isolation and characterization of lipid rafts in emiliania huxleyi: a...

Isolation and characterization of lipid rafts in Emilianiahuxleyi: a role for membrane microdomains inhost–virus interactions

Suzanne L. Rose,1† James M. Fulton,2

Christopher M. Brown,1 Frank Natale,1

Benjamin A. S. Van Mooy2 and Kay D. Bidle1*1Institute of Marine and Coastal Sciences, RutgersUniversity, New Brunswick, NJ, USA.2Department of Marine Chemistry and Geochemistry,Woods Hole Oceanographic Institution, Woods Hole,MA, USA.

Summary

Coccolithoviruses employ a suite of glycosphingo-lipids (GSLs) to successfully infect the globallyimportant coccolithophore Emiliania huxleyi. Lipidrafts, chemically distinct membrane lipid microd-omains that are enriched in GSLs and are involved insensing extracellular stimuli and activating signallingcascades through protein–protein interactions, likelyplay a fundamental role in host–virus interactions.Using combined lipidomics, proteomics and bioin-formatics, we isolated and characterized the lipid andprotein content of lipid rafts from control E. huxleyicells and those infected with EhV86, the type strainfor Coccolithovirus. Lipid raft-enriched fractions wereisolated and purified as buoyant, detergent-resistantmembranes (DRMs) in OptiPrep density gradients.Transmission electron microscopy of vesicle mor-phology, polymerase chain reaction amplification ofthe EhV major capsid protein gene and immuno-reactivity to flotillin antisera served as respectivephysical, molecular and biochemical markers. Sub-sequent lipid characterization of DRMs via highperformance liquid chromatography-triple quadra-pole mass spectrometry revealed four distinctGSL classes. Parallel proteomic analysis confirmedflotillin as a major lipid raft protein, along with avariety of proteins affiliated with host defence, pro-grammed cell death and innate immunity pathways.

The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs atthe interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associatedproteins.

Introduction

The coccolithophore Emiliania huxleyi (Lohmann) Hay &Mohler (Prymnesiophyceae, Haptophyta) forms massivespring blooms in the North Atlantic that are routinelyinfected and terminated by lytic, double-stranded DNA-containing viruses (EhVs) (Bratbak et al., 1993; Jordanand Chamberlain, 1997; Tyrell and Merico, 2004; Allenet al., 2006a). As members of the Phycodnaviridae, EhVsare giant microalgal viruses (diameter ∼180 nm) withan extensive genetic capability (∼407 kb genomes) tomanipulate host metabolic pathways for their replication(Castberg et al., 2002; Schroeder et al., 2002; Van Ettenet al., 2002; Wilson et al., 2005). Given the array of sen-sitive and resistant host strains in culture along with anumber of genetically diverse EhVs, the E. huxleyi–EhVhost–virus system has emerged as one of the best char-acterized model systems to investigate marine algal host–virus interactions and the cellular processes mediatinginfection dynamics (Mackinder et al., 2009; Bidle andVardi, 2011).

The determinants of host susceptibility or resistanceto viral infection, especially the factors involved in theinitial cellular response, are of fundamental interestin host–virus interactions. Coccolithoviruses employ asophisticated, co-evolutionary biochemical ‘arms race’ tomanipulate host lipid metabolism, alter glycosphingolipid(GSL) production and ultimately regulate cell fate viainduction of a programmed cell death (PCD) pathway [viareactive oxygen species, metacaspase expression andcaspase activity (Bidle et al., 2007; Vardi et al., 2009;2012; Bidle and Vardi, 2011)] in a manner reminiscent ofthe ‘Red Queen’ dynamic (VanValen, 1973) driving plant-and animal-pathogen interactions (Staskawicz et al.,2001). Subtle differences in the physiological regulation ofthe PCD machinery appear to be critical in host suscep-tibility (Bidle and Kwityn, 2012), perhaps as part of aninnate immune response to determine cell fate. Yet, little is

Received 20 June, 2013; accepted 5 December, 2013. *Forcorrespondence. E-mail [email protected]; Tel. (+1) 848932 3467; Fax (+1) 732 932 4083. †Present address: University ofNebraska – Lincoln, Department of Biochemistry, Lincoln, NE,USA.

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Environmental Microbiology (2014) doi:10.1111/1462-2920.12357

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd

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known about the cellular determinants of infectivity asthey relate to lipid dynamics within host cell membranes.

The cell membrane fluid mosaic model originally pro-posed in 1972 (Singer and Nicolson, 1972) has evolvedover the past decade leading to the contemporary char-acterization of biological membranes as having discreteorganizational regions, or microdomains (Brown andLondon, 2000; Edidin, 2003; Lingwood and Simons,2010). Coined as ‘lipid rafts’, these microdomains areenriched in sphingolipids, GSLs and sterols forming aliquid-ordered phase distinct from the phospholipid con-taining liquid-disordered phase of the surrounding mem-brane (Van Meer and Simons, 1988; Rajendran andSimons, 2005; Hancock, 2006; Hakomori, 2008). Thedistinct biochemical nature of lipid rafts allows for theirisolation due to their insolubility in cold non-ionicdetergents (e.g. Brij-96 and Triton-X) and ability toform buoyant ‘detergent-resistant membranes’ (DRMs)(Radeva and Sharom, 2004; Borner et al., 2005) withlipid raft domain sizes ranging from ∼150 to 250 nm indiameter (Pike, 2003), perhaps deriving from smaller,∼50 nm diameter assemblies that coalesce in responseto different cellular or environmental conditions (Simonsand Ehehalt, 2002).

The ability of lipid rafts to incorporate or excludespecific proteins and facilitate selective protein–proteininteractions provides an important platform for the regu-lation of a wide range of cellular processes, includingcytoskeleton organization, cell adhesion, signal trans-duction pathways and apoptosis (Brown and London,2000; Munro, 2003; Morrow and Parton, 2005; Hancock,2006; Lingwood and Simons, 2010). Small individual lipidraft size (∼10–50 nm) suggests their role in the regulationof raft-associated proteins, whereby signalling proteinsare physically separated and thereby held in an inacti-vated state (Simons and Ehehalt, 2002). Subsequentclustering of lipid rafts into a larger morphological platform(∼200 nm) in turn facilitates protein–protein interactionsthat are essential to trigger pathways involved in cellgrowth, stress response, death and survival (Simonsand Ehehalt, 2002). Lipid rafts have been found to beenriched in glycosylphosphatidylinositol (GPI)-anchoredproteins, Src family kinases, heterotrimeric G proteins andstomatin, prohibitin, flotillin and HflC/K (SPFH) superfam-ily proteins, all of which are targeted to lipid rafts viaessential lipid modifications (Morrow and Parton, 2005;Rajendran and Simons, 2005; Rivera-Milla et al., 2006).Flotillin, an evolutionary conserved SPFH domain-containing protein, has been proposed to function as alipid raft organizer, acting as a scaffold and linking theaforementioned lipid raft domains and raft-dependent cel-lular process (Simons and Toomre, 2000; Langhorst et al.,2005; Morrow and Parton, 2005). Flotillin-enriched lipidrafts appear to be involved in host invasion indicating

a possibly important role in host–pathogen sensitivityor resistance (Rivera-Milla et al., 2006; Babuke andTikkanen, 2007; del Cacho et al., 2007).

Lipid rafts are targeted and usurped by a vast numberof pathogens as sites of host attack in higher eukaryotes(van der Goot and Harder, 2001). In the E. huxleyi–EhVsystem, they are hypothesized to function as specificpoints of viral attachment and entry, as well as in virusassembly and budding (Mackinder et al., 2009; Bidle andVardi, 2011). EhVs induce and employ a unique suite ofvirus-encoded GSLs (vGSLs) to successfully infectE. huxleyi (Vardi et al., 2009; 2012; Bidle and Vardi,2011). Furthermore, EhV86 is an icosahedral virus that isenveloped with a lipid membrane composed of variousGSLs (Vardi et al., 2009; Fulton et al., 2014) and containsa majority (23 of 28) of putative membrane proteinswith transmembrane domains (Allen et al., 2008). Thus,lipids and lipid-associated proteins appear to play a criti-cal role in EhVs and their ‘animal-like’, envelope fusion-based infection strategy for entering and exiting infectedhost cells (Mackinder et al., 2009). To date, very littleis known about lipid raft characteristics in unicellularmarine phytoplankton, much less this globally importantcoccolithophore.

In this study, we investigated the lipid and protein com-position of lipid rafts in E. huxleyi, and their potentialinvolvement in E. huxleyi–EhV host–virus interactions.Building off of previously published methods in highereukaryotes (Radeva and Sharom, 2004; Macdonald andPike, 2005) and utilizing a flotillin-like protein in E. huxleyias a biochemical marker, we developed a protocol for theisolation of lipid rafts and associated proteins from bothuninfected and infected cell populations and character-ized their dynamics and composition through genome-enabled proteomics and lipidomics. Our results provideinsight into the initial biochemical and biophysical basis ofviral–host interactions and provide a better mechanisticunderstanding of the cell surface properties that serve toregulate host response to viral infection.

Results and discussion

A primary goal of this study was to isolate and character-ize lipid rafts, here defined as insoluble DRMs, particularlytheir associated proteomes and lipidomes, in bothuninfected and EhV86-infected cell populations. Ourapproach was to use buoyant densities and vesicle mor-phology, along with flotillin and GSL species as respectiveprotein and lipid biochemical markers, to inform our tar-geted isolation of lipid rafts for more detailed characteri-zation. Flotillins are expressed in a wide variety ofeukaryotes and prokaryotes and play a central role in lipidraft generation (protein–lipid binding domain), as well asin the stabilization of raft-associated proteins (protein–

2 S. L. Rose et al.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

protein binding domain), both of which are fundamentalin the regulation of diverse processes (i.e. signaltransduction, membrane trafficking and pathogen entry)(Borner et al., 2005; Morrow and Parton, 2005).Bioinformatic analysis of the E. huxleyi CCMP1516annotated proteome (http://genome.jgi-psf.org/Emihu1/Emihu1.home.html) revealed a 306 amino acid, 33.5 kDa‘flotillin-like’, SPFH domain-containing protein (Protein ID363433; Accession# EOD34547) that was also character-ized by ‘Band_7’ protein domains (IPR001107 andcd03407; e-value = 1.42e-93) diagnostic of other flotillin(reggie)-like proteins. This particular Band_7 subgroup(cd03407) contains proteins similar to SPFH and podicin,many of which are lipid raft-associated. The putativeE. huxleyi flotillin-like protein also showed high sequenceidentity to a ‘hypersensitive-induced response protein’ inArabidopsis thaliania (NP_177142; e-value of 6e-88)(Theologis et al., 2000) that is linked to PCD pathway inhigher plants (Lam et al., 2001).

