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. 2010 Jul 2;405(1):253–258. doi: 10.1016/j.virol.2010.05.038

Proteomic analysis of Chilo iridescent virus

İkbal Agah İnce a,c,d, Sjef A Boeren b, Monique M van Oers a,⁎, Jacques JM Vervoort b, Just M Vlak a
PMCID: PMC7111926  PMID: 20598335

Abstract

In this first proteomic analysis of an invertebrate iridovirus, 46 viral proteins were detected in the virions of Chilo iridescent virus (CIV) based on the detection of 2 or more distinct peptides; an additional 8 proteins were found based on a single peptide. Thirty-six of the 54 identified proteins have homologs in another invertebrate and/or in one or more vertebrate iridoviruses. The genes for 5 of the identified proteins, 22L (putative helicase), 118L, 142R (putative RNaseIII), 274L (major capsid protein) and 295L, are shared by all iridoviruses for which the complete nucleotide sequence is known and may therefore be considered as iridovirus core genes. Three identified proteins have homologs only in ascoviruses. The remaining 15 identified proteins are so far unique to CIV. In addition to broadening our insight in the structure and assembly of CIV virions, this knowledge is pivotal to unravel the initial steps in the infection process.

Keywords: Chilo iridescent virus, Invertebrate iridovirus 6, Proteomics, LC-MS/MS

Introduction

Chilo iridescent virus (CIV), also known as Invertebrate iridescent virus 6, belongs to the family Iridoviridae and is the type species of the genus Iridovirus (Fauquet et al., 2005, Williams, 1996, Williams et al., 2005, Willis, 1990). Iridoviruses are large, cytoplasmic, icosahedral viruses with a linear double-stranded DNA genome, which is both circularly permuted and terminally redundant (Darai et al., 1983, Goorha & Murti, 1982). The CIV virion consists of an unusual three layer structure containing an outer proteinaceous capsid, an intermediate lipid membrane, and a core DNA–protein complex containing the 212, 482 bp genome (Jakob et al., 2001, Williams, 1996, Williams et al., 2005). Up to now, thirteen complete sequences of iridovirus genomes have been published, including CIV (Huang et al., 2009, Williams et al., 2005). The availability of the CIV sequence facilitates the identification and functional analysis of the proteome of CIV virions. Replication of CIV occurs in the nucleus of infected cells and the assembly takes place in the cytoplasm (Goorha and Murti, 1982).

Many questions remain to be answered concerning the structure and scaffolding of the virus particles, the nature of virus–host interactions and the initial steps in virus infection, including the mechanism behind the onset of transcription of CIV genes. Viral structural proteins are likely to play crucial roles in these processes. Initiation of viral transcription for instance requires one or more virion proteins, since CIV DNA alone is not infectious, similar to what has been shown for the vertebrate iridovirus Frog virus 3 (Willis and Granoff, 1985). In previous studies, efforts have been made to characterize the polypeptides in CIV virions by one- or two-dimensional SDS-PAGE. The presence of 21–28 polypeptides was revealed by one-dimensional SDS-PAGE, while 35 polypeptides were observed in two-dimensional SDS-PAGE (Barray & Devauchelle, 1979, Barray & Devauchelle, 1985, Cerutti & Devauchelle, 1985, Kelly & Tinsley, 1972, Orange & Devauchelle, 1987). The size of these polypeptides ranged from 11 to 300 kDa. However, most of these proteins were not further characterized and it is unknown, except for the major capsid protein MCP, by which CIV genes they are encoded.

In the current study we identified the CIV virion proteins by a proteomic approach, based on a combination of one-dimensional SDS-PAGE and Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS). The data obtained were analyzed by searches against a CIV ORF database. This provided a fast and highly sensitive method for the identification of genes through the sequences of the encoded proteins (Pandey and Mann, 2000).

