Proteomic analysis of the Spodoptera frugiperda ascovirus 1a virion reveals 21 proteins

Abstract

The Spodoptera frugiperda ascovirus 1a (SfAV-1a) is a double-stranded DNA virus that attacks lepidopteran larvae, in which it produces enveloped virions with complex symmetry which have an average diameter of 130 nm and length of 400 nm. Here, we report identification of 21 SfAV-1a-encoded proteins that occur in the virion, as determined by nano-liquid chromatography/tandem mass spectrometry. These included a helicase (ORF009), nuclease (ORF075), ATPase (ORF047), serine/threonine-like protein kinase (ORF064), inhibitor of apoptosis-like protein (ORF015), thiol oxidoreductase-like protein (ORF061), CTD phosphatase (ORF109), major capsid protein (ORF041) and a highly basic protein, P64 (ORF048). The latter two were the most abundant. Apart from ascoviruses, the closest orthologues were found in iridoviruses, providing further evidence that ascoviruses evolved from invertebrate iridoviruses. These results establish a foundation for investigating how ascovirus virion proteins interact to form their complex asymmetrical structure, as well as for elucidating the mechanisms involved in SfAV-1a virion morphogenesis.

The authors thank Dr Songqin Pan of the W. M. Keck Proteomics Laboratory, University of California, Riverside, for the peptide sequences obtained by nano-LC-MS/MS. This research was supported in part by grant INT-9726818 from the US National Science Foundation to B. A. F. and grants to Y. B. from NATO and C.N.R.S. (PICS ° 3434), France.

Footnotes

^,†, Dennis K. Bideshi¹^,2 †These authors contributed equally to this work.

The family Ascoviridae comprises large double-stranded DNA viruses with circular genomes ranging from 100 to 186 kb (Asgari et al., 2007; Bideshi et al., 2006; Bigot et al., 1997a, b; Wang et al., 2006). Four species of ascoviruses (AVs) are recognized, Spodoptera frugiperda AV (SfAV-1), the type species, Trichoplusia ni AV (TnAV-2), Heliothis virescens (HvAV-3) and Diadromus pulchellus AV (DpAV-4). Another species, TnAV-6a (previously TnAV-2c), has been proposed (Wang et al., 2006; Bigot et al., 2008). Ascoviruses are transmitted by parasitoid wasps to lepidopteran larvae and pupae in which they cause a chronic, fatal disease (Federici & Govindarajan, 1990). In susceptible hosts, ascovirus pathology includes stunted larval growth and prolonged development and a novel cytopathology characterized by a modified form of virus-induced programmed cell death, in which apoptotic bodies are rescued as they develop and are converted into virion-containing vesicles. After cleavage from the cell, these dissociate and accumulate in the haemolymph, changing its colour from translucent green to opaque white (Bideshi et al., 2005; Federici, 1983; Federici et al., 2005).

The ultrastructure of AV virions shows they are complex in symmetry and depending on the species, vary in shape. For example, virions of SfAV-1a are bacilliform, whereas those of TnAV-2a and HvAV-3a are allantoid, and those of DpAV-4a are ovoid. AV virions are typically 130–150 nm in diameter at their widest, and their length ranges from 200 nm for DpAV-2a to 300–400 nm for SfAV-1a, TnAV-2a and HvAV-3a (Federici, 1983; Cheng et al., 2000; Federici et al., 2005). With respect to ultrastructure, AV virions consist of an outer envelope and an inner particle that contains a DNA/protein core, which apparently also contains a lipid bilayer. In negatively stained preparations, AV virions exhibit a unique reticulate appearance that distinguishes them structurally from virions of other virus families.

Although ultrastructural studies have revealed unique features of AVs, little is known of the proteins that compose their virions. In a recent report, Cui et al. (2007) identified seven structural proteins associated with the TnAV-6a virion. Of these, four, including the capsid protein, had homologues in SfAV-1a and HvAV-3c. However, the complex symmetry, large size and ultrastructure of AV virions suggest that they are composed of more than seven structural proteins. Here, we report the results of a proteomic analysis of SfAV-1a virion proteins identified using nano-liquid chromatography/tandem mass spectrometry (nano-LC-MS/MS) (Mueller et al., 2007). Twenty-one virus-coded proteins associated with the SfAV-1a virion were identified. These include four SfAV-1a homologues [open reading frames (ORFs) 041, 048, 064 and 084] of TnAV-6a (ORFs 153, 141, 115 and 43, respectively) that were identified by Cui et al. (2007). Moreover, comparative analyses revealed that the TnAV-6a genome encoded 13 additional apparent homologues of the SfAV-1a virion proteins, whereas a total of 20 were encoded by HvAV-3e.

