglutinin cellattachment protein, a minor capsid protein that determines serotype-specifichumoral response and is also involved in modulating the apoptoticresponse in reovirus-infected cells (10, 42).

SfAV ORF 46 also contains a stretch of amino acid residues withhigh homology to the cytoskeletal intermediate-filament proteinsignature domain (50). The critical role of cytoskeletal proteins,particularly the involvement of intermediate filaments in virionmaturation and release from infected cells, is well established(28, 49, 76). The assembly of large DNA viruses, such as poxviruses,iridoviruses, and the closely related African swine fever virus,occurs in the cytoplasm in viral factories and aggresomes thatrecruit mitochondria, causing the rearrangement of intermediatefilaments and the collapse of vimentin into cage-like structures(49). Cordo and Candurra (25) have also shown that intermediatefilament integrity is essential for replication of the Juninvirus. As apoptosis, which includes cytoskeletal disintegrationand reorganization, proceeds rapidly during SfAV infection,the cytoskeletal network that could be essential for SfAV maturationmay not be available. Thus, the putative intermediate filamentprotein encoded by SfAV ORF 46 could play an essential rolein forming scaffolding networks for viral assembly and maturation.Viral gene homologues of SfAV ORF 46 occur in granuloviruses(family Baculoviridae) of Cydia pomonella (U82510) and Phthorimaeaoperculella (AF499596), viruses that replicate in the cytoplasmof infected cells.

Comparison of SfAV-1a and TnAV-2c genomes. The genomic sequence of Trichoplusia ni ascovirus 2c (TnAV-2c)has recently been published (107). The genome of TnAV-2c iscircular, consists of 174,059 bp, with a G+C content of 35.4%,and putatively encodes 165 proteins. This makes the TnAV-2cgenome approximately 11% larger than that of SfAV-1a. Basedon the putative annotated proteins coded for by TnAV-2c andSfAV-1a, homologues shared by both viruses include those involvedin virion structure (major capsid protein), nucleic acid metabolism(delta DNA polymerase, RNA polymerase, ATPases, RNase III, andnucleases), and inhibitor of apoptosis (IAPs) (Table 3). Withrespect to the unique cytopathology of ascoviruses, TnAV-2calso contains ORFs that putatively encode enzymes involved inpromoting apoptosis and viral vesicle formation, as describedabove for SfAV. These proteins include a caspase, cathepsinB, and enzymes involved in lipid metabolism (patatin-like phospholipase,PlsC phosphate acyltransferase, fatty acid elongase, and lipase)(Table 3). Apparent genes that occur in SfAV but not in TnAV-2cinclude those that putatively encode SbcC and RecD V exonucleases,a UrvD/Rep helicase, serine/threonine kinases, an executionercaspase, and a PutA/proline dehydrogenase.

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TABLE 3. Putative ORFs common to SfAV-1a and TnAv-2c^a

In summary, the genome of SfAV encodes proteins that would beexpected based on what is known about the biochemical characteristicsof the virions and the replication of this virus initially inthe nucleus and then in a mixture of nucleoplasm and cytoplasm.These proteins include various DNA and RNA polymerases, helicases,kinases, and structural proteins, such as the capsid protein.In addition, various other proteins consistent with the uniquecytopathology of ascoviruses that are either rare or absentfrom other known viruses were present. These include an executionercaspase, cathepsin B, and a complex of enzymes that modify lipids.The presence of genes for these enzymes, which are absent orrare in other viruses and do not occur as a group, suggeststhat they are directly involved in rescuing apoptotic bodiesand converting them into virion-containing vesicles. Althoughthe interactions of this diverse set of enzymes and structuralproteins are obviously complex, the availability of the SfAVgenomic sequence provides a foundation upon which the role ofthese various proteins in viral reproduction and pathogenesiscan be studied. Aside from contributions to virology, such studiesshould contribute to our understanding of the regulation andmanipulation of apoptosis as well as cell biology and lateralgene transfer during ascovirus evolution. With respect to thelast, the relationship we detected between SfAV-1a and ChiloIV, an iridovirus that infects lepidopterans, suggests thatmany genes coding for proteins involved in ascovirus vesicleformation or their homologs may have been acquired from a lepidopteranhost, as CIV does not form such vesicles. An alternative hypothesisis that these genes originated from the genome of a parasiticwasp, such as the ichneumonid D. pulchellus, that transmitsDpAV-4a, the genome of which has been detected in nuclei ofits wasp vector without causing any apparent pathology (17).These hypotheses are clearly speculative and will be answeredonly upon more experimental research with ascovirus and thedetermination of full genomic sequences for several endoparasiticwasp and lepidopteran species. Nevertheless, as no other virusundergoes the apoptotic-like process characteristic of ascoviruses,it is probable that the key genes involved in this process originatedby lateral gene transfer from either a lepidopteran or a hymenopteranhost.

ACKNOWLEDGMENTS

This research was funded in part by grants to B. A. Federicifrom the United States National Science Foundation (INT-9726818)and to Y. Bigot from the CNRS, the Groupement de Recherche CNRS2157, the Institut Fédératif de Recherche 136,the Ministère de l'Education Nationale, de la Rechercheet de la Technologie, and the University François Rabelaisof Tours.

We thank J. J. Johnson for his assistance throughout our investigations.

FOOTNOTES

* Corresponding author. Mailing address: Department of Entomology, University of California—Riverside, Riverside, CA 92521. Phone: (951) 827-5006. Fax: (951) 827-3086. E-mail: brian.federici{at}ucr.edu.

Published ahead of print on 20 September 2006.

REFERENCES

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