Results

To identify the virion proteins of CIV, the proteins of purified virion particles were separated by one-dimensional SDS-PAGE. Staining of the gel with colloidal blue revealed at least 21 proteins ranging from 10 to 250 kDa (Fig. 1 ) much in line to what has been found previously (Barray & Devauchelle, 1979, Barray & Devauchelle, 1985, Cerutti & Devauchelle, 1985, Kelly & Tinsley, 1972, Orange & Devauchelle, 1987). The gel lane was divided into 6 slices containing proteins with a molecular mass lower than 26 kDa, ranging from 26–34 kDa, 34–43 kDa, 43–55 kDa or 55–95 kDa and higher than 95 kDa, respectively. The proteins were digested with trypsin and analyzed by LC-MS/MS. A decoy database strategy (Elias and Gygi, 2007) was used which, after applying the appropriate filters, resulted in 89 protein hits: 54 CIV proteins, 34 contaminants and 1 decoy hit giving a False Discovery Rate of 1.1%. Out of the 54 CIV proteins, 46 of the more abundant proteins were identified with 2 or more peptides (Table 1 ), while relatively small proteins like ORFs 342R, 227L or 104L as well as some less abundant proteins could be identified with one peptide only (Table 2 ). The proteins with one hit were manually verified to correlate well to the theoretical b+y ion spectrum and to be unique for one protein only (see also Supplementary Material S1).

Fig. 1.

Fig. 1

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SDS-PAGE profile and LC-MS/MS identification results of purified CIV virion proteins. CIV proteins were separated by 12% one-dimensional SDS-PAGE and stained with colloidal blue. The SDS-PAGE gel was divided into 6 slices, which, based on comparison to a molecular marker, ranged from higher than 95 kDa, 55–95 kDa, 43–55 kDa, 34–43 kDa, 26–34 kDa and lower than 26 kDa. Proteins were in-gel-digested with trypsin, extracted and subjected to LC-MS/MS. The column on the right indicates the relative abundance of the proteins as visualized by SDS-PAGE. The boxes on the left give the ORF numbers of the identified proteins in a particular gel slice in order of the predicted mass (see Table 1, Table 2). Underlined numbers represent single peptide hits. Indications R and L point towards the direction of transcription from the CIV genome (see Fig. 2).

Table 1.

Structural proteins of CIV identified by LC-MS/MS with 2 or more distinct peptides.. The ORFs are ordered by the mass of the encoded proteins (column 3).

ORF NCBI accession No Mol. mass (kDa) Protein coverage (% by amino acids) Peptide hits on first rank Relative abundancea (% peak area) Predicted domains/function
443R AAK82303 237.22 8.10 15 0.30
295L AAK82156 156.42 24.70 43 0.22 Bipartite nuclear localization signal
179R AAB94478 137.93 14.70 24 0.06 CAP10, Putative lipopolysaccharide-modifying enzyme, tyrosine protein kinase
022L AAD48148 135.34 32.20 34 0.20 Putative nucleoside triphosphatase I; DEXDc; DEAD-like helicase superfamily
261R AAK82122 129.06 2.70 30 2.20 Potential repetitive protein
209R AAK82071 118.34 39.20 52 0.69 Serine/threonine protein kinase
396L AAK82256 111.28 21.90 29 0.16 Potential repetitive protein
268L AAK82129 83.22 46.10 74 2.06
149L AAB94464 76.41 36.60 72 0.91
232R AAK82093 75.56 49.00 75 1.63 DNA polymerase (viral) N terminal domain, 2-cysteine adaptor domain, OTU like cysteine protease
439L AAK82299 63.45 12.10 8 0.02 Protein kinase domain
361L AAK82221 60.58 50.70 55 1.06 Peptidase_C1A_CathepsinB
380R AAK82240 59.91 54.50 73 2.05 S_TKc, Serine or threonine-specific kinase subfamily
213R AAK82075 58.42 29.70 22 0.16 Putative peptidoglycan bound protein
118L AAB94444 55.29 55.10 65 1.77 Putative envelope protein
198R AAK82060 52.15 42.60 26 0.32
274L AAK82135 51.29 63.20 157 17.97 Major capsid protein
229L AAK82090 50.64 22.30 15 0.25
337L AAK82199 46.13 27.20 25 0.21 Poxvirus protein of unknown function
159L AAB94468 45.76 34.90 58 3.98
329R AAK82190 42.74 28.80 16 0.29
219L AAK82081 34.64 19.00 5 0.01
142R AAB94459 33.64 33.60 16 0.25 dsRNA-specific ribonuclease
457L AAK82317 33.13 25.90 47 2.97
155L AAB94465 29.81 39.20 23 0.40
401R AAK82261 28.23 25.50 11 0.04 HMG-box superfamily of DNA-binding proteins
117L AAB94443 27.45 29.70 43 0.93
415R AAK82275 26.66 63.20 70 1.34
309L AAK82170 24.83 70.00 12 0.10
422L AAK82282 22.73 49.50 19 0.20 Cydia pomonella granulovirus ORF34
378R AAK82238 22.21 47.70 12 0.10 2-cysteine adaptor domain
355R AAK82216 22.01 52.70 10 0.02 Catalytic domain of ctd-like phosphatases
234R AAK82095 21.09 62.70 63 3.07
111R AAB94438 20.01 35.40 9 0.05
096L AAB94430 19.69 33.30 11 0.05 Fasciclin domain
374R AAK82234 19.12 22.40 3 0.00 Bat coronavirus spike protein
325L AAK82186 18.91 24.50 5 0.08
203L AAK82065 18.53 18.80 7 0.02
084L AAB94426 18.40 25.50 15 0.04
061R AAB94416 17.91 31.60 17 0.01 Lysosome associate membrane glycoproteins
123R AAB94448 16.38 7.70 3 0.00 Dual specificity phosphatases
453L AAK82313 15.91 26.10 12 0.05 Protein disulfide isomerase
034Rb AAK81969 14.63 16.40 5 0.03
366R AAK82226 13.66 17.50 2 0.01
138R AAB94455 13.03 16.70 7 0.02
312R AAK82173 10.60 20.70 3 0.01
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a