To determine the protein components, SfAV-1a virions were isolated from the haemolymph of early fourth instar Spodoptera exigua larvae, 5–7 days post-infection by isopycnic centrifugation on caesium chloride gradients (Federici et al., 1990). The purity of virion preparations was assessed by electron microscopy (Fig. 1). Purified virion proteins were fractionated by SDS-PAGE (Laemmli, 1970) and stained with Coomassie brilliant blue (Fig. 2a). Contiguous sections spanning the complete gel lane that contained at least 21 visible bands were excised for nano-LC-MS/MS peptide sequence analysis (Mueller et al., 2007; Pan et al., 2003) at the W. M. Keck Proteomics Laboratory (University of California, Riverside, USA). Peptide sequences were compared against GenBank/EMBL/DDBJ databases and coding sequences of the SfAV-1a genome (GenBank accession no. AM398843[GenBank] ).

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Fig. 1. Purification of SfAV-1a virions used for mass spectrometry analysis. (a) Virions prepared from SfAV-1a-infected S. exigua haemolymph were purified by caesium chloride isopycnic gradient centrifugation; a single distinct band (arrow) was observed after centrifugation for 40 h at 149 000 g. (b, c) Micrographs showing SfAV-1a virions in samples prepared from the single band shown in (a). Bar, 0.2 µm (b) and 100 nm (c).

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Fig. 2. Protein profiles of purified SfAV-1a virions. (a) SfAV-1a virions were purified by caesium chloride gradient centrifugation and virion proteins were fractionated by using 10 % SDS-PAGE and stained with Coomassie brilliant blue. (b) Relative location of 21 SfAV-1a-encoded virion proteins identified by MS. MW, Molecular mass standard markers.

In Table 1 we present a list of these peptide sequences, the corresponding SfAV-1a ORFs, the molecular masses of the putative proteins encoded by these ORFs, their closest viral homologues and their putative functions. SfAV-1a-encoded proteins were identified by 100 % homology with at least two peptide sequences derived by mass spectrometry (MS), although most were identified using the deduced MS sequences of six or more peptides. Of the 21 SfAV-1a virion proteins identified (Fig. 2b), 15 migrated in the SDS-PAGE gel within ±3 kDa of their empirically predicted position. In order of decreasing predicted molecular masses from 119.3 to 14.4 kDa, these were ORFs 084, 009, 047, 064, 027, 048, 015, 041, 054, 033, 003, 061, 109, 038 and 091. The remaining six, predicted to range from 29.6 to 8.6 kDa, had apparent molecular masses that were significantly different from their deduced amino acid sequences. For these, the predicted and apparent molecular masses, respectively, were 29.6 versus 40 kDa for ORF075; 26 versus 8 kDa for ORF043; 25.6 versus 51 kDa for ORF035; 20.1 versus 45 kDa for ORF036; 13.7 versus 18 kDa for ORF002; and 8.6 versus 5 kDa for ORF060. The differences in predicted and apparent molecular masses suggest that these proteins undergo post-translational modification, including glycosylation, phosphorylation and/or proteolytic cleavage (Witze et al., 2007).

Table 1. SfAV-1a virion proteins identified by MS

The two most abundant proteins were the major capsid protein (ORF041), previously characterized by Stasiak et al. (2003), and P64 (ORF048; 64.6 kDa), an unusually basic protein with a predicted pI of 12.2. Arginine (19.8 %) and lysine (6.2 %) composed 26 % of the residues in P64 and an additional 24.1 % of this protein was composed of serine (18.9 %) and threonine (5.2 %). Two domains spanning residues 1–263 (amino-terminal) and 265–565 (carboxy-terminal) were predicted by the SCRATCH protein predictor program (). The amino-terminal contained four well-conserved virus-specific 2-cysteine adaptor domains (P08793.1; Iyer et al., 2006) at positions 13–50, 58–94, 141–177 and 182–218. These domains are known to be fused to ovarian tumour/A20-like peptidases and serine-threonine protein kinases. Although its biochemical function is unknown, the occurrence of the 2-cysteine adaptor in these proteins suggests that they could function as viral adaptors that facilitate SfAV-1a virion morphogenesis. The carboxy-terminal portion of P64 shared no homology with virus proteins of known function. However, its residue from 269–365 shared a high level of identity and similarity (53 and 61 %, respectively) with the sperm nuclear basic protein PL-1 isoform (GenBank accession no. AAT45385[GenBank] ) of the mollusk Spisula solidassina. The function(s) of P64 is unknown. However, because it is an unusually basic virion structural protein, it is possible that it could function in condensing and packaging DNA during SfAV-1a assembly. It must be noted that viral core basic DNA-binding proteins typically have low molecular masses, for example, the small arginine-rich protein homologues among baculoviruses represented by the 6.9 kDa protein in virions of Autographa californica nucleopolyhedrovirus (AcMNPV) (Wilson et al., 1987). Thus, P64 could represent a novel DNA-binding core protein among viruses.