Secondary structure analysis of the E. huxleyi flotillin-like protein revealed the evolutionary conserved SPFH

domain (aa residues 4–166; Fig. 1A). This conserveddomain contains two hydrophobic sphingolipid bindingdomains (aa residues 13–41 and 133–151) along with aconserved palmitoylation site, both of which contributeto membrane association (Morrow et al., 2002; Morrowand Parton, 2005). The required post-translationalpalmitoylation of flotillins at the N-terminus follows theirsynthesis on ribosomes followed by an unusual Golgi-independent trafficking pathway that targets the proteinto the plasma membrane (Morrow et al., 2002). TheC-terminal end of the E. huxleyi flotillin-like protein pos-sessed a predicted α-helix coil (aa residues 266–286;Fig. 1A), which is a characteristic of all classic flotillins andis thought to oligomerize with other flotillin molecules(Morrow and Parton, 2005; Solis et al., 2007). The pres-ence of three adjacent α-helix coils within this domainallow for the oligomerization of flotillin in highereukaryotes (Solis et al., 2007). Exposed within this proteinare lysine residues thought to facilitate a high-affinity,stable covalent linkage between flotillin monomers (Soliset al., 2007). Modelling of the tertiary structure of the

MW hc Ehux

kDa

50

37*

A

B C

MEACGCVITSQDERKVVERCGKFEDVLDAGFSCVLPCLCQFVKGSISTRIQMLEINADTKTKDNVFVGIK70

VAVQYQVNGDSQSIQDAMYKLTNPRAQIESYVLDVVRSSVPKIDLDNVFLEKEEIAASIKEMLGETMGRF140

GYSILATPVTDIEPNLEVKRAMNEINKAKRLRQAAVDEGEAIKIRSIKEAEAEAARTEIQAKADAEAKFM210

QGQGIARQRQAIVSGLRDSVNCFKADVAGVDSKQVMDLLLVTQYFDMMKDVGGNSRSNAVFMNHSPGALQ280

DLTQAIQGGFMSSLPAAPSFAQAQRH306

predicted α-helix which can dimerize flotillin monomers via a triple coil

hydrophobic Sphingolipid Binding Domains (13-41 aa; 133-151 aa)

conserved palmitoylation site

Fig. 1. Analysis of a flotillin-like protein in Emiliania huxleyi CCMP1516.A. The 306 amino acid, 33.5 kDa putative flotillin-like protein (Protein ID 363433) contains notable lipid raft-associated domain characteristics.Predicted secondary structural elements consist of the evolutionary conserved prohibitin homology (PHB)/stomatin prohibitin flotillin and HflC/K(SPFH) domain residues (aa 4–166; underlined) and the hydrophobic sphingolipid binding domain (SBD) regions (aa 13–41; aa 133–151),along with the conserved palmitoylation site, which contributes to membrane association and where α-helix coils are predicted to facilitatedimerization of flotillin proteins.B. Tertiary structure prediction (Phyre2 PDB SCOP code c3bk6C membrane protein; viewed with PyMOL software), with visualization of aα-helix coil tail (orange-red).C. Western blot analysis of E. huxleyi cell extracts challenged with a human anti-flotillin antibody displayed strong immunohybridization with a67 kDa protein consistent with the predicted size of the dimerized protein (Protein ID 363433). Immunoblot control consisted of the predicted42 kDa human flotillin (asterisk in lane ‘hc’; NCBI Accession #AAF17215; 253 aa) from human A-375 whole cell lysate of malignant melanomacells. Molecular weight markers (MW; kDa) are indicated.

Lipid rafts in Emiliania huxleyi 3


http://genome.jgi-psf.org/Emihu1/Emihu1.home.html


E. huxleyi flotillin-like protein using Phyre2 and PyMOLsoftware packages (Fig. 1B) successfully visualizedthis dimerizing α-helix coil tail. Furthermore, Westernimmunoblot analysis with a monoclonal antibody raisedagainst human flotillin-2 (NCBI Accession #Q14254) suc-cessfully detected the expression of a prominent 67 kDaband in E. huxleyi CCMP1516, consistent with the pre-dicted dimer size of the flotillin-like protein (Fig. 1C).Immunoblot analysis also successfully detected the pre-dicted 42 kDa human flotillin in human A-375 whole celllysate of malignant melanoma cells, thereby serving as apositive control for our analysis.

Lipid raft dynamics, isolation and characterization

We investigated lipid raft dynamics and composition bothin control host cells (uninfected) and during host–virusinteractions at 2, 24, 48 and 72 h post-infection (hpi) to notonly determine the lipid raft landscape in healthy cells, butto also determine if and how lipid rafts are altered by virusinfection. We first confirmed successful lytic infection ofE. huxleyi CCMP1516 with EhV86, which resulted in anotable reduction of host cell abundance over 72 h com-pared with uninfected control cells (Fig. 2A); infected cellpopulations only reached host cell abundances of

∼1.25 × 106 cells ml−1 at 72 h, while uninfected cell abun-dance reached 2.5 × 106 cells ml−1. Uninfected E. huxleyicells continued to increase in abundance over the timecourse of the experiment. EhV86 abundance increasedby over two orders of magnitude in infected culturesbetween 24 and 72 hpi (Fig. 2B), concomitant withdecreases in host growth (Fig. 2A).

We developed a purification method, based onpublished techniques (Radeva and Sharom, 2004;Macdonald and Pike, 2005) to isolate lipid rafts fromhigher eukaryotes, employing lysis and homogenizationof cells in a non-ionic detergent (Brij-96) followed byultracentrifugation in a discontinuous (5–45%) OptiPrepdensity gradient (Fig. 3A and B). Detergent-resistant lipidrafts from higher eukaryotic cells have been isolated usingthe detergent Triton X-100 followed by sucrose gradientfractionation, with fractions enriched in sphingolipids, cho-lesterol, flotillin and GPI-anchored proteins distributed inthe top few, less dense fractions (Munro, 2003; Pike,2003; Radeva and Sharom, 2004; Macdonald and Pike,2005). Here, Brij-96 detergent treatment was used toproduce ‘outside-in’ DRMs for subsequent identificationand characterization of lipid raft-associated lipids and pro-teins found on the extracellular plasma membrane ofE. huxleyi uninfected and EhV86-infected cell popula-tions. Direct visualization of lipid vesicles in select frac-tions via transmission electron microscopy (TEM) showedthe presence of prominent, 0.2 μm DRM vesicles inbuoyant Fraction 2 (1.055 g cm−3) for both uninfected con-trols and EhV86-infected samples (Fig. 3C). This is con-sistent with published characteristics of DRMs in highereukaryotes (Pike, 2003; Radeva and Sharom, 2004). Nolipid vesicles were seen in higher density fractions foreither treatment.

Polymerase chain reaction (PCR) amplification of theEhV86 major capsid protein (MCP) was also used as aproxy to identify the distribution of EhVs (and associatedDNA) in OptiPrep density gradient fractions from EhV86-infected cell populations at 2, 24, 48 and 72 hpi (Fig. 4).This helped provide an initial characterization of theOptiPrep density fractions and important context onwhere to expect viruses (and their associated GSLs andproteins). Successful amplification of the expected 248 bpMCP amplicon was only detected in fractions 6–12 forboth 2 and 24 hpi, indicating the selective presence ofEhVs in higher density, soluble fractions (Fig. 4). The dra-matic increase in EhV abundance (10- to 100-fold) duringlate lytic phase (48–72 hpi; Fig. 1) was clearly reflected inthe increased intensity MCP amplification in fractions6–12 (Fig. 4). At the same time, a weaker, yet notable,amplification signal of EhV MCPs was also seen in lightDRM fractions (1–5) indicative of an accumulation ofEhVs with distinct lipid characteristics. Our MCP amplicondata could represent the distribution of intact EhV virions

1.0E+06

1.0E+07

1.0E+08

1.0E+09

0 24 48 72 96

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

3.0E+06

Uninfected Control

+EhV86

A

B

Time (hours post infection)

EhV

abundance

(virus m

l-1)

E.h

uxle

yi c

ell

abundance

(cells

ml-1

)

Fig. 2. Dynamics of EhV86 infection of E. huxleyi CCMP1516.Time course of host (A) and virus (B) abundance over 96 h foruninfected control and EhV86-infected (+EhV86) cultures. Errorbars represent standard deviation for triplicate measurements and,where not discernible, are smaller than symbol size.

4 S. L. Rose et al.


or the released EhV genomic DNA within the OptiPrepfractions.

We also tried to locate and quantify EhV abundance inOptiPrep fractions via SYBR-Green staining and flowcytometry, but were unable to clearly discern the distinctEhV population signal in gradient fractions (data not

shown). Brij-96 treatment of a freshly generated EhV86lysate was found to compromise the SYBR-Green, flowcytometry signal of EhVs; it wasn’t as focused asuntreated lysates and had a detectable increase in the520 nm fluorescence signal (Supporting InformationFig. S1). Nonetheless, most EhVs (60.5% of the

5%

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45% +

E. huxleyi cell lysate

Optiprep™ gradient

Fraction 1

Fraction 12

(39,000 x g, 4°C, 16 h)

F3

F4

F11

F2

0.2

0.2

0.2

0.2

F2control +EhV86

A

B C

g/cm3

1.0551.055

1.2501.190

1.0601.0651.065

1.2601.2901.3001.3101.360

0.2

Fig. 3. Lipid raft isolation and characterizationin E. huxleyi CCMP1516.A. Schematic of the OptiPrep density gradientprocedure used to isolate lipid raft fractions.Corresponding fraction densities (g cm−3) areindicated.B. Picture of OptiPrep gradientscorresponding to the 2 (#1), 24 (#2), 48 (#3)and 72 (#4) h samples for control E. huxleyicultures. Gradients were also performed forEhV86-infected cell populations at the sametime points (not shown).C. Direct visualization of lipid vesicles inselect fractions (F2, F3, F4 and F11) viatransmission electron microscopy (TEM).Images in the left column correspond touninfected control cells; image in rightcolumn corresponds to fraction 2 forEhV86-infected-cells. Note the presence ofprominent detergent-resistant lipid vesiclesonly in Fractions 2 and 3. No lipid vesicleswere observed in lower fractions for eithertreatment.