The relative abundance was calculated by Bioworks as % peak area over all peaks (including contaminants observed) shown after applying the following filter settings: ΔCn > 0.08, Xcorr > 1.5 for charge state 2+, Xcorr > 3.3 for charge state 3+ and Xcorr > 3.5 for charge state 4+, Sf > 0.6.

b

This protein was identified in the 34–43 kDa gel piece.

Table 2.

Structural proteins of CIV identified by LC-MS/MS with 1 peptide. The ORFs are ordered by the mass of the encoded proteins (column 3).

ORF NCBI Accession No Molecular mass (kDa) Peptide sequence Protein coverage (% by amino acids) MH+ Delta m/z (ppm) z Xcorr
317L AAK82178 43.95 IVNLIPQGQFQAK 3.11 1455.832 −0.30 2 1.77
130R AAB94451 23.18 ICFSEQPLLDDFSNK 7.46 1812.847 1.04 2 2.86
307L AAK82168 22.86 LKPLGYLNSLQ 5.58 1245.720 0.33 2 1.81
395R AAK82255 17.28 YAINNENQYR 6.62 1284.597 −0.72 2 2.54
010R AAK81948 12.84 TGSMVCSSTR 8.33 1085.471 3.19 2 2.34
342R AAK82203 9.33 IQAQNYATMGIYN-QGSQIR* 21.59 2156.055 2.74 2 3.73
227L AAK82088 7.72 TFAYEVPIRa 14.30 1095.583 1.49 2 2.61
104L AAB94434 7.05 RVACSPR* 12.30 845.441 2.01 2 2.78
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a

The same peptide was measured multiple times in different gel slices.

The proteins identified are indicated in Fig. 1. A genomic map of CIV ORFs that encode polypeptides represented in the proteome of CIV particles is shown in Fig. 2 . For individual CIV virion proteins, 2.7% to 70% of the amino acid sequence was covered with peptides retrieved from the analysis. The major capsid protein (MCP) encoded by ORF 274L is one of the most abundant CIV proteins (Barray & Devauchelle, 1979, Barray & Devauchelle, 1985, Cerutti & Devauchelle, 1985, Kelly & Tinsley, 1972) and this is clearly reflected by its relative abundance in the current analysis compared to all other CIV proteins (Table 1). The nature of the other major band is not clear at this moment.