At least five SfAV-1a virion proteins (ORFs 009, 047, 075, 091 and 109) were identified that contained conserved domains in protein families known to be involved in nucleic acid metabolism. ORF009 contains conserved DEADc (cd00046) and SNF2_N (P00176[GenBank] ) box motifs, in addition to sequences conserved in the HepA (COG0553) and SrmB (COG0513) protein superfamilies. Collectively, these proteins are known to function as DNA/RNA helicases, and participate in DNA replication, recombination and repair, transcription and translation. They are also known to be involved with pre-mRNA splicing, ribosome biogenesis, RNA decay and nucleocytoplasmic transport. The ORF047 protein contains multiple conserved domains, including Smc (COG1196), HEC1 (COG5185) and MukB (COG3096), known to function as chromosome segregation and partitioning ATPases, and SMC_N, RecF/RecN/SMC amino-terminal domains (P02463) and SbcC (COG0419), known to be involved in DNA replication, recombination and repair. ORF0047 contains a myosin tail 1 domain (P1576) with a calcium-binding coiled-coil domain (P07888). The SfAV-1a ORF075 contains a conserved motif from members of the S1/P1 nuclease family (P02265). Members in this family contain both S1 and P1 nucleases (EC:3.1.30.1) known to cleave RNA and single-stranded DNA with no base specificity. The SfAV-1a ORF091 protein contains a single high mobility group (HMG)-box/HMGB_UBF_HMG-box motif (P00505; cd01390) and an overlapping NHP6B motif (COG5648). These motifs are present in a wide variety of eukaryotic chromosome-associated proteins and transcription factors. HMGs bind to the minor groove of DNA. Three distinct classes of HMGs are recognized. Class I proteins bind DNA in a sequence-specific manner; class II (e.g. non-histone chromosomal proteins HMG1 and HMG2) and class III proteins (HMGB-UBF, nucleolar and mitochondrial transcription factors) bind DNA non-specifically and contain two or more tandem HMG boxes. Lastly, ORF109 contains a catalytic domain of CTD-like phosphatases (CPDc, smart00577), nuclear interacting factor-like phosphatases (NIF, NLI, P03031) and transcription factor TFIIF-interacting CTD-phosphatases (FCP1, COG5190). Current data suggest that, in addition to their roles in transcription, emerging roles of CTD-like phosphatases include chromatin remodelling and modification, DNA repair and mRNA packaging, editing and nuclear export (Meinhart et al., 2005). Therefore, ORF109 could have multiple functions in the metabolism of nucleic acids, although most likely, its primary role involves mRNA biogenesis during early virogenesis.

The ORF061 protein contains an Erv1/Alr conserved domain (COG5054) found in mitochondrial thiol oxidases, such as thiol oxidase that is involved in the biogenesis and maturation of cytosolic iron/sulfur proteins that play roles in a number of biochemical processes, including post-translation modification and protein turnover. ORF061 also shares 46 % homology at residues 21–120 with the Kil-9GL protein (GenBank accession no. AAF27964[GenBank] ) of the African swine fever virus. The poxvirus structural E10R and G4L proteins also contain the Erv1/Alr conserved domain. The thiol transferase activity of G4L is required for formation of disulfide bonds and it is known that both E10R and G4L facilitate disulfide bond formation between conserved cysteine residues of viral membrane-bound structural proteins, and are essential for poxvirus virion morphogenesis (Senkevich et al., 2002a, b; White et al., 2002). Thus, ORF061 may participate in SfAV-1a virion maturation.

Thirteen of the SfAV-1a virion proteins that were identified lacked conserved domains, suggestive of their potential functions. ORF015 shares homology in different regions with a number of proteins that contain zinc-finger/RING finger motifs, with conserved cysteine and histidine residues at the corresponding positions. These include cell growth regulator, mindbomb ubiquitin 3 ligase and a putative inhibitor of the apoptosis protein in Arabidopsis thaliana (46, 41 and 53 %, and GenBank accession nos AAH37677[GenBank] , AAN18023[GenBank] and AAC79106[GenBank] , respectively). Interestingly, ORF015 shares a high level of identity and similarity (57 and 68 %) with ORF084 (GenBank accession no. NP_037844[GenBank] ) of the Spodoptera exigua nucleopolyhedrovirus (SeMNPV). The function of ORF084 in SeMNPV is unknown.

