Virion proteome of Cafeteria roenbergensis virus strain BV-PW1
This project describes the protein composition of the Cafeteria roenbergensis virus (CroV, strain BV-PW1: TaxID 693272) particle, a giant marine DNA virus that infects the heterotrophic nanoflagellate microeukaryote C. roenbergensis. CroV is a member of the Nucleo-Cytoplasmic Large DNA Virus clade and related to Acanthamoeba polyphaga mimivirus. CroV possesses a DNA genome of ~730 kilobase pairs that encodes 544 predicted proteins. We analyzed the protein composition of purified CroV particles by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified 141 virion-associated CroV proteins. Predicted functions could be assigned to 37% of these proteins, which include structural proteins as well as enzymes for transcription, DNA repair, redox reactions and protein modification. Homologs of 36 CroV virion proteins have previously been found in the virion of Acanthamoeba polyphaga mimivirus. This study shows that giant DNA virus particles contain more than one hundred viral proteins that include specific enzymatic functions.
Sample Processing Protocol
Extracellular CroV particles were produced by infecting C. roenbergensis strain E4-10 with CroV strain BV-PW1. Lysates were concentrated by tangential flow filtration and CroV virions were purified by iodixanol density gradient ultracentrifugation. A 20 μl sample containing ~3E+10 virions was solubilized in SDS sample buffer with 2.5x10-3 U/μl benzonase nuclease added and allowed to sit for 1 h at room temperature. Proteins were resolved on a 10% SDS-polyacrylamide gel that was subsequently stained with blue-silver colloidal Coomassie. The gel lane was cut into 10 slices and proteins were reduced with 10 mM dithiothreitol for 45 min at 56°C, alkylated with 55 mM iodoacetamide for 30 min at 37°C and proteolyzed to peptides with 12.5 ng/μl trypsin for 15 h at 37°C.The samples were desalted with a STop-And-Go Extraction (STAGE) tip and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS setup consisted of a linear trapping quadrupole-Orbitrap (LTQ-OrbitrapXL), which was coupled on-line to a 1100 Series nanoflow high performance liquid chromatography system with a nanospray ionization source and a 20 cm long and 50 μm inner diameter holding column packed in-house with ReproSil-Pur C18-AQ, 3 μm, 120 A. The trap column contained AQUA C18, 5 μm, 200 A. Buffer A (0.5% acetic acid) and buffer B (0.5% acetic acid, 80% acetonitrile) were run in gradients of 10% B to 32% B over 57 min, 32% B to 40% B over 5 min, 40% B to 100% B over 2 min, 100% B for 2 min, and finally 0% B for another 10 min to recondition the column. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution from 350 to 1500 thomson (Th) in the Orbitrap and to simultaneously fragment the top five peptide ions in each cycle in the LTQ.
Data Processing Protocol
A custom protein database was first compiled from Genbank and consisted of 544 predicted CroV protein-coding sequences (CDSs, Genbank accession NC_014637), 1352 CroV unidentified reading frames (URFs), 20 Mavirus CDSs (Genbank accession NC_015230), and 53 Mavirus URFs, as well as all predicted proteins from the C. roenbergensis transcriptome and the C. roenbergensis mitochondrial DNA (Genbank accession NC_000946). MaxQuant (v188.8.131.52) was used to identify peptides from the fragment spectra using the above database and default parameters. Trypsin was specified as the enzyme, with up to one missed cleavage, cysteine carbamidomethylation was defined as a fixed modification while methionine oxidation and deamidation of asparagine and glutamine were allowed as variable modifications. A 1% false discovery rate (at the protein level) was used to determine whether a protein was confidently identified. The intensity values were calculated as the average intensity of the five-most intense ions detected for each protein.