Mimivirus Giant Particles Incorporate a Large Fraction of Anonymous and Unique Gene Products^{▿ †}

Sample preparation for bidimensional (2D) gel electrophoresis.

Acanthamoeba polyphaga mimivirus (23) was purified through a sucrose gradient (25%) and washed twice with phosphate-buffered saline (PBS) in the presence of protease inhibitors (Complete; Roche, Mannheim, Germany). The resulting pellet was solubilized in 40 mM Tris-HCl, pH 7.5, supplemented with 2% (wt/vol) sodium dodecyl sulfate (SDS; Sigma-Aldrich) and 60 mM dithiothreitol (DTT), followed by 5 min of heating at 95°C. The insoluble fraction was removed by centrifugation (12,000 × g, 4°C, 10 min), and soluble proteins were precipitated using a PlusOne 2-D cleanup kit (Amersham Biosciences) to remove SDS. The final pellet was resuspended in solubilization buffer {7 M urea, 2 M thiourea, 4% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)} and stored at −80°C until isoelectric focusing (IEF) was performed.

2D gel electrophoresis and silver staining.

Immobiline DryStrips (18 cm, pH 4 to 7 or 6 to 11; Amersham) were rehydrated overnight using 350 μl rehydration buffer (8 M urea, 2% [wt/vol] CHAPS, 60 mM DTT, 2% [vol/vol] Immobiline pH gradient (IPG) buffer [Amersham]) containing 200 μg of solubilized mimivirus proteins, and IEF was carried out according to the manufacturer's protocol (Multiphor II; Amersham). Before the second-dimension electrophoresis was performed, strips were equilibrated twice in 10 ml equilibration buffer (30% [vol/vol] glycerol, 2% [wt/vol] SDS, 6 M urea, 50 mM Tris-HCl, bromophenol blue, pH 8.8) for 15 min. This buffer was supplemented with 65 mM DTT for the first equilibration and with 100 mM iodoacetamide for the second one. The strips were then embedded in 0.5% agarose, and the proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Ettan DALT; Amersham) at 5 W/gel for 30 min, followed by 4 to 5 h at 17 W/gel. Following migration, gels were stained by a method compatible with mass spectrometry (36). Spots excised from the gel were stored at −20°C until identification. For accuracy, the spectra of at least two separate samples of each protein were analyzed and compared.

In-gel digestion and MALDI-TOF mass spectrometry (MS).

Spots excised from silver-stained gels were destained and subjected to in-gel digestion with trypsin (sequencing-grade modified porcine trypsin; Promega, Madison, WI) (36). Tryptic peptides were then extracted from the gel by successive treatments with 5% formic acid and 50% acetonitrile-5% formic acid. Extracts were pooled and dried in a Speedvac evaporator. Peptides resuspended in an α-cyano-4-hydroxycinnamic acid matrix solution (prepared by diluting 3 times a saturated solution in 50% acetonitrile-0.3% trifluoroacetic acid) were then spotted on the matrix-assisted laser desorption ionization (MALDI) target. Mass analyses were performed on a MALDI-time of flight (MALDI-TOF) Brüker Ultraflex spectrometer (Brüker Daltonique, Wissembourg, France). Mass spectra were internally calibrated using autolytic peptides from trypsin. Tryptic peptide mass lists were used to identify the proteins, using Mascot software. Searches were performed against all available sequences in public databases, including those for eukaryotes (http://www.matrixscience.com).

Immunization and Western blotting.

Acanthamoeba polyphaga mimivirus proteins resolved by 2D gel electrophoresis were transferred onto nitrocellulose membranes (Semi-Phor unit; Hoefer Scientific, San Francisco, CA). Membranes were then blocked in PBS supplemented with 0.2% Tween 20 and 5% nonfat dry milk (PBS-Tween-milk) for 1.5 h before incubation with anti-Acanthamoeba polyphaga mimivirus sera. The sera were obtained from BALB/c mice immunized by three intraperitoneal injections (with 15-day intervals between injections) of 5 μg of purified viral particles resuspended in CpG as an adjuvant. After 1 h of incubation (1:6,400 dilution in PBS-Tween-milk), membranes were washed three times with PBS-Tween and probed with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (1:1,000; Amersham). Detection was achieved by chemiluminescence (ECL; Amersham).

SDS-PAGE coupled with electrospray ionization-ion-trap MS/MS analysis.