SfAV-1a ORFs 002, 003, 027, 033, 035, 036, 038, 043, 054, 060, 064 and 084 do not have conserved domains or motifs, although ORF084 and ORF064 share low levels of sequence similarities with dynein-like beta chain and serine-threonine kinase, respectively (Bideshi et al., 2006). Apparent homologues and orthologues are present in HvAV-3e, TnAV-6a and iridoviruses (Table 1; Bideshi et al., 2006). However, at present, their putative roles in SfAV-1a virion structure and virogenesis could not be assigned due to a lack of information regarding the function(s) of related proteins in their respective hosts.

In many large DNA viruses, virus-encoded DNA polymerases are known to be present in their virions. For example, proteomic analyses have shown that DNA polymerase is present in virions of the AcMNPV and Helicoverpa armigera NPV baculoviruses (Braunagel et al., 2003; Deng et al., 2007), vaccinia virus (Resch et al., 2007) and human cytomegalovirus (Huang & Johnson, 2000). The peptide sequences obtained in the present study did not match or show significant homology with the predicted SfAV-1a ORF001 DNA polymerase protein sequence (125.8 kDa). Furthermore, a protein band occurring in the predicted range for ORF001 was not observed using SDS-PAGE. Whether ORF001 is present in SfAV-1a virions at quantities insufficient for detection by the methods used is not known. Further studies are required to determine whether ORF001 is required for SfAV-1a genome replication or if its corresponding gene is transcribed following infection of susceptible cells. The TnAV-6a virus-coded DNA polymerase (ORF001; Wang et al., 2006) was also not identified as a structural protein in virions of this ascovirus (Cui et al., 2007).

Recently, Cui et al. (2007) used a similar approach to identify seven structural proteins of the TnAV-6a virion (ORFs 153, 147, 142, 141, 115, 043 and 002). Of these, only four (ORFs 153, 141, 115 and 043) had protein homologues in SfAV-1a virions (ORFs 041, 048, 064 and 084, respectively) and gene homologues were also found in HvAV-3e (ORFs 056, 061, 077 and 146). As with SfAV-1a, the capsid (ORF041) and P64 (ORF048) homologues were also the most prevalent species in TnAV-6a (ORFs 153 and 141), as observed in SDS-PAGE profiles (Cui et al., 2007).

The identification of 21 SfAV-1a virion proteins in this study, compared with only seven for TnAV6a reported by Cui et al. (2007), suggests that SfAV-1a is structurally more complex than TnAV-6a. However, further studies are required to confirm this speculation. For example, it is not known whether the complete SDS-PAGE virion protein profile was reported for TnAV-6a, as the proteins presented ranged from 31 to 220 kDa, so representation of the protein profile below 31 kDa, if known, was not shown (Cui et al., 2007). Considering this, 10 of the 21 SfAV-1a virion proteins identified by SDS-PAGE migrated below the 31 kDa standard molecular marker and three of these (ORFs 043, 060 and 091) were prominent species occurring below the 14 kDa standard marker (Fig. 2b). Thus, whether homologues of these 10 proteins occur in TnAV-6a virions is not known based on available data.

Aside from these technical reasons for the larger number of proteins detected in the SfAV-1a virion, the presence of the distinctive vesicular occlusion body produced by this virus could be responsible for some of these additional proteins (Federici et al., 1990). This structure is not produced by any of the other known ascoviruses. So, although the occlusion body may contain some SfAV-1a virion proteins, it is a structure separate from virions and we can not therefore rule out the possibility that it contains non-virion proteins that could have contaminated our virion preparations. These proteins would have appeared as minor proteins in the proteomic results, but the corresponding genes would also be present in the SfAV-1a genome, and as a result would be inadvertently identified as virion proteins.

Finally, previous phylogenetic studies on ascoviruses were based on comparative analyses of capsid, DNA polymerase, thymidine kinase and ATPase III proteins, and provided evidence that these viruses evolved from invertebrate iridoviruses (Stasiak et al., 2003). Indeed, analysis of genomic sequences of the SfAV-1a, HvAV-3e and TnAV-6a showed that these viruses contained an abundance of gene orthologues in iridoviruses compared with other virus families (Asgari et al., 2007; Bideshi et al., 2006; Wang et al., 2006). The results reported here showed that 10 of the 21 SfAV-1a virion structural proteins (ORFs 009, 035, 041, 048, 054, 061, 064, 084, 091 and 109) had orthologues in iridoviruses, whereas only one orthologue (ORF015) was found in baculoviruses. Therefore, in addition to comparative genomics, the proteomic data reported here provide additional strong evidence that ascoviruses evolved from invertebrate iridoviruses.

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Received 25 July 2008; accepted 5 October 2008.