MW nt h v 1 2 3 4 5 6 7 8 9 10 11 12

OptiPrep fraction

300

300

300

3002

24

48

72

Ho

urs

po

st in

fectio

n

Fig. 4. Distribution of EhVs in OptiPrepdensity gradients. PCR of the EhV MCP (seeExperimental procedures) was used as aproxy to identify the distribution of EhVs (andassociated DNA) in density gradients ofEhV86-infected cell fractions for 2, 24, 48 and72 hpi post infection samples. Successfulamplification of the expected 248 base pairamplicon was detected in fractions for eachtime point. Note that at early time points(2–24 hpi), MCP was distributed betweenfractions 6–10. Subsequent time points (48and 72 hpi) displayed intense amplicons inFractions 6–12 and weaker amplification inFractions 1–5; this corresponded with 10- to100-fold increase in EhV abundance (seeFig. 1B). EhV86 viral lysate served as apositive control (‘v’; upper panel). No template(nt) and genomic DNA for Emiliania huxleyi(h) were used as negative controls (upperpanel). Molecular weight (bp) markers areindicated.



population) localized within the diagnostic side scatter(SSC) vs 520 nm fluorescence gate (Supporting Informa-tion Fig. S1) and were likely intact. Viruses subjected toOptiPrep ultracentrifugation gradients have reportedbuoyant densities of 1.14–1.22 g cm−3 (Lawrence andSteward, 2010), including a purified Aureococcusanaphagefferens virus (AaV), a large dsDNA virus infect-ing the Pelagophyte Aureococcus anophageferrens that,like EhV, is a member of the nucleocytoplasmic large DNAvirus (NCLDV) family (M. Moniruzzaman, G.R. LeCleir,C.M. Brown, C.J. Gobler, W.H. Wilson, K.D. Bidle, andS.W. Wilhelm, in preparation). Based on this information,one would expect intact EhVs to be concentrated betweenFractions 5 and 8 (1.065–1.260 g cm−3). Notably, thisindeed corresponds with distribution of a majority of EhVMCP amplicons in Fig. 4 (especially for 2 and 24 hpi timepoints). Given reported buoyant densities of DNA at 1.69–1.71 g cm−3 (Smith, 1977), the presence of EhV MCPamplicons in more dense fractions (Fractions 10–12) atlater time points (48–72 hpi) suggests that some EhVshad indeed been disrupted and released genomic DNA.Taken together, these flow cytometry, PCR and densitydata argue that most EhVs (and associated proteins)were not present in more buoyant fractions (e.g. Fraction2) containing DRMs and suggest that a subpopulation ofEhVs have a membrane envelope structure (Mackinderet al., 2009) that is susceptible to disruption andsolubilization by non-ionic detergent treatment.

EhV replication appears to be uniquely characterizedby two distinct modes of infection, consisting of a ‘chronic-like’ phase through budding and a lytic phase (Mackinderet al., 2009). Most EhV progeny emerge through lyticphase [Fig. 1; (Bidle et al., 2007; Mackinder et al., 2009;Vardi et al., 2009)], as is evidenced by the concomitantdisappearance of host cells (= lysis) and exponentialincrease in EhVs (Fig. 1). EhVs produced through thesetwo fundamentally different modes might be expected toproduce distinct virus morphologies and detergent sensi-tivities, with lytic EhVs perhaps possessing an inner mem-brane like the other Phycodnaviridae (Van Etten et al.,2002). Fulton and colleagues (2014) recently updatedthe two-step model for EhV acquisition of lipids originallyproposed by Mackinder and colleagues (2009), and sug-gested that EhVs acquire an inner membrane in associa-tion with intracellular lipid bodies and then acquire anouter membrane, which is potentially more GSL rich andpresumably more detergent resistant, via budding throughlipid rafts in the host plasma membrane. Together with theresults of Fulton and colleagues (2014), our findings pointtowards a bimodal distribution of EhVs, where onesubpopulation emerges from the host via budding andpossess a detergent-resistant outer membrane, whileanother detergent-susceptible subpopulation is releasedvia host lysis and lacks an outer membrane.

Anti-flotillin immunoblot analysis was performed on 2and 48 h control and EhV86-infected E. huxleyi cellextracts to identify flotillin-containing fractions. Strongimmunohybridization was observed to a prominent67 kDa flotillin-like protein in both E. huxleyi control andEhV86-infected fractions at both time points (Fig. 5).Uninfected E. huxleyi fractions showed strong immuno-hybridization of the 67 kDa flotillin-like protein amonga broad range of low density, DRM-containing fractions1–5 (Fig. 5, upper panels). The pattern of anti-flotillinimmunohybridization was dramatically different in EhV86-infected cell populations, instead being characterizedby a much more defined and focused hybridization signalin fractions 2 and 3 at 2 hpi and 48 hpi respectively(Fig. 5, lower panels). These results clearly demonstratethat the interaction of E. huxleyi host cells with EhV86virions, even for short time periods (2 hpi), has a pro-nounced effect on the physical attributes of flotillin-associated DRMs. Taken together, our physical (buoyantdensities, TEM, SYBR-Green), molecular (distribution ofMCP amplicons) and biochemical (anti-flotillin westernimmunoblot analysis) results highlighted fraction 2 as astrong candidate for subsequent characterization of thelipidome and proteome associated with isolated lipid rafts.

Distribution and composition of GSLs

GSLs are polar lipids that critically ‘lubricate’ E. huxleyi–EhV interactions both in culture (Vardi et al., 2009; Bidleand Vardi, 2011) and in natural systems (Vardi et al.,2009; 2012). Indeed, host-specific glycosphingolipids(hGSLs), consisting of palmitoyl-based GSLs (Vardi et al.,2012) and viral glycosphingolipids (vGSLs), consisting ofmyristoyl-based GSLs (Vardi et al., 2009; 2012), playimportant functional roles in the infection of E. huxleyi andeven serve to trace both host and virus populationsrespectively (Vardi et al., 2012). Furthermore, a novelclass of sialic acid glycosphingolipids (sGSLs) with2-Keto-3-deoxynonic acid as the headgroup appear to bepromising biomarkers for E. huxleyi populations suscepti-ble to EhV infection (Fulton et al., 2014). Knowing thatGSLs are classically thought to play prominent roles inlipid rafts of higher eukaryotes, we examined their rela-tionship to DRMs in E. huxleyi and determined whichGSLs, if any, coincide with the flotillin biochemical proteinmarker of lipid rafts.

Using high-performance liquid chromatography-triplequadrapole mass spectrometry, we examined the abun-dance and chemical identity of GSLs in the OptiPrepfractions from control and EhV86-infected cell populationsat 2, 48 and 72 h (Fig. 6). A variety of GSL species weredetected in our analysis, which were grouped into fourgeneral component classes: ‘hGSLs’, hGSLs with a d19:3long-chain base (LCB) and either a C22:3 hydroxy- or

6 S. L. Rose et al.


C22:2 hydroxy-fatty acid (Vardi et al., 2012); ‘vGSLs’,EhV-derived, myristoyl-based GSLs with C15–C24 satu-rated fatty acids and C20–C24 monounsaturated fattyacids (Vardi et al., 2009) that increased substantially withEhV infection; ‘sGSLs’, sGSLs (monosialylceramides)containing a palmitoyl-based LCB (d18:2 or d18:1) anda C22:0 fatty acid (Fulton et al., 2014), that increasedwith infection; and ‘rGSLs’, raft GSLs with a glycosylheadgroup and a primary neutral mass loss of −162 Da,indicative of an LCB similar to vGSL (Vardi et al., 2009).rGSLs were detected as a sequence with relatively lowmolecular weights (m/z 704, 718, 732 and 746) that likelydiffered in fatty acid chain length, and based on their earlyelution times, we predict that the rGSL LCB was longer,saturated and less hydroxylated than that of vGSL; rGSLsdid not increase with infection. Total GSL concentration(normalized to the glycolipid recovery standard DNP-PE;see Experimental procedures) was generally confined tohigher density, lower fractions (6–12; Fig. 6), indicatingthat most GSLs were not associated with DRMs. We werespecifically interested in the relative percentage of indi-vidual GSL classes in each fraction to get a better senseof their individual distribution and whether any specificclasses are enriched in flotillin-associated DRMs in bothcontrol and EhV-challenged cells.

Among the different GSL classes in uninfected controlcultures (Fig. 6; left column), hGSLs were generally con-fined to the lower fractions (5–12) and comprised a rela-

tively high percentage (30–98%) of GSLs in uninfectedcontrol cells within each of these more dense fractions.The sGSL class, which was only recently described(Fulton et al., 2014), showed a more even distribution inOptiPrep density gradients than hGSLs, but there wasevidence of a high relative sGSL percentage in morebuoyant DRMs, with sGSLs making up to 94% and 72% ofthe GSLs in fractions 1 and 2 at 72 h. rGSLs showed avery different distribution in the OptiPrep density gradients(Fig. 6) instead being preferentially concentrated in theupper, low density fractions (1–5) and accounting for up to87% of the GSLs in these respective fractions. These hostrGSLs clearly behaved differently in the density gradientsthan the other three GSL classes, and more closely cor-responded to other lipid raft physical (presence of DRMs,Fig. 3) and biochemical markers (distribution of flotillin,Fig. 4) and, thereby, more likely represent the lipid raftlipidome. Not surprisingly, no chemical signatures of‘vGSLs’ were detected in uninfected control cells at alltime points suggesting that this class of GSLs is indeedderived from EhV biosynthetic machinery (Monier et al.,2009; Vardi et al., 2009).

Viral infection triggered a massive upregulation (3–10-fold) in the normalized total GSL production at 48 h andespecially 72 h post-infection, with most GSLs beingconfined to fractions 5–10 (Fig. 6, right column; Support-ing Information Table S1). Our results further supportexisting data that EhVs induce GSL biosynthesis during

37

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hc 1 2 3 4 5 6 7 8 9 10 11 12

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hc 1 2 3 4 5 6 7 8 9 10 11 12

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2 h 48 h

control

+EhV86

Fig. 5. Distribution of the E. huxleyi flotillin-like protein in OptiPrep density gradients. Anti-flotillin Western immunoblot analysis of densitygradient fractions at 2 and 48 h for uninfected (control; top row) and EhV86-infected (+EhV86; bottom row) cells. Note that, while theprominent flotillin-like protein is distributed among most control fractions, it is concentrated within fractions 1–5. In contrast, EhV86-infectionconsiderably focused this protein to within fractions 2–3. Flotillin control immunoblot analysis recognized the predicted 42 kDa human flotillin(NCBI Accession# AAF17215; 253 aa) in human A-375 whole cell lysate of malignant melanoma cells. Molecular weight markers (MW; kDa)are indicated.