Fig. 2.

Fig. 2

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Linearized genomic presentation of the 54 CIV structural protein ORFs determined by LC-MS/MS. Arrows indicate the positions and the direction of gene transcription (R or L). Red arrows are ORFs unique to CIV, green arrows represent ORFs present in all sequenced iridovirus genomes. The yellow and the white ORFs, have an entomopox- and baculovirus homolog, respectively. The remaining ORFs are indicated in blue. Genomic positions are indicated on the right in base pair number.

Functional domains alluding to possible functions were found in fifteen other identified virion proteins, including three putative serine/threonine kinases (ORFs 209R, 380R and 439R), one dual specificity phosphatase (123R), a protein with homology to the N-terminal domain of viral DNA polymerases (232R), carboxy-terminal domain (CTD) phosphatase (355R), nucleoside triphosphatase (NTP I) (22L), fasciclin (96L), ribonuclease III (142R), tyrosine protein kinase (179R), cathepsin (361L), DNA binding protein (401R), protein disulfide isomerase (453L), lysosome associate membrane glycoprotein (061R), and a ranavirus envelope protein homolog (118L). For the 38 remaining proteins in the virion, we have no clear idea about their specific function at this moment (Table 1, Table 2). Some of these show partial homology to viral proteins of poxvirus, coronavirus or baculovirus origin.

Recent cryoelectron microscopy studies on the capsid of CIV revealed, in addition to MCP, a group of relatively less abundant capsid proteins (Yan et al., 2009). These proteins form a complex which contains a “finger” protein, a “zip” protein, a pentameric complex and an anchor protein. The molecular mass estimations for the finger and zip proteins, the anchor protein and the monomer of the pentameric complex were estimated to be 19.7, 11.9, 32.4 and 39.3 kDa, respectively. For the finger protein the standard deviation was 1.5 kDa, giving a size range of 18.2–21.2 kDa (Yan et al., 2009). Based on this range, seven candidate genes for the finger protein were found in the CIV proteome: ORFs 234R, 111R, 096L, 374L, 325L, 203L, and 084L from large to small (Table 1). The zip protein with an expected size range of 10.5 to 13.3 kDa (1.4 kDa standard deviation) may correspond to three candidate ORFs represented in the proteome: 010R, 138R and 321R. The monomer of the pentameric complex estimated at 39.3 kDa corresponds most closely in size to ORFs 329R and 219L. Anchor protein candidate genes in the CIV proteome could be 457L or 142R, with sizes close to 32.4 kDa (Table 1).

Discussion

The CIV proteome revealed 54 proteins. The genes encoding these virion proteins are scattered over the genome (Fig. 2). It is not known which of the identified proteins are engaged in the scaffolding and assembly of CIV virions, and which are not essential for building the virion structure, but may be important for other aspects, such as the initial stages of the infection process and the regulation of gene expression. It is possible that one of these additional proteins is involved in chaperoning the viral DNA into the nucleus to initiate DNA replication (Willis and Granoff, 1985). To get a better clue about their importance, the conservation of the CIV virion protein genes in the complete genomes of members of the family Iridoviridae as well as Ascoviridae was assessed. The latter family was included since a common ancestry between iridoviruses and ascoviruses has been inferred from phylogenetic analysis (Stasiak et al., 2000).

Of the 54 ORFs encoding CIV virion proteins identified in the current study, thirty-four have homologs in Invertebrate iridovirus 3 (IIV3), which belongs to the genus Chloriridovirus (Table 3 , column 2) (Chen et al., 2008, Song et al., 2004). Fifteen of the 34 ORFs with homologs in IIV3, also have homologs in one or more vertebrate iridoviruses. The CIV proteome shares five ORFs with all iridoviruses: 022L, 118L, 142L, 274L (MCP) and 295L, and these may be considered to belong to the iridovirus core genes. The Rana gryliovirus (RGV) ORF 53R, which is a homolog of the putative core gene 118L, has been shown to encode a novel iridovirus envelope protein (Zhao et al., 2008). The CIV proteome shares thirteen viral protein homologs with Singapore grouper iridovirus (SGIV) virion proteins identified by two independent mass spectrometric approaches (Chen et al., 2008, Song et al., 2004).