An Acanthamoeba polyphaga mimivirus sample (175 μg) prepared as described above was separated by SDS-PAGE on an 11% acrylamide gel (22), and the proteins were revealed afterwards by Coomassie blue R-250 staining. Bands of 1-mm thickness were systematically cut from the gel, resulting in 49 pieces to be analyzed. These bands were reduced and alkylated before trypsin digestion, and the resulting peptides were analyzed through liquid chromatography (LC) (Finnigan Surveyor HPLC system; Thermo Electron, San Jose, CA) coupled to an electrospray ionization-ion-trap mass spectrometer (LCQ-Deca XP; Thermofinnigan). Tryptic peptides were resuspended in 20 μl formic acid at 5% in water, and 1/20 of the whole extract obtained from each piece of gel was desalted on a C₁₈ trapping column (Zorbax 300SB-C18 [5-μm inner diameter, 5 by 0.3 mm]; Agilent) by a 20-min washing step with 0.1% formic acid. Peptide separation was achieved by a 60-min linear gradient of acetonitrile (0 to 65%) in 0.2% formic acid on a C₁₈ reverse-phase column (PicoFrit column [5-μm BioBasic C₁₈, 300-Å pore size, 75-μm inner diameter, 100-mm long, 15-μm tip]). The peptides were then ionized at a capillary temperature of 160°C with a 2-kV spray voltage. A collision energy of 35% was applied for the MS/MS scan events. The MS/MS spectrum of the three most intense peaks was obtained after each full MS scan. The dynamic exclusion features were set at a repeat count of 2 within 0.5 min, with an exclusion duration of 3 min.

Protein identification was performed using the TurboSequest algorithm (11) in the Bioworks 3.2 software package (Thermo Electron) and the Acanthamoeba polyphaga mimivirus database (33). TurboSequest automatically identifies proteins by matching a peptide's experimental MS/MS spectrum to a predicted mass spectrum for amino acid sequences within databases. Identified peptides were evaluated using the cross-correlation number (X_corr) versus the charge state. Criteria for positive identification of peptides were X_corr values of >1.8 for singly charged ions, >2.5 for doubly charged ions, and >3.5 for triply charged ions. Only the best match was considered, with two or more unique peptides identifying a protein. Four proteins with masses below 19 kDa were assigned by a single peptide after visual inspection of the MS/MS spectrum.

Protein glycosylation analysis.

A purified pellet of Acanthamoeba polyphaga mimivirus was resuspended in β-mercaptoethanol (Sigma-Aldrich) and incubated overnight at 4°C. The insoluble fraction was removed by centrifugation (12,000 × g, 4°C, 10 min). Proteins present in the supernatant were separated by SDS-PAGE on a 15% acrylamide gel (22) and visualized by Coomassie R-250 staining. Glycoproteins were revealed using a GlycoProfile III kit (Sigma-Aldrich), which allows specific and sensitive in-gel fluorescence detection of glycoproteins.

Sequence annotation and ORF functional predictions.

ORF sequences were annotated using homology searches against all available databases, as previously described (33). In addition, anonymous (homologous to proteins of unknown function) and orphan sequences were reanalyzed using the 3D structure-based homology program FUGUE (37), generating more tentative structural and/or functional assignments (indicated within parentheses in Table 1). Only assignments scored as “likely” or “certain” were reported.

Immunogold labeling.

Immunolabeling of ultrathin sections for electron microscopy was performed as previously described (24). Briefly, ultrathin sections (70 nm) were cut using an ultramicrotome (Ultracut E; Leica), collected on 300-mesh nickel grids without Formvar (Electron Microscopy Sciences), and then incubated in PBS supplemented with 0.2% bovine serum albumin (Roche Diagnostics). The sections were then treated with 0.05 M lysine (Sigma-Aldrich) in order to neutralize putative residual aldehyde groups. After repeated washings with PBS, the sections were then incubated for 3 h at 37°C under a humidified atmosphere with a monoclonal anti-Mimi_L725 antibody (Mab6E2; 1:100) (23) diluted in PBS supplemented with 3% nonfat dry milk. The secondary reaction used goat anti-mouse antibodies conjugated to 25-nm colloidal gold particles (Aurion EM Reagents, The Netherlands), followed by washings with PBS and H₂O. Gold particles were visualized using an R-GENT SE-EM silver enhancement kit (Aurion) according to the instructions of the manufacturer. After washings and aldehyde fixation, a treatment with 5% uranyl acetate (ICN Biomedicals) was done to produce intense electron-opaque staining, and samples were viewed with a Philips electron microscope (MORGAGNI 268D) at 80 kV. A negative control experiment was performed with preimmune serum.

Global proteomic analysis of mimivirus particle by LC-MS/MS.

