Lipidomic study of intracellular Singapore grouper iridovirus

Abstract

Singapore grouper iridoviruses (SGIV) infected grouper cells release few enveloped extracellular viruses by budding and many unenveloped intracellular viruses following cell lysis. The lipid composition and function of such unenveloped intracellular viruses remain unknown. Detergent treatment of the intracellular viruses triggered the loss of viral lipids, capsid proteins and infectivity. Enzymatic digestion of the viral lipids with phospholipases and sphingomyelinase retained the viral capsid proteins but reduced infectivity. Over 220 lipid species were identified and quantified from the viruses and its producer cells by electrospray ionization mass spectrometry. Ten caspid proteins that dissociated from the viruses following the detergent treatments were identified by MALDI-TOF/TOF-MS/MS. Five of them were demonstrated to be lipid-binding proteins. This is the first research detailing the lipidome and lipid-protein interactions of an unenveloped virus. The identified lipid species and lipid-binding proteins will facilitate further studies of the viral assembly, egress and entry.

Fig. 1

Electron micrographs of intracellular SGIV particles. (a) Intracellular viral particles observed before purification; (b) and (c) viral particles were purified from cell pellet collected at 3 days after infection by self-generated iodixanol gradients. The layer depicted by an arrow head in (b) is the viral capsid shell. (d) Viral particles after 75% ether treatment; (e) viral particles after 0.4 mM n-dodecyl-α-d maltoside treatment. All those viral preparations were stained with 1% phosphotungstic acid followed by examination under a transmission electron microscope (JEOL 100 CXII). (f) Western blotting analyzed host cellular protein actin and viral structure protein ORF075, showing the amount of actin in purified viruses decreased with each step of purification (bar = 100 nm).

Fig. 2

Abundance of phosphoinostides (PIPn). Phosphoinostides enriched lipid samples were extracted in the acidic condition and quantified as described in Materials and methods. The total amount of PI, PIP and PIP2 was set at one. Bar graph shows relative abundance of glycerophosphatidylinositol (PI), glycerophosphatidylinositol phosphate (PIP) and glycerophosphatidylinositol biphosphate (PIP2) in SGIV and its host cells. All data are mean ± standard deviation (n = 3).

Fig. 3

Effects of solvent, detergent and lipase treatments on viral lipid composition and viral infectivity. Viral infectivity was determined by TCID₅₀ assay. The data represents the means of three independent experiments. Error bars represent standard deviation of the mean. (a) Viruses were treated with Triton, ether, n-dodecyl-α-d maltoside (DDM) and n-octyl-β-d-glucopyranoside (OG). Viral infectivity is expressed as the percentages of the untreated control. (b) Pre-treated intracellular viruses with 0.2 mM DDM were incubated with liposome prepared from host cell lipids to rescue viral infectivity. The rescued viruses show a 3-fold higher infectivity than the pre-treated ones. (c) Viruses were treated with phospholipase C (PLC), phospholipase A2 (PLA2) and sphingomyelinase (Smase). Viral infectivity is expressed as the percentages of the untreated control. (d) Depletion of viral lipids by various treatments was quantitatively analyzed as described in Materials and methods. The abundance of each lipid group in treated viruses is shown as the percentage of the corresponding lipid group in untreated control, which was set to 100.

Fig. 4

SDS-PAGE analysis and Western blot detection of purified recombinant proteins. Lane A, bovine serum albumin (BSA); lane B–H, recombinant proteins of ORF18R, 26R, 75R, 89L, 90L, 101R, and 158L. Two microgram of BSA was loaded on the gel to provide a reference for protein quantification. All purified recombinant proteins show a clear major band (4a) and could be detected by the corresponding antibodies (4b).

Fig. 5

Lipid–protein interaction assay. 0.01 mM of each viral protein was incubated with PIP-Strips (echelon), showing their binding abilities to the different lipid molecules (right). The first strip was a positive control using one μg/ml PIP2 Grip™ protein. ORF18R and -158L did not show binding affinity to the lipids (data not shown). A diagram in the left shows lipid molecules on the PIP strip. PtdIns represents phosphatidylinositol.

Fig. 6

Apoptosis induced by SGIV infection. Light micrographs of SGIV infected cells show cell shrinkage (a, solid arrows) and apoptotic bodies (b). Fluorescent microscopic analysis of apoptotic cells stained with FITC annexin V/propidium iodide (c and d). Apoptotic cells are denoted by open arrows while dead or necrotic cells are denoted by arrow heads. Necrotic cell nuclei permitted staining propidium iodide.