Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host–virus interactions

Abstract

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins.

Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga

“…In agreement, vGSLs have been found to act as signaling lipids to induce algal host PCD (35). Moreover, EhV have been shown to interact with E. huxleyi lipid rafts, which are membrane microdomains enriched with GSLs, sterols, and proteins (59). These findings provide an interesting link between the unique structure of SLs induced by EhV and their involvement in the timely induction of cell death processes during viral infection.…”

The Molecular Ecophysiology of Programmed Cell Death in Marine Phytoplankton

“…EhVs may employ an entry and exit strategy through lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signaling cascades through protein-protein interactions. Combined lipidomic, proteomic, and bioinformatic analyses of purified lipid rafts from EhV-infected E. huxleyi cells revealed two distinct GSL classes that are preferentially enriched in these specialized membrane features [sialic acid GSLs (sGSLs) and raft GSLs (rGSLs)], along with a variety of proteins affiliated with host defense, PCD, and innate immunity pathways (Rose et al 2014). Key proteins included calmodulin-binding DENN/MADD domain-containing proteins that are involved in mitogen-activated protein kinase induction (Schievella et al 1997); proline-rich extensins (PRICHEXTENSN), which function in the signal transduction of pathogen defense after the cell wall structure has been compromised (Sanabria et al 2010, Silva & Goring 2002; and Toll/interleukin 1 receptor (TIR) and leucine-rich repeat (LRR) domain proteins, which are often connected by a nucleotide-binding (NB) domain and mediate pathogen recognition/resistance and activate host cell defense responses (Peart et al 2005, Swiderski et al 2009).…”

Genome of brown tide virus (AaV), the little giant of the Megaviridae, elucidates NCLDV genome expansion and host–virus coevolution

“…Heparan sulfate, a sulfated carbohydrate, is known to be a surface receptor for a number of viruses including Vaccinia and Herpes Simplex (Zhu et al, 2011). In the case of E. huxleyi, EhV-86 encodes C-type lectin-containing protein that associates with purified lipid rafts from 2 h postinfected host cells, arguing that EhV infection occurs at the interface between virus proteins and host lipid-raft sugar-lipid moieties (Rose et al, 2014). No studies have been conducted on the molecular mechanisms of AaV-host interactions yet, so whether sulfated carbohydrates have any role in such interaction remains an open question.…”