FractionTotalGSL* hGSL† sGSL#vGSL‡

% GSLa

2 h

48 h

Control + EhV86

72 h

123456789101112

123456789101112

123456789101112

a - calculated based on realtive peak areas of individual GSL classes* - expressed as the normalized (%) peak area, compared to a DNP-PE recovery standard (see Experimental procedures) † - based on the d19:3 long chain base and either a C22:3 hydroxy- or C22:2 hydroxy-fatty acid (Vardi et al., 2012)‡ - EhV-derived myristoyl-based GSLs with C15-C24 saturated fatty acids and C20-C24 monounsaturated fatty acids (Vardi et al., 2009)# - GSL with a sialyl (KDN) headgroup, d18:1 or d18:2 LCB and C22:0 fatty acid (Fulton et al., 2014)§ - Low molecular weight GSLs with a glycosyl headgroup; differ from vGSLs in the LCB length and number and/or positions of hydroxyls

0.0 11 0 19 700.0 19 0 16 660.1 0 0 13 870.1 31 0 30 400.3 52 0 13 341.2 62 0 32 62.1 86 0 7 71.9 68 0 31 12.3 70 0 27 22.3 64 0 34 11.0 62 0 37 10.5 70 0 25 5

rGSL§TotalGSL* hGSL† sGSL#vGSL‡

% GSLa

rGSL§

0.1 18 0 1 810.1 21 0 21 580.1 9 0 48 440.1 35 0 50 150.6 90 0 9 24.9 68 0 32 17.4 68 0 09.3 60 0 40 110.0 56 0 44 07.2 76 0 24 03.3 76 0 24 02.3 71 0 29 0

0.1 6 0 94 00.5 2 0 72 260.3 53 0 46 10.6 11 0 40 497.7 33 0 67 015.4 68 0 32 012.0 59 0 40 112.5 60 0 39 114.5 50 0 50 012.7 54 0 46 04.2 47 0 53 03.7 55 0 44 2

32

0.1 11 0 13 760.2 5 0 12 840.0 0 0 16 840.1 0 0 6 940.3 29 0 8 630.1 15 0 0 851.2 92 0 6 21.5 74 23 0 31.6 98 0 0 22.5 78 18 3 11.6 37 53 4 70.7 85 0 1 13

0.4 5 80 0 150.7 16 78 1 60.2 18 53 11 190.7 4 54 4 381.9 50 35 1 25

14.6 51 42 6 116.5 61 36 3 013.6 55 41 5 018.7 44 37 18 0

9.6 61 27 13 06.0 60 38 2 03.6 59 29 12 0

6.0 2 94 1 325.6 8 90 2 012.1 3 94 4 010.8 5 94 1 047.9 21 68 11 0

100.0 13 83 5 070.6 17 70 14 063.2 20 59 21 073.2 21 68 11 049.3 20 71 9 015.0 22 74 4 0

9.6 13 74 13 0

Fig. 6. Distribution of total glycosphingolipids (GSLs) and the corresponding relative percentages of individual GSL classes (hGSLs, vGSLs,sGSLs and rGSLs) in OptiPrep density gradient fractions (1–12) for uninfected control and EhV86-infected Emiliania huxleyi CCMP1516 at 2,48 and 72 h. Definitions of the four GSL classes are provided.

8 S. L. Rose et al.


infection (Vardi et al., 2009; 2012; Bidle and Vardi, 2011;Fulton et al., 2014). The hGSL distribution and percentcontribution at 2 hpi was even more concentrated indenser gradient fractions (6–10) compared with controlcells. Infection with EhV86 subsequently downregulatedhGSL production at 48 hpi and even more so at 72 hpi(peak lytic phase), whereby hGSLs comprised only13–22% of GSLs in fractions 5–12. For comparison,hGSLs accounted for 47–68% of GSLs for uninfectedcontrol fractions at this time point. Likewise, virus infectiondownregulated the relative contribution of sGSL and rGSLspecies, especially at 72 hpi, where rGSLs were almostcompletely removed from much of the fractionatedlipidome and sGSLs were < 20%. sGSLs were, however,relatively enriched in buoyant DRM fractions (1–5) at2 hpi. This fractionation with DRMs (fractions 1–5) waseven more apparent for rGSLs at 2 hpi comprising63–94% of the fraction-specific GSLs. In stark contrast,vGSLs dominated the GSL composition as infection pro-gressed, and were generally distributed throughout all 12fractions at 48 and 72 hpi (Supporting InformationTable S1). At 72 hpi, vGSLs accounted for an impressive59–94% of GSLs in all fractions. This distribution ofvGSLs corresponded quite well with our PCR-basedamplification of the EhV MCP gene, indicating thatgenomic DNA and viral membranes (which consist largelyof vGSLs) co-localized in our density gradients lendingfurther support that most EhVs were intact.

Characterizing the lipid raft proteome

Given the physical (via TEM), biochemical (via flotillinimmunoreactivity) and lipid (GSL species) distribution pat-terns, we targeted fraction 2 from both control and EhV86-infected samples at 2 hpi for proteomic analyses in orderto provide insight into the key protein players in initialviral–host interactions (Allen et al., 2006b; Mackinderet al., 2009) and provide a better understanding of the cellsurface proteins that serve to mediate viral infection andhost cell response. The liquid chromatography-tandemmass spectrometry (LC-MS/MS) peptide fragment datawere searched against the common Repository of Adven-titious Proteins (cRAP; http://www.thegpm.org/crap/),which consists of proteins commonly found in proteomicsexperiments through unavoidable contamination withcommon laboratory proteins and/or protein standards,and the respective E. huxleyi CCMP1516 and EhV86annotated proteome databases with amino acid sequen-ces parsed from Uniprot database. A total of 86 proteinswere identified in the uninfected control E. huxleyi cellssample, whereas 116 total proteins were found in theEhV86-infected sample. An analysis of the top 20 proteinhits (protein log-e rank order) for both control and EhV86-infected samples (Tables 1–2 respectively) indentifiedmany common proteins, most notably a Band_7, flotillin-like protein (Protein ID 363433; log e = −109.1; 34 uniquepeptides) with the predicted SPFH-containing domain,

Table 1. Top 20 protein hitsa detected in proteomic analysis of lipid raftsc for uninfected Emiliania huxleyi cells at 2 h.

Protein IDb Protein description Protein log e# of unique peptidesdetected

ProteinMW (kDa)

444491 Outer membrane protein porin −160.6 19 31.6363433 Band_7; flotillin-like protein −109.1 14 33.5444671 Protein of unknown functione −73.5 11 19.6366130 Chlorophyll a/b binding protein −72.4 9 20.2432658 Mitochondrial import receptor subunit −67 7 30.9440685d High affinity nitrate transporter −61.1 9 73.3436031d Delta-carbonic anhydrase −58.9 8 72.4446310 Chlorophyll a/b binding protein −58.2 6 22.6441334 Type 1 actin −56.2 8 41.6358662 Chloroplast light harvesting protein −55.9 5 21.9420185 Chloroplast light harvesting protein −53.1 3 15.6433847 Chloroplast light harvesting protein −53 7 30360139 Mitochondrial substrate carrier −49.9 6 36.7246130 Histone H4 −46.8 7 11.4439254d Nitrite transporter −35.5 5 29.346509 Chloroplast light harvesting protein −35.2 5 18434557 Protein of unknown functione −31.8 4 16.5444211d Protein of unknown functione −27.8 4 30.6460471 Protein of unknown functione −26.8 4 31.2447939d Na+/Ca2 ± K + exchanger −19.8 3 67.9

a. Based on rank order of Protein log e.b. JGI (filtered best models; http://genome.jgi-psf.org/).c. Fraction #2 from 2 h.d. Contains predicted transmembrane domains.e. No Gene Ontology, Conserved Domains, or BlastP hits with e-value < 0.



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thus corroborating our anti-flotillin Western blot analysesand further supporting fraction 2 as a representative bonafide lipid raft fraction. Our proteomic analysis also identi-fied several known membrane proteins, with predictedtransmembrane domains, including an outer membraneporin, nitrate/nitrite transporters, an import receptor, asodium/calcium exchange membrane protein and a sub-strate carrier protein. Carrier proteins are integral mem-brane proteins that are involved in the movement of ions,small molecules or macromolecules, such as anotherprotein, across biological membranes. These all providedconfidence in the membrane-containing nature of our lipidraft proteome. Interestingly, we also detected a variety ofchlorophyll a/b binding proteins among our top 20 proteinhits. The chlorophyll pigment distribution in OptiPrepdensity gradients did not coincide with lipid raft, DRMfractions (Supporting Information Fig. S2), indicating thatthese fractions were not contaminated with bulk chloro-plast material. Instead, these findings suggest that lipidrafts (and associated proteins) are also features ofsubcellular organelle membranes, not surprising given theroles that lipid rafts play in normal cell trafficking.

We wanted to better differentiate the ‘basal’ host lipidraft proteome from a virus-manipulated one by identifyingproteins that were uniquely associated with respectiveuninfected (Supporting Information Table S2) and EhV86-infected (Tables S3) cell states. We were particularlyinterested in the pool of stress-, PCD- and/or innate

immunity-related proteins associated with lipid rafts giventhe established connections of these cellular processeswith EhV infection (Bidle et al., 2007; Vardi et al., 2009;Bidle and Vardi, 2011; Bidle and Kwityn, 2012). Althoughour analysis admittedly included many singleton proteinfragments with low Blast-e confidence scores, it nonethe-less revealed that E. huxleyi cells not only have a diverse,basal lipid raft proteome composition, but that EhVs fun-damentally alter this composition during early infectionprocesses.