A critical parameter for every proteome project is the purity of the sample to be analyzed. This is particularly true when considering intracellular pathogens. For this study, the absence of major eukaryotic debris was assessed by negative-staining electron microscopy (Fig. 1A). Mimivirus proteins separated by one-dimensional SDS-PAGE (Fig. 1B) were fractionated in 49 pieces of gel, and corresponding tryptic digests were analyzed by capillary LC-MS/MS. Alternatively, mimivirus samples were resolved by 2D gel electrophoresis (Fig. 2), and protein identification was achieved by MALDI-TOF-MS. These complementary approaches allowed 114 proteins to be identified. These are listed in Table 1, together with their predicted functions (where applicable), and are classified into broad functional categories. The diversity of functions represented in the viral particle is quite large, with bona fide “structural” proteins amounting for a small fraction of the number of proteins. We considered only five candidates, whose sole role is to constitute the virion particle, as belonging to such a class. They are an A16L-like virion-associated membrane protein, the major core protein, the capsid protein D13L, a putative spore coat assembly factor, and a lipocalin-like outer membrane lipoprotein. Transcriptional enzymes and factors (12 gene products) constitute the largest functional category associated with the viral particle. This set includes all five DNA-directed RNA polymerase subunits as well as two helicases, one mRNA guanylyltransferase, and four transcription factors. Incidentally, one of the best identification scores was obtained with the β-subunit of the RNA polymerase (MIMI_R501), where the intron computationally identified within the gene was found to be excised as predicted (33). The next largest functional group is constituted of nine gene products, all of which are associated with oxidative pathways. Some of these enzymes might help the virus to cope with the oxidative stress generated by the host defense. The protein/lipid modification functional category is also well represented, including a phosphoesterase and a lipase, which are eventually used to digest the cell membrane, two protein kinases, and a protein phosphatase. Finally, five proteins associated with DNA topology and damage repair were identified, including the topoisomerase IA (MIMI_L221) and a DNA UV damage repair endonuclease (MIMI_L687), which were never described before as viral proteins. Other identified proteins belonging to this category are a tRNA methyltransferase and a putative mannose-6-phosphate isomerase (see Table S1 in the supplemental material). Yet the most abundant class of proteins associated with the mimivirus particle corresponds to those with unknown function (65 proteins), among which are 45 ORFans exhibiting no convincing sequence match in the databases. We noticed that the fraction of ORFan gene products found to be associated with the virion (45/114 [39.47%]) is equivalent to their proportion within the whole mimivirus genome (39%), a finding consistent with the notion that these genes are in no way different from other mimivirus genes and probably all encode bona fide proteins of importance to virus physiology. In a landmark study, Iyer et al. (16) identified a set of nucleocytoplasmic large DNA virus core genes that they further classified into four classes according to their decreasing levels of conservation across the main nucleocytoplasmic large DNA virus clades. We found that mimivirus particles incorporated four of the nine class I core gene products (including the major capsid protein), none of the class II core gene products, five of the class III gene products, and three of the class IV gene products (see Table S1 in the supplemental material). Finally, a small fraction (13/114) of the products originated from genes preceded by the highly conserved predicted mimivirus promoter (found in front of about half of all mimivirus genes) (42), confirming its putative role in governing the transcription of early or late-early genes (see Table S1 in the supplemental material).

Posttranslational modifications.

As shown on 2D gels, most of the mimivirus proteins were not resolved into single spots but rather as a train of spots (Fig. 2). This was the case, for instance, for the capsid protein D13L (spots 6 to 9), which is glycosylated (Fig. 3). We observed that 66% of ORFan-encoded proteins exhibited molecular weights corresponding to their in silico predictions. Others, such as the MIMI_L442 ORFan gene product, were not located at their expected locations on the gel. Extensive analysis of the 11 corresponding isoforms showed that tryptic peptides from spot 23 matched exclusively with the central part of the protein, while spots 24 to 26 and 30 to 36 matched the N- and C-terminal extremities of the full-length 140-kDa precursor, respectively. In other cases, both the mature protein and cleavage products were identified. Other cases of discrepancy might correspond to computational errors in the identification of the true N-terminal boundary of the protein (i.e., the bona fide initiation codon). The same problem was noticed for proteins with functional attributes. According to both the areas and intensities of silver-stained spots, we can assume that some of the ORFan-encoded proteins are strongly expressed (spots 47 to 51, MIMI_L725; spots 52 to 53, MIMI_L688; and spot 61, MIMI_R653). Only four proteins not detected by LC-MS/MS were identified through 2D electrophoresis coupled with MALDI-TOF-MS.

Antigenic properties of ORFan-encoded mimivirus proteins.

To investigate the antigenicity of mimivirus proteins, immunoblots were performed on samples resolved by 2D-PAGE, using sera from mice immunized with the whole particle (Fig. 4). Interestingly, two ORFan-encoded proteins, namely, MIMI_L724 (218 amino acids) and MIMI_L725 (224 amino acids), were specifically recognized, while no protein was recognized by preimmune sera. Both proteins are rich in cysteine residues and might be involved in a cross-linked structure accessible at the periphery of the particle. Immunogold labeling using an anti-MIMI_L725 monoclonal antibody further revealed gold particles surrounding the capsid core structure of mimivirus, confirming the exposure of the corresponding epitope to the surface (Fig. 5).