Table 3.

List of CIV virion proteins identified by LC-MS/MS ordered by mass with homolog in other iridoviruses and/or ascoviruses.⁎

Invertebrate
 Vertebrate
 Ascoviridae‡
Irido-virus
 Chlorirido-virus
 Ranavirus
 Lymphocystivirus
 Megalocytivirus
CIV IIV3 ATV TFV FV3 SGIV GIV STIV LCDV-C LCDV-1 ISKNV RBIV OSGIV
443R 91L
295L 16R 72R 45R 41R 57L 29L 45R 234R 92R 76L 72L 75L a144R,b43R,c84L
179R 35R 60R 29R 27R 78L 44R 31R 172R 110R a129L, b58R, c90R
022L 87L 7L 9L 9L 60R 30L 11L 75L 70L 63L 59L 63L a15R,b161R,c9R
261R 91L
209R a76R, b115L, c64R
396L 91L, 8L
268L 74L
149L 113L
232R 84L 84L 21R b141R
439L 35R 110R 114L 111L c90R
38R
98L
361L 24R 223L 23R a101L, b102R, c114R
380R 10L 84L 19R 19R 39L 17L 21R 13L, 45R, 149R, 165L 174R,184R, 200L 5L, 42R, 47R, 50R, 51R, 88L
11L 150L+ 83L
213R 51L
118L 6R 53L 55R 53R 88L 49L 55R 157R 35L 7L 8L 8L b157L,d5L
198R 69L
274L 14L 14L 96R 90R 72R 39R 96R 43L 80L 6L 7L 7L a55R,b153R,c41R,d19R
229L 46R 3R 4R 229L 16L 2L 5R
337L 47R 1L 2L 2L 19R 4R 2L 38R 89L 85L b129L, c54R
329R 99R
219L 36R, 91L
142R 101R 25R 85L 80L 84L 46L 87L 186R 74R 87R 83R 85R a26R,b8R,c22R,d18L
155L 113L
401R 68R
117L 107R 83L 20R 20R 038L 16L 23R 73R 109R
415R 18L
309L 63R
422L d8R, d9R, d14L
307L 33L 11R 100L 94L 98R 56R 152L 9R 86R a142R, c86L, d4R
378R 100L 84L 19R 19R 39L 17L 21R 13L 50R b141R
355R 104L 67R 40R 37R 61R 31L 41R 147L 43L 5L 6L a108R, b93L, c109L
374R b1R
203L 85L
395R 1R
453L 41R
366R 63R 33R 32R 35R
010R 43R
342R 115R
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⁎ORFs in bold are conserved in all analyzed iridio- and ascovirus genomes.

‡ The a-d indices for the ascovirus ORFs refer to the following species: a HvAV3e, Heliothis virescens ascovirus 3e (Asgari et al., 2007), b TnAV2c, Trichoplusia ni ascovirus 2c (Wang et al., 2006), c SfAV1a, Spodoptera frugiperda ascovirus 1a (Bideshi et al., 2006), d DpAV4a, Diadromus pulchellus ascovirus 4a (Bigot et al., 2008, Stasiak et al., 2000). The names of the other viruses are abbreviated as follows; CIV, Chilo iridescent virus (Jakob et al., 2001); IV3, Aedes taeniorhynchus iridescent virus (Delhon et al., 2006); ATV, Ambystoma tigrinum stebbensi virus (Jancovich et al., 2003); TFV, Tiger frog virus (He et al., 2002); FV3. Frog virus 3 (Tan et al., 2004); SGIV, Singapore grouper iridovirus (Song et al., 2004); GIV, Grouper iridovirus (Tsai et al., 2005); STIV, Soft-shelled turtle iridovirus (Huang et al., 2009); LCDV-C, Lymphocystis disease virus - isolate China (Zhang et al., 2004); LCDV-1, Lymphocystis disease virus 1 (Tidona and Darai, 1997); ISKNV, Infectious spleen and kidney necrosis virus (He et al., 2001); RBIV, Rock bream iridovirus (Do et al., 2004); OSGIV, Orange-spotted grouper iridovirus (Lü et al., 2005).