The ‘basal’ lipid raft proteome (Supporting InformationTable S2) of healthy E. huxleyi cells encompassed a widerange of proteins, whose homologues and evolutionaryconserved domains are widespread in higher eukaryotesand are involved in the regulation of normal cell function.These included nutrient transporters, DNA- and RNA-binding proteins, protein kinases and phosphatases,a cell division regulator protein, fibronectin, ferredoxin,tetratricopeptide repeat proteins and ankyrins. We alsoidentified a WD40-containing Sec31-like secretory proteinthat is integral to specialized membrane domains and actas transporters, scaffolds or adaptors to mediate protein–protein interactions, intracellular trafficking or vesicletransport (Mohler et al., 2002; Cortajarena and Regan,2006; Suman et al., 2011). Proteins with notable stressand defence domain functions were also associated withhealthy E. huxleyi lipid rafts, such as phosphoinositide-binding Phox, a U-Box/Ring finger, patatin and RAP RNA-

Table 2. Top 20 protein hitsa detected in proteomic analysis of lipid raftsc for EhV86-infected E. huxleyi cells at 2 h post infection.

Protein IDb Protein description Protein log e

# of uniquepeptidesdetected

ProteinMW (kDa)

444491 Outer membrane protein porin −160.6 19 31.6363433 Band_7; flotillin-like protein −109.1 14 33.5444671 Protein of unknown functione −73.5 11 19.6366130 Chlorophyll a/b binding protein −72.4 9 20.2439740d membrane-bound proton-translocating pyrophosphatase −71.2 9 93.7432658 Mitochondrial import receptor subunit −67 7 30.9442092 Heat shock protein 70 −62 9 71.8440685d High affinity nitrate transporter −61.1 9 73.3437063 Mitochondrial carrier protein −59.8 7 33.3436031d Delta-carbonic anhydrase −58.9 8 72.4446310 Chlorophyll a/b binding protein −58.2 6 22.6441334 Type 1 actin −56.2 8 41.6358662 Chloroplast light harvesting protein −55.9 5 21.9420185 Chloroplast light harvesting protein −53.1 3 15.6433847 Chloroplast light harvesting protein −53 7 30360139 Mitochondrial substrate carrier −49.9 6 36.7246130 Histone H4 −46.8 7 11.4439254d Nitrite transporter −35.5 5 29.346509 Chloroplast light harvesting protein −35.2 5 1875032d H ± translocating pyrophosphatase −32.9 5 75.8

a. Based on rank order of Protein log e.b. JGI (filtered best models; http://genome.jgi-psf.org/).c. Fraction #2 from 2 h.d. Contains predicted transmembrane domains.e. No Gene Ontology, Conserved Domains, or BlastP hits with e-value < 0.

10 S. L. Rose et al.



binding domains. Phox-like proteins possess an ability togenerate superoxide (Kawahara and Lambeth, 2007) andplay an important role in host defence through amicrobiocidal complex. U-box domain proteins, most com-monly found in plants, act as negative regulators in celldeath signalling (Zeng et al., 2008). Patatin proteins, agroup of glycoproteins also found in plants, have lipolyticactivity and participate in defence and signal transductionpathways (Banerji and Flieger, 2004). RAP RNA-bindingdomains are important in various parasite–host cell inter-actions in a variety of eukaryotic host species.

Of the 73 proteins that were uniquely associated withlipid rafts of the 2 hpi EhV86-infected cell state (Support-ing Information Table S3), several notable candidateshave putative function in lipid trafficking, signal trans-duction processes, pathogen defence and innate immuneresponse. CRAL/TRIO domain-containing proteins regu-late intracellular trafficking of hydrophobic ligands bybinding small lipophilic molecules (Panagabko et al.,2003). The calmodulin-binding DENN/MADD domain-containing proteins are involved in mitogen-activatedprotein (MAP) kinase induction (Schievella et al., 1997), aknown mediator of stress-induced PCD, and they serveas regulators of Rab GTPases (Marat et al., 2011), whichcontrol membrane trafficking. Proline-rich extensins(PRICHEXTENSN) function in the signal transduction ofpathogen defence upon compromised cell wall structure(Silva and Goring, 2002; Sanabria et al., 2010). Toll inter-leukin 1 receptor (TIR) and leucine rich repeat (LRR)domain proteins, which are often connected through anucleotide-binding (NB) domain, collectively mediatepathogen recognition/resistance and activate host celldefence responses (Peart et al., 2005; Swiderski et al.,2009). TIR-NB-LRR proteins have actually been shown tospecially recognize viral membrane proteins throughligand–receptor interaction resulting in the stress-induced, plant hypersensitive PCD response (Heath,2000; Nimchuk et al., 2003). Membrane-associatedproteophosphoglycans and regulator of chromosomecondensation 1 proteins are also particularly interestingcandidates given their respective involvement in parasiteevasion of host cell immune systems (Aebischer et al.,1999) and regulation of parasite virulence through con-trols on efficient nuclear trafficking (Frankel et al., 2007).

The detection of an EhV86 C-type lectin 1 domain-containing membrane protein (ehv149; Q4A2Y5) amongthe EhV86-infected lipid rafts directly implicates lipidraft domains as viral entry and/or exit sites. This proteinwas among the 23 putative, transmembrane domain-containing proteins detected in the mature EhV virionproteome (Allen et al., 2008). Denaturing polyacrylamidegel electrophoresis of the mature EhV virion proteomedetected a prominent ∼40 kDa band (Allen et al., 2008)that could be composed of a combination of four proteins:

ehv067, ehv100, ehv149 and ehv175 with predictedweights of 41.9, 40.0, 40.0, 40.6 kDa respectively. Con-sequently, EhV virions could conceivably be enriched inehv149. We wanted to constrain that ehv149 and othermature EhV proteins would not migrate to buoyant frac-tions on their own accord upon Brij-96 treatment. Themost abundant EhV protein is the MCP (Allen et al.,2008), accounting for ∼40% of total virome protein mass inPhycodnaviridae (Nandhagopal et al., 2002). If EhV pro-teins, like ehv149, migrated to Fraction #2 on their ownaccord, the MCP protein should have been detected in ourproteomic analysis, but it was not. Furthermore, it is highlyunlikely that any protein would migrate to Fraction #2 onits own accord, based on the inherent biophysical rela-tionship between protein buoyant densities and molecularweight. Using a comprehensive biophysical dataset ofprotein mass density of proteins, Fischer and colleagues(2004) showed that the mass density of proteins rangesfrom 1.34 g cm−3 (∼160 kDa proteins) to 1.42 g cm−3

(< 20 kDa proteins), inversely scaling with molecularweight. Proteins of ∼40 kDa have an inherent massdensity of ∼1.37 g cm−3 that would place ehv149 at thebottom of the OptiPrep gradient (see Fig. 3A), alongwith all other free proteins. The only conceivable waythat ehv149 could migrate to buoyant Fraction #2(1.055 g cm−3) is if it was associated with DRMs/lipid rafts.

The C-type lectin-containing proteins are classic ligand-binding partners for toll-like receptors/TIR (Neilan et al.,1999). C-type lectin-containing proteins, involved in cell–cell adhesion and endocytosis, have been reported inpoxviruses and African swine fever virus mediating theearly events involving host cell attachment and subse-quent internalization (Neilan et al., 1999; Cambi et al.,2005). Microarray analysis has classified expression ofehv149 transcripts to a 2 hpi ‘window’ (Allen et al.,2006b), and laser confocal microscopy has confirmed anearly stage (< 4 hpi), ‘chronic-like’ replication and releaseof EhV virions (Mackinder et al., 2009), so it is unclear ifour proteomic analysis represents entry or exit of EhVs.Nonetheless, it implicates a possible protein–protein(TIR-NB-LRR/C-type Lectin) specific binding interactionbetween E. huxleyi CCMP1516 and EhV86 for the suc-cessful attachment to host cell membranes for viralentry or exit through lipid rafts (Neilan et al., 1999;Nimchuk et al., 2003; Cambi et al., 2005; Swiderski et al.,2009). Notably, the other C-type lectin 2 domain-containing protein (ehV060) in the mature EhV proteome(Allen et al., 2008) was not detected in our lipid raftproteome analysis so it is unclear whether it also interactswith lipid rafts. A truncated version of this particularprotein (ehv060) resides in EhV163 (Allen et al., 2006b),another Coccolithovirus that, unlike EhV86, is unableto infect E. huxleyi CCMP2090 (previously E. huxleyiCCMP1516B). In light of our lipid raft proteome data, it is



tempting to speculate that the interactions of these C-typelectin-containing proteins with lipid raft proteins are keyconduits to successful infection and targets of cellularresistance mechanisms.

Conclusion

We demonstrated the successful isolation of lipid raftsusing host-derived GSLs and flotillin as respective lipidand protein biomarkers, in order to characterize raft-associated proteins involved in host–virus interactions.Our data provide the first direct biomolecular evidence ofEhV interaction with lipid rafts, whereby they profoundlyalter and shape their lipid and protein composition, in partthrough the induction of PCD- and innate immuneresponse-related proteins. Further research on the func-tionality of raft-associated proteins and their response tovirus infection will elucidate cross-talk pathways betweenthese proteins and others triggering downstream PCDand/or innate immunity pathways, and/or in facilitatingviral entry and exit strategies. The application of methodsused in this study to various sensitive and resistant strainsof E. huxleyi to EhV86 infection will likely further implicateraft-associated proteins as critical determinants host–viralinteractions.

Experimental procedures

Bioinformatic analysis

The completed Emiliania huxleyi CCMP1516 genomeassembly [http://genome.jgi-psf.org/Emihu1/Emihu1.home.html; v1.0; (Read et al., 2013)] was used to search, identifyand characterize the E. huxleyi flotillin-like protein (Protein ID363433). The EhV86 genome (accession# NC 007346) wasaccessed through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990) wasused for initial protein characterizations, with subsequentprotein sequence analyses used the Simple Modular Archi-tecture Research Tool (http://smart.embl.de/) (Shultz et al.,1998; Letunic et al., 2012). Secondary and tertiary structureanalyses employed Protein Homology/Analog Y RecognitionEngine V2.0 (PHYRE2) (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Kelly and Sternberg, 2009) forprotein fold recognition and PyMOL molecular visualiza-tion system (http://www.pymol.org/) for modelling three-dimensional structures.

Host and virus growth and maintenance

Emiliania huxleyi strain CCMP1516 (Ehux1516) wasobtained from the Provasoli-Guillard Center for Culture forMarine Phytoplankton and grown in f/2-Si (minus silica) under14:10 (L:D) cycle, 200 μmol photons m−2 s−1 at 18°C withconstant aeration. Cell abundance was determined using aCoulter Multisizer II (Beckman Coulter, Fullerton, CA, USA)

or flow cytometry (details below). EhV86 was propagatedusing Ehux1516 as previously described (Bidle et al., 2007).Virus abundance was determined by SYBR Gold staining andanalytical flow cytometry (details below).

Virus infection

Experiments were designed to harvest biomass fromboth uninfected, ‘control’ Ehux1516 and EhV86-infectedEhux1516 at 2, 24, 48 and 72 h post-infection (hpi). Expo-nentially growing cultures (5 L; ∼2 × 105 cells per ml) ofEhux1516 were infected with viral strain EhV86 at a multi-plicity of infection (MOI) of 2. A control culture without additionof EhV86 was performed in parallel. Cultures were grownaccording to the conditions mentioned above. For each timepoint, 1 L from each culture was harvested via vacuum filtra-tion, snap frozen in liquid N2 and stored at −80°C until pro-cessed. Samples were also collected for cell and viral countsfor each time point and analysed daily by analytical flowcytometry (details below).

Flow cytometry analysis

Samples from each time point were counted on an InfluxMariner 209 s Flow Cytometer and High Speed Cell Sorter(BD Biosciences, San Jose, CA, USA) at the RutgersMicrobial Flow Sort Lab (http://marine.rutgers.edu/flowsort/index.html). Emiliani huxleyi cells were counted using chlo-rophyll fluorescence (692/40 nm) after triggering with forwardscatter (granularity). EhV86 abundance was determined byfixation and staining with SYBR Gold, as previouslydescribed (Brussaard et al., 2000). Briefly, samples (40 μl)were fixed in 1% glutaraldehyde at 4°C for 15 min, −80°C for5 min, and then room temperature, followed by staining in0.5X SYBR Gold (Life Technologies, Grand Island, NY, USA)in TE buffer [10 mM Tris-Cl (pH 7.8), 1 mM EDTA)] at 80°Cfor 10 min in the dark. Stained samples were then countedusing green fluorescence (520 nm) after triggering with SSC(size).

Lipid raft isolation

Frozen pellets from 2, 24, 48 and 72 h time points werebrought to 4°C on ice and suspended in 1 ml lysis buffer(0.5% Brij-96, 25 mM Tris-HCl pH 8.0, 140 mM NaCl pH 8.0,1 mM PMSF), which used anon-ionic detergent (Brij-96;Sigma-Aldrich, St. Louis, MO, USA) to solubilize all mem-brane and a protease inhibitor to prevent protein degradation.Samples were placed on ice for 30 min followed by lysateclarification for 30 min via centrifugation at 4°C (4000 g). Theresulting supernatants were removed and stored at −20°C foruse in OptiPrep (Sigma) density gradient centrifugation. Cor-responding pellets were stored at −80°C.

DRM fractions from total cell lysates were isolated using5–35% discontinuous OptiPrep density gradients. The super-natant from each time point was added to OptiPrep to obtaina 45% solution and placed at the bottom of 13 ml ultra-clearcentrifuge tubes (Beckman #344059). The 45% OptiPrep/sample mixture was sequentially overlaid with 35% and 5%OptiPrep solutions respectively. A blank density gradient (with





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http://www.pymol.org/

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no added cell lysate to the 45% OptiPrep solution) was run inparallel. Density gradients were centrifuged using a SW40 TiBeckman rotor at 39 000 × g, 4°C, for 16 h (protocol adaptedfrom Macdonald and Pike, 2005). Twelve 1 ml fractions werecollected by pipette in a top down fashion. Individual fractions(1–12) were collected from each time point (200 μl) andstored at −80°C for subsequent proteomic, lipidomic, Westernimmunoblotting, PCR and TEM analyses.

Western blot analysis

Equal volumes of density gradient subsamples were loadedand separated by SDS-PAGE (Criterion TGX 4–20% gels;Bio-Rad Laboratories, Inc., Hercules, CA, USA) andeletrophoretically transferred to PVDF membranes in trans-fer buffer (200 ml methanol, 3.03 g Tris, 14.4 g glycine per1 L of MilliQ H20) at 100 V for 1 h on ice. Membranes wereprobed with an anti-human flotillin 2 monoclonal antibody(1:500; #sc-25507; Santa Cruz Biotechnology, Santa Cruz,CA, USA) followed by polyclonal goat anti-rabbit secondaryantibody (1:20 000; #sc-2004; Santa Cruz Biotechnology)and horseradish peroxidase chemiluminescence detection(SuperSignal; Pierce, Rockford, IL, USA) using ChemiDocMP System imager and Image Lab Software version 4.0.1(Bio-Rad Laboratories, Inc.). Protein Plus (Bio-Rad #161–0374) was used as a protein molecular weight standard.Human A-275 whole cell lysate of malignant melanomacells (#sc-3811; Santa Cruz Biotechnology) was used as apositive control.

TEM

TEM was used to visualize the presence and size of DRMvesicles. Fractions were dialysed against 200 mM Hepesbuffer (pH 7.5). Dialysis of each fraction (200 μl) wasachieved using a Pierce Slide-A-Lyzer 10 K (10 000 mwco)dialysis cassette with 0.1–0.5 ml sample volume (ThermoScientific, Rockford, IL, USA) following manufacturer’s proto-col. Samples were prepared for TEM using an adapted nega-tive staining method. Formvar/carbon-coated mesh grids(Electron Microscopy Sciences, Hatfield, PA, USA) were sub-jected to ultraviolet light to remove static interference enhanc-ing sample binding then placed on top of the dialysed samplefor 1 min. Excess sample was wicked off using Whatmanpaper (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA)and then placed sample side down onto 1% uranyl acetatesolution for 1 min. Excess stain was wicked off and gridswere allowed to air dry 5–10 min before visualization.Samples were visualized at the Rutgers Electron ImagingFacility (http://cbn.rutgers.edu/emlab/emlab.html) using aJEOL 100 CX Electron microscope (JOEL Ltd, Tokyo, Japan)operating at 80 kV. Digitized images were viewed and cap-tured using a Gatan CCD 673-0200 camera (Gatan Inc,Warrendale, PA, USA) connected to two high-resolutionvideo monitors.

PCR amplification

PCR was used to amplify the EhV MCP using previouslypublished primer sequences [(Schroeder et al., 2003); EhV-

MCP-F1, 5′-GTC TTC GTA CCA GAA GCA CTC GCT-3′;EhV-MCP-R1 5′-ACG CCT CGG TGT ACG CAC CCT CA-3′].PCR reaction conditions were as follows: 5 μl of each fractionwas directly added to a 15 μl reaction cocktail mixture con-taining 1 U RedTaq DNA polymerase (Sigma), 1 × PCR reac-tion buffer (Promega, Madison, WI, USA), 0.25 mM dNTPsand 30 pmol of each primer. PCR reactions were run in aMastercycler EPgradient S (Eppendorf, NY, USA) with aninitial high temperature cycle to lyse intact viral capsids (8°C,65°C, 97°C × 3 cycles) followed by an initial denaturing stepof 94°C (5 min) and 30 cycles of denaturation at 94°C (30 s),annealing at 55°C (30 s), and extension at 72°C (45 s). PCRamplicons were run on a 1% agarose gel (w/v) in 1 × TAE at80 V for 1.5 h, stained with ethidium bromide (0.5 μg ml−1)and visualized using ChemiDoc MP System imager andImage Lab Software version 4.0.1 (Bio-Rad Laboratories). EZLoad 100 bp molecular weight marker (Bio-Rad; Cat #170–8352) was used as a DNA sizing standard. Purified E. huxleyigenomic DNA and fresh EhV86 viral lysate served as nega-tive and positive template controls respectively.

Lipidomic analysis

Subsamples (100 μl) from OptiPrep gradient fractions wereanalysed for intact polar lipids using a Thermo TSQ Vantagetriple quadrupole mass spectrometer with electrospray ioni-zation at the Woods Hole Oceanographic Institution (WoodsHole, MA, USA). Lipids were extracted using a modifiedBligh–Dyer method, as previously described (Bligh and Dyer,1959; Popendorf et al., 2013). Cellular polar membrane lipidswere separated by high-performance liquid chromatography(HPLC) as described (Sturt et al., 2004; Popendorf et al.,2013) using an Agilent 1200 HPLC. Brij-96 and OptiPrepreagents at high concentrations interfere with the detection ofIPL molecular ions using typical ion trap MS (ITMS) methods(Sturt et al., 2004; Van Mooy et al., 2011) thereby necessitat-ing detection and quantitation by characteristic MS to MS2

neutral losses by triple quadrapole mass spectrometry(Popendorf et al., 2011; 2013). The total lipid extracts frominfected and uninfected E. huxleyi CCMP1516 cultures wereused for the initial identification and tuning parameters ofhGSL (neutral loss of 180 Da), vGSL (−162 Da) and sGSL(−268 Da), which were detected previously by HPLC-ESI-ITMS (Vardi et al., 2009; Vardi et al., 2012; Fulton et al., 2014#2218}. rGSL was detected only in low density raft fractionsusing the parameters for vGSL, but due to its relatively earlyelution time, it is thought to be structurally distinct from vGSL.A subset of the samples were analysed using identical HPLCconditions on a Thermo LTQ FT Ultra high-resolution Fourier-transform ion cyclotron resonance mass spectrometer forconfirmation of the elemental formulas of hGSL, vGSL andsGSL molecular ions and MS2 fragment ions.

Chlorophyll a was detected as an early eluting peakat the beginning of the intact polar lipid normal phase chro-matographic method. It was identified by its characteristicphotodiode array ultraviolet (UV)-visible spectrum withabsorbance maxima at 431 nm (Soret band) and 665 nm (Qy

band), as well as smaller peaks at 617, 580 and 535 nm. Wedid not detect pheophytin a or any other chlorophyll degrada-tion products and assume that any degradation from samplestorage in dichloromethane under Argon or low pigment



http://cbn.rutgers.edu/emlab/emlab.html

yields due to inefficient extraction by the Bligh and Dyermethod are proportional in all samples. We present relativequantitation based on chromatographic peak area integrationat 665 nm. UV-visible light spectra showed that there wereno interfering compounds absorbing in the Qy band. The peakareas were normalized to our internal recovery standard,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4-dintrophenyl) (DNP-PE), which elutes later and has an absorb-ance maximum at 340 nm.