Previous phylogenetic studies on ascoviruses were based on comparative analyses of the capsid protein, DNA polymerase, thymidine kinase, and ATPase III, and led to the hypothesis that ascoviruses may have evolved from invertebrate iridoviruses (Stasiak et al., 2003). The proteomic analysis of CIV performed here showed that 16 ORFs encoding CIV virion proteins have homologs in one or more ascoviruses (Asgari et al., 2007, Bideshi et al., 2006, Bigot et al., 2008, Stasiak et al., 2000, Wang et al., 2006). Nine CIV structural proteins have homologs in Heliothis virescens ascovirus 3e (HvAV3e), thirteen have homologs in Trichoplusia ni ascovirus 2c (TnAV2c), eleven in Spodoptera frugiperda ascovirus (SfAV1a) and six in Diadromus pulchellus ascovirus 4a (DpAV4a). The gene products of six of the eleven SfAV1a homologs were also found in the proteome of SfAV1a virions (Tan et al., 2009a). A homolog of the SfAV1a virion protein P64, which was recently shown to be a major DNA binding protein with proposed DNA condensing activity (Tan et al., 2009b) is not encoded in the CIV genome.

Three of the identified CIV virion ORFs are found in one or more ascoviruses, but not in other iridoviruses (209T, 422L and 374R). One of these (422L) is the only CIV virion ORF with a baculovirus homolog (Cydia pomonella granulovirus ORF34; genus Betabaculovirus). ORF 337L has homology to an entomopoxvirus gene (Table 1, Fig. 2). These results underscore the evolutionary distance of iridoviruses from both baculoviruses and entomopoxviruses and the closer relation to ascoviruses. Despite the proposed close evolutionary relation between the symbiotic ascovirus DpAV4a and Chilo iridescent virus (Bigot et al., 2009) the number of CIV virion proteins with homologs in DpAv4a is limited in comparison to the other ascoviruses.

Although the morphology of the virions of members of the family Ascoviridae differs considerably from that of viruses of the family Iridoviridae, evidence is mounting that the ascoviruses and iridoviruses shared a common ancestor. Phylogenetic analyses based on proteins found in most enveloped dsDNA viruses provide strong evidence that ascoviruses evolved from iridoviruses, despite the marked differences in the characteristics of the virions belonging to these two families and differences in their cytopathology (Bigot et al., 2008). The conservation of structural proteins between CIV and ascoviruses further supports the hypothesis of common ancestry.

In conclusion, this is the first detailed study towards the determination of the virion proteins of an invertebrate iridovirus. This study will contribute to a better understanding of the molecular mechanisms underlying CIV virion assembly, CIV entry into cells, the initial steps of early iridovirus gene expression and the cell to cell movement of this virus.

Materials and methods

Preparation of virus particles and gel electrophoresis

CIV was propagated in larvae of the wax moth, Galleria mellonella, isolated as described by Marina et al. (1999) and further purified by 25–65% sucrose density gradient centrifugation. The purified CIV particles were checked for quality by transmission electron microscopy and quantified by UV spectroscopy. The purified particles were denatured and the proteins were separated by 12% one-dimensional SDS-PAGE. The gel was stained with colloidal blue and the gel lane containing the virion proteins was cut into six segments based on a comparison with molecular markers. Each gel piece was sliced and dehydrated with acetonitrile (100%) (ACN). After vacuum drying, the gel segments were incubated in 10 mM dithiothreitol in 50 mM ammonium bicarbonate (ABC buffer) at 57 °C for 1 h and subsequently in 55 mM iodoacetamide (Sigma) in ABC buffer at room temperature for 1 h. After a final wash step with ABC buffer the gel material was dried.