Proteomic analysis

Frozen lipid raft fractions were sent to the Biological MassSpectrometry Facility co-run by the UMDNJ-Robert WoodJohnson Medical School and Rutgers’ Center for AdvancedBiotechnology and Medicine (http://www3.cabm.rutgers.edu/home.php) for proteomic analysis via LC-MS/MS. Proteinswere concentrated by vacuum aspiration (200 μl sampleswere reduced to 20 μl) and run ∼1 cm into a Novex gelBis-Tris 10% gel (Life Technologies, Carlsbad, CA, USA).The entire gel band was excised and proteins therein werereduced, carboxymethylated and digested with trypsin usingstandard facility protocols. Peptides were extracted, solub-ilized in 0.1% trifluoroacetic acid and analysed by nanoLC-MS/MS using a RSLC system (Dionex, Sunnyvale, CA, USA)interfaced with a Velos-LTQ-Orbitrap (ThermoFisher, SanJose, CA, USA). Samples were loaded onto a self-packed100 μm × 2 cm trap packed with Magic C18AQ, 5 μm 200 A(Michrom Bioresources, Auburn, CA, USA) and washed with‘Buffer A’ (0.2% formic acid) for 5 min with flow rate of10 μl min−1. The trap was brought in-line with the homemadeanalytical column (Magic C18AQ, 3 μm 200 A, 75 μm ×50 cm) and peptides fractionated at 300 nL min−1 usingmulti-step gradients of ‘Buffer B’ (0.16% formic acid 80%acetonitrile) consisting of 4–25% over 60 min and 25–55%over 30 min). Mass spectrometry data were acquired using adata-dependent acquisition procedure with a cyclic series ofa full scan acquired in Orbitrap with resolution of 60 000followed by MSMS scans (acquired in linear ion trap) of 20most intense ions with a repeat count of two and the dynamicexclusion duration of 60 s.

The LC-MS/MS data were searched against the completeannotated E. huxleyi CCMP1516 (filtered best models; http://genome.jgi-psf.org/) and EhV86 protein databases, with thelatter being parsed from Uniprot database and cRAP (http://www.thegpm.org/crap/). Analyses used a local version of theGlobal Proteome Machine (GPM cyclone, Beavis Informatics,Winnipeg, Canada) with carbamidomethyl on cysteine asfixed modification and oxidation of methionine and tryptophanas variable modifications using a 10 ppm precursor ion toler-ance and a 0.4 Da fragment ion tolerance.

Acknowledgements

We thank Haiyan Zheng at the Rutgers University BiologicalMass Spectrometry Facility for assistance in proteomic analy-ses and Dr. Helen Fredricks (Woods Hole OceanographicInstitution) for her assistance and guidance with HPLC/TQ-MS analyses. This study was supported by funding fromthe National Science Foundation (OCE-1061883 to KDB and

BVM) and in part by The Gordon and Betty Moore Founda-tion. The authors have no conflicts of interest with regard tothis research.

References

Aebischer, T., Harbecke, D., and Ilg, T. (1999) Proteo-phosphoglycan, a major secreted product of intracellularLeishmania mexicana amastigotes, is a poor B-cell antigenand does not elicit a specific conventional CD4+ T-cellresponse. Infect Immun 67: 5379–5385.

Allen, M., Howard, J., Lilley, K.S., and Wilson, W.H. (2008)Proteomic analysis of the EhV-86 virion. Proteome Sci 6:11.

Allen, M.J., Schroeder, D.C., Holden, M.T., and Wilson, W.H.(2006a) Evolutionary history of the Coccolithoviridae. MolBiol Evol 23: 86–92.

Allen, M.J., Forster, T., Schroeder, D.C., Hall, M., Roy, D.,Ghazal, P., and Wilson, W.H. (2006b) Locus-specific geneexpression pattern suggests a unique propagation strategyfor a giant algal virus. J. Virology 80: 7699–7705.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman,D.J. (1990) Basic local alignment search tool. J Mol Biol215: 403–410.

Babuke, T., and Tikkanen, R. (2007) Dissecting the molecularfunction of reggie/flotillin proteins. Eur J Cell Biol 86: 525–532.

Banerji, S., and Flieger, A. (2004) Patatin-like proteins: a newfamily of lipolytic enzymes present in bacteria? Microbiol.150: 522–525.

Bidle, K.D., and Kwityn, C.J. (2012) Assessing the role ofmetacaspase expression and caspase activity on viral sus-ceptibility of the coccolithophore, Emiliania huxleyi. JPhycol 48: 1079–1089.

Bidle, K.D., and Vardi, A. (2011) A chemical arms race at seamediates algal host–virus interactions. Curr Opin Microbiol14: 449–457.

Bidle, K.D., Haramaty, L., Barcelos-Ramos, J., andFalkowski, P.G. (2007) Viral activation and recruitment ofmetacaspases in the unicellular coccolithophorid, Emilianiahuxleyi. Proc Natl Acad Sci USA 104: 6049–6054.

Bligh, E.G., and Dyer, W.J. (1959) A rapid method of total lipidextraction and purification. Can J Physiol Pharm 37: 911–917.

Borner, G.H., Sherrier, D.J., Weimar, T., Michaelson, L.V.,Hawkins, N.D., MacAskill, A., et al. (2005) Analysis ofdetergent-resistant membranes in Arabidopsis. Evidencefor plasma membrane lipid rafts. Plant Phys 137: 104–116.

Bratbak, G., Egge, J.K., and Heldal, M. (1993) Viral mortalityof the marine alga Emiliania huxleyi (Haptophyceae) andtermination of algal blooms. Mar Ecol Prog Ser 93: 39–48.

Brown, D.A., and London, E. (2000) Structure and function ofsphingolipid- and cholesterol-rich membrane rafts. J BiolChem 275: 17221–17224.

Brussaard, C.P.D., Marie, D., and Bratbak, G. (2000) Flowcytometric detection of viruses. J Virol Methods 85: 175–182.

del Cacho, E., Gallego, M., Sanchez-Acedo, C., and Lillehoj,H.S. (2007) Expression of flotillin-1 on Eimeria tenella



http://www3.cabm.rutgers.edu/home.php

http://www3.cabm.rutgers.edu/home.php





sporozoites and its role in host cell invasion. J Parasitol 93:328–332.

Cambi, A., Koopman, M., and Figdor, C.G. (2005) HowC-type lectins detect pathogens. Cell Microbiol 7: 481–488.

Castberg, T., Thyrhaug, R., Larsen, A., Sandaa, R.-A.,Heldal, M., Van Etten, J.L., and Bratbak, G. (2002) Isolationand characterization of a virus that infects Emiliania huxleyi(Haptophyta). J Phycol 38: 767–774.

Cortajarena, A.L., and Regan, L. (2006) Ligand binding byTPR domains. Protein Sci 15: 1193–1198.

Edidin, M. (2003) The state of lipid rafts: from model mem-branes to cells. Annu Rev Biophys Biomol Struct 32: 257–283.

Fischer, H., Polikarpov, I., and Craievich, A.F. (2004) Averageprotein density is a molecular-weight-dependent function.Protein Sci 13: 2825–2828.

Frankel, M.B., Mordue, D.G., and Knoll, L.J. (2007) Discoveryof parasite virulence genes reveals a unique regulator ofchromosome condensation 1 ortholog critical for efficientnuclear trafficking. Proc Natl Acad Sci USA 104: 10181–10186.

Fulton, J.M., Fredricks, H.F., Bidle, K.D., Vardi, A., Kendrick,J., DiTullio, G.R., and Van Mooy, B.A.S. (2014) Novelmolecular determinants of viral susceptibility and resist-ance in the lipidome of Emiliania huxleyi. Environ Microbioldoi:10.1111/1462-2920.12358.

van der Goot, F.G., and Harder, T. (2001) Raft membranedomains from a liquid-ordered membrane phase to a site ofpathogen attack. Semin Immunol 13: 89–97.

Hakomori, S. (2008) Structure and function of glyco-sphingolipids and sphingolipids: recollections and futuretrends. Biochim Biophys Acta 1780: 325–346.

Hancock, J.F. (2006) Lipid rafts: contentious only from sim-plistic standpoints. Nature 7: 456–462.

Heath, M.C. (2000) Hypersensitive response-related death.Plant Mol Biol 44: 321–334.

Jordan, R.W., and Chamberlain, A.H.L. (1997) Biodiversityamong haptophyte algae. Biodivers Conserv 6: 131–152.

Kawahara, T., and Lambeth, J.D. (2007) Molecular evolutionof Phox-related regulatory subunits for NADPH oxidaseenzymes. BMC Evol Biol 7: 178.

Kelly, L.A., and Sternberg, M.J.E. (2009) Protein structureprediction on the web: a case study using the Phyre server.Nat Prot 4: 363–371.

Lam, E., Kato, N., and Lawton, M. (2001) Programmedcell death, mitochondria and the plant hypersensitiveresponse. Nature 411: 848–853.

Langhorst, M.F., Reuter, A., and Stuermer, C.A.O. (2005)Scaffolding microdomains and beyond: the function ofreggie/flotillin proteins. Cell Mol Life Sci 62: 2228–2240.

Lawrence, J.E., and Steward, G.F. (2010) Purification ofviruses by centrifugation. In Manual of Aquatic ViralEcology. Wilhelm, S., Weinbauer, M., and Suttle, C. (eds).Waco, TX: American Society of Limnology and Oceanog-raphy, Chapter 17, pp. 166–181.

Letunic, I., Doerks, T., and Bork, P. (2012) SMART 7: recentupdates to the protein domain annotation resource. NucleicAcids Res 40: D302–D305.

Lingwood, D., and Simons, K. (2010) Lipid rafts as amembrane-organizing principle. Science 327: 46–50.

Macdonald, J.L., and Pike, L.J. (2005) A simplified method forthe preparation of detergent-free lipid rafts. J Lipid Res 46:1061–1067.

Mackinder, L.C., Worthy, C.A., Biggi, G., Hall, M., Ryan, K.P.,Varsani, A., et al. (2009) A unicellular algal virus, Emilianiahuxleyi virus 86, exploits an animal-like infection strategy. JGen Virol 90: 2306–2316.

Marat, A.L., Dokainish, H., and McPherson, P.S. (2011)DENN domain proteins: regulators of Rab GTPases. J BiolChem 286: 1379–13800.

Mohler, P.J., Gramolini, A.D., and Bennett, V. (2002)Ankyrins. J Cell Sci 115: 1565–1566.

Monier, A., Pagarete, A., Vargas, C.D., Allen, M.J., Read, B.,Claverie, J.-M., and Ogata, H. (2009) Horizontal genetransfer of an entire metabolic pathway between aeukaryotic alga and its DNA virus. Genome Res 19: 1441–1449.

Morrow, I.C., and Parton, R.G. (2005) Flotillins and the PHBdomain protein family: rafts, worms and anaesthetics.Traffic 6: 725–740.

Morrow, I.C., Rea, S., Martin, S., Prior, I.A., Prohaska, R.,Hancock, J.F., et al. (2002) Flotillin-1/reggie-2 traffics tosurface raft domains via a novel golgi-independentpathway. Identification of a novel membrane targetingdomain and a role for palmitoylation. J Biochem 277:48834–48841.

Munro, S. (2003) Lipid rafts: elusive or illusive? Cell 115:377–388.

Nandhagopal, N., Simpson, A.A., Gurnon, J.R., Yan, X.,Baker, T.S., Graves, M.V., et al. (2002) The structure andevolution of the major capsid protein of a large, lipid-containing DNA virus. Proc Natl Acad Sci USA 99: 14758–14763.

Neilan, J.G., Borca, M.V., Lu, G., Kutish, G.F., Kleiboeker,S.B., Carrillo, C., et al. (1999) An African swine fever virusORF with similarity to C-type lectins is non-essential forgrowth in swine macrophages in vitro and for virus viru-lence in domestic swine. J Gen Virol 80: 2693–2697.

Nimchuk, Z., Eulgem, T., Holt, B.F., III, and Dangl, J.L. (2003)Recognition and response in the plant immune system.Ann Rev Genet 37: 579–609.

Panagabko, C., Morley, S., Hernandez, M., Cassolato, P.,Gordon, H., Parsons, R., et al. (2003) Ligand specificity inthe CRAL-TRIO protein family. Biochemistry 42: 6467–6474.

Peart, J.R., Mestre, P., Lu, R., Malcuit, I., and Baulcombe,D.C. (2005) NRG1, a CC-NB-LRR protein, together with N,a TIR-NB-LRR, mediates resistance against tobaccomosaic virus. Curr Biol 15: 968–973.

Pike, L.J. (2003) Lipid rafts: bringing order to chaos. J LipidRes 44: 655–657.

Popendorf, K.J., Lomas, M.W., and Van Mooy, B.A.S. (2011)Microbial sources of intact polar diacylglycerolipids in theWestern North Atlantic Ocean. Org Geochem 42: 803–811.

Popendorf, K.J., Fredricks, H.F., and Van Mooy, B.A.S.(2013) Molecular ion-independent quantification of polarglycerolipid classes in marine plankton using triplequadrupole MS. Lipids 48: 185–195.



Radeva, G., and Sharom, F.J. (2004) Isolation and characteri-zation of lipid rafts with different properties from RBL-2H3(rat basophilic leukaemia) cells. J Biochem 380: 219–230.

Rajendran, L., and Simons, K. (2005) Lipid rafts and mem-brane dynamics. J Cell Sci 118: 1099–1102.

Read, B.A., Kegel, J., Klute, M.J., Kuo, A., Lefebvre, S.C.,Maumus, F., et al. (2013) Pan genome of the phytoplank-ton Emiliania underpins its global distribution. Nature 499:209–213.

Rivera-Milla, E., Struermer, C.A.O., and Malaga-Trillo, E.(2006) Ancient origin of reggie (flotillin), reggie-like, andother lipid-raft proteins: convergent evolution of the SPFHdomain. Cell Mol Life Sci 63: 343–357.

Sanabria, N.M., Huang, J.-C., and Dubery, I.A. (2010) Self/nonself perception in plants in innate immunity anddefense. Self Nonself 1: 40–54.

Schievella, A.R., Chen, J.H., Graham, J.R., and Lin, L.-L.(1997) MADD, a novel death domain protein that interactswith the type I tumor necrosis factor receptor and activatesmitogen-activated protein kinase. J Biol Chem 272:12069–12075.

Schroeder, D.C., Oke, J., Malin, G., and Wilson, W.H. (2002)Coccolithovirus (Phycodnaviridae): characterization of anew large dsDNA algal virus that infects Emiliania huxleyi.Arch Virol 147: 1685–1698.

Schroeder, D.C., Oke, J., Hall, M., Malin, G., and Wilson,W.H. (2003) Virus succession observed during anEmiliania huxleyi bloom. Appl Environ Microbiol 69: 2484–2490.

Shultz, J., Milpetz, F., Bork, P., and Ponting, C.P. (1998)SMART, a simple modular architecture research tool: iden-tification of signaling domains. Proc Natl Acad Sci USA 95:5857–5864.

Silva, N.F., and Goring, D.R. (2002) The proline-rich,extensin-like receptor kinase-1 (PERK1) gene is rapidlyinduced by wounding. Plant Mol Biol 50: 667–685.

Simons, K., and Ehehalt, R. (2002) Cholesterol, lipid rafts,and disease. J Clin Invest 110: 597–603.

Simons, K., and Toomre, D. (2000) Lipid rafts and signaltransduction. Nat Rev Mol Cell Biol 1: 31–41.

Singer, S.J., and Nicolson, G.L. (1972) The fluid mosaicmodel of the structure of cell membranes. Science 175:720–731.

Smith, H. (ed.) (1977) The Molecular Biology of Plant Cells.Berkeley, CA: University of California Press.

Solis, G.P., Hoegg, M., Munderloh, C., Schrock, Y.,Malaga-Trillo, E., Rivera-Milla, E., and Stuermer, C.A.O.(2007) Reggie/flotillin proteins are organized into stabletetramers in membrane microdomains. Biochem J 403:313–322.

Staskawicz, B.J., Mudgett, M.B., Dangl, J.L., and Galan, J.E.(2001) Common and contrasting themes of plant andanimal diseases. Science 292: 2285–2289.

Sturt, H.F., Summons, R.E., Smith, K., Elvert, M., andHinrichs, K.-U. (2004) Intact polar membrane lipidsin prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionizationmultistage mass spectrometry – new biomarkers forbiogeochemistry and microbial ecology. Rapid CommunMass Sp 18: 617–628.

Suman, S.K., Mishra, A., Ravindra, D., Yeramala, L., andSharma, Y. (2011) Evolutionary remodelling of theBetagamma-crystallins for domain stability at the cost ofCa2+-binding. J Biol Chem 286: 43891–43901.

Swiderski, M.R., Birker, D., and Jones, J.D.G. (2009) The TIRdomain of TIR-NB-LRR resistance proteins is a signalingdomain involved in cell death induction. Mol Plant MicrobeIn 22: 157–165.

Theologis, A., Ecker, J.R., Palm, C.J., Federspiel, N.A., Kaul,S., White, O., et al. (2000) Sequence and analysis of chro-mosome 1 of the plant Arabidopsis thaliana. Nature 408:816–820.

Tyrell, T., and Merico, A. (2004) Emiliania huxleyi: bloomobservations and the conditions that induce them. InCoccolithophores: From molecular processes to globalimpact. Thierstein, H.R., and Young, J.R. (eds). BerlinHeidelberg: Springer-Verlag, pp. 75–97.

Van Etten, J.L., Graves, M.V., Muller, D.G., Boland, W., andDelaroque, N. (2002) Phycodnaviridae- large DNA algalviruses. Arch Virol 147: 1479–1516.

Van Meer, G., and Simons, K. (1988) Lipid polarity andsorting in epithelial cells. J Cell Biochem 36: 51–58.

Van Mooy, B.A.S., Hmelo, L.R., Sofen, L.E., Campagna,S.R., May, A.L., Dyhrman, S.T., et al. (2011) Quorumsensing control of phosphorus acquisition in Tricho-desmium consortia. ISME Journal 6: 422–429.

Van Valen, L. (1973) A new evolutionary law. Evol. Theory 1:1–30.

Vardi, A., Van Mooy, B.A.S., Fredricks, H.F., Popendorf, K.J.,Ossolinski, J.E., Haramaty, L., and Bidle, K.D. (2009) Viralglycosphingolipids induce lytic infection and cell death inmarine phytoplankton. Science 326: 861–865.

Vardi, A., Haramaty, L., Van Mooy, B.A.S., Fredricks, H.F.,Kimmance, S.A., Larsen, A., and Bidle, K.D. (2012) Host–virus dynamics and subcellular controls of cell fate in anatural coccolithophore population. Proc Natl Acad SciUSA 109: 19327–19332.

Wilson, W.H., Schroeder, D.C., Allen, M.J., Holden, M.T.G.,Parkhill, J., Barrell, B.G. et al. (2005) Complete genomesequence and lytic phase transcription profile of aCoccolithovirus. Science 309: 1090–1092.

Zeng, L.R., Park, C.H., Venu, R.C., Gough, J., and Wang,G.L. (2008) Classification, expression pattern, and E3ligase activity assay of rice U-box-containing proteins. MolPlant 1: 800–815.

Supporting information

Additional Supporting Information may be found in the onlineversion of this article at the publisher’s web-site:

Fig. S1. Flow cytometry plots showing the relationshipbetween side scatter (SSC) and fluorescence (520 nm) forSYBR-Green-stained, untreated (upper panel) or Brij-treated(lower panel) EhV virions.Fig. S2. Chlorophyll a distribution in OptiPrep density gradi-ent fractions (1–12) for uninfected control and EhV86-infectedEmiliania huxleyi at 2, 48 and 72 h post infection. Chlorophylla was detected as an early eluting peak at the beginning of theintact polar lipid normal phase chromatographic method (seeExperimental procedures). Note that Chlorophyll a distribution



is preferentially enriched in fractions 5–10 and does notoverlap with buoyant DRM, lipid raft fractions.Table S1. Detailed breakdown of the fraction distribution ofvGSL species# in OptiPrep density gradients for EhV86-infected E. huxleyi cells at 2, 48 and 72 h post infection.

Table S2. List of proteins uniquely associated# with lipidrafts‡ of control Emiliania huxleyi cells.Table S3. List of proteins uniquely associated# with lipidrafts‡ for EhV86-infected Emiliania huxleyi cells at 2 h postinfection.



isolation and characterization of lipid rafts in emiliania huxleyi: a...

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