Trypsin digestion and LC-MS/MS

In-gel protein digestions were performed using sequencing grade modified porcine trypsin (Promega, Madison, WI) in ABC buffer at 37 °C for 15 h, after which the digests were centrifuged at 6000 g. The supernatants were collected, and the remaining gel pieces were extracted with 5% triflouroacetic acid (TFA) and then with 15% ACN /1% TFA. The extracts were combined with the supernatants of the original digests, vacuum-dried, and the dried material was dissolved in 20 μl 0.1% formic acid in water. The peptides resulting from this digestion were analyzed by LC-MS/MS. To this aim, 18 μl of the samples were concentrated over a 0.10 × 32 mm Prontosil 300-5-C18H (Bischoff, Germany) pre-concentration column at a flow of 6 μl/min for 5 min. Peptides were eluted from the pre-concentration column and loaded onto a 0.10 × 200 mm Prontosil 300-3-C18H analytical column with a gradient of 10% to 35% ACN in 0.1% formic acid at a flow of 0.5 μl/min for 50 min. After that the percentage of ACN was increased to 80% (with 0.1% formic acid) in 3 min as a column cleaning step. Between the pre-concentration and analytical column, an electrospray potential of 3.5 kV was applied directly to the eluent via a solid 0.5 mm platina electrode fitted into a P875 Upchurch microT. Full scan positive mode Fourier transform mass spectra (FTMS) were measured between mass-to-charge ratios of 380 and 1400 with a LTQ-Orbitrap spectrometer (Thermo electron, San Jose, CA, USA). MS/MS scans of the four most abundant doubly and triply charged peaks in the FTMS scan were recorded in a data dependent mode in the linear trap (MS/MS threshold = 10.000). All MS/MS spectra obtained with each run were analyzed with Biowork 3.1.1 software (Thermo Fisher Scientific, Inc.). A maximum of 1 allowed differential modification per peptide was set for oxidation of methionines and de-amidation of asparagine and glutamine residues. Carboxamidomethylation of cysteines was set as a fixed modification. Trypsin specificity was set to fully enzymatic and a maximum of 3 missed cleavages with monoisotopic precursor and fragment ions. The mass tolerance for peptide precursor ions was set to 10 parts per million (10 ppm = 0.01 op m/z 1000 amu) and for MS/MS fragment ions to 0.5 Da. An Invertebrate iridescent virus 6 protein database was used for the analysis (AF303741; created July 31, 2001; downloaded from www.ncbi.nlm.nih.gov/sites/entrez) after adding a list of commonly observed contaminants like: BSA (P02769, bovine serum albumin precursor), trypsin (P00760, bovine), trypsin (P00761, porcin), keratin K22E (P35908, human), keratin K1C9 (P35527, human), keratin K2C1 (P04264, human) and keratin K1CI (P35527, human). A decoy database was created by adding the reversed sequences using the program SequenceReverser from the MaxQuant package (Cox and Mann, 2008), resulting in a total of 1058 proteins in the database. To identify the proteins in the CIV virions, the MS/MS spectra obtained from the LC-MS/MS were searched against the CIV ORF database using Bioworks 3.3.1 (Table 1). The peptide identifications obtained were filtered in Bioworks with the following filter criteria: ΔCn > 0.08, Xcorr > 1.5 for charge state 2+, Xcorr > 3.3 for charge state 3+ and Xcorr > 3.5 for charge state 4+ (Peng et al., 2003). Only those proteins that showed a Bioworks Score factor (Sf) larger then 0.6 were considered.

Acknowledgments

This research was supported by a grant from the Scientific and Technological Research Council of Turkey and a Research Project Grant from the Graduate School for Production Ecology and Resource Conservation of Wageningen University, the Netherlands, to İkbal Agah İnce. Monique M. van Oers was supported by a MEERVOUD grant from the Research Council of Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO). All proteomic LC-MS/MS measurements were done at Biqualys Wageningen (www.biqualys.nl).

Footnotes

Appendix A

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2010.05.038.

Appendix A. Supplementary data

Supplementary material S1

Spectra of single hit peptides, arranged according to predicted molecular mass of corresponding proteins.

mmc1.doc (115KB, doc)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material S1

Spectra of single hit peptides, arranged according to predicted molecular mass of corresponding proteins.

mmc1.doc (115KB, doc)

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