Endoparasitoid wasp bracovirus-mediated inhibitionof hemolin function and lepidopteran hostimmunosuppression

Vassiliki Labropoulou,* Vassilis Douris,†

Dimitra Stefanou, Christiana Magrioti, Luc Sweversand Kostas IatrouInsect Molecular Genetics and Biotechnology Group,Institute of Biology, National Centre for ScientificResearch ‘Demokritos’, 153 10 Aghia Paraskevi Attikis(Athens), Greece.

Summary

Successful embryonic development of parasitoidwasps in lepidopteran hosts is achieved throughco-injection of polydna viruses whose gene productsare thought to target the immune responses of thehost. One gene product of the endosymbiont bracovi-rus of the parasitic wasp Cotesia rubecula, CrV1, hasbeen reported to inhibit the immune responses of itsendoparasitized lepidopteran host through inter-ference with the haematocyte cytoskeletal structure.Here we establish that CcV1, the Cotesia congregatabracovirus orthologue of CrV1, is also uptaken bylepidopteran haemocytes and haemocyte-like estab-lished cell lines, but we also report on a differentfunction of CcV1, which is highly relevant to the inhi-bition of the host immune responses and is based onits direct interaction with the pattern recognition mol-ecule hemolin. Recombinant CcV1 inhibits hemolinfunctions, such as lipopolysaccharide binding andbacterial agglutination as well as bacterial phagocy-tosis by haemocytes and haemocyte-like cell lines,producing functional phenotypes equivalent to thoseobserved to arise from RNAi-based inhibition ofhemolin gene expression. Finally, we show that CcV1and hemolin colocalize on the membrane surface ofhemolin-expressing cells, a finding suggesting thatCcV1 may be uptaken by haemocytes and inhibithaemocyte function as a result of its interaction withmembrane-anchored hemolin.

Introduction

Polydnaviruses (PDVs) are vertically transmitted insectviruses with segmented, double-stranded DNA genomes(Turnbull and Webb, 2002; Webb et al., 2006; Beck et al.,2007). They are associated with certain types of parasi-toid wasps of the families Braconidae and Ichneumonidaefrom which they derive their names, Bracoviruses (BVs)and Ichnoviruses respectively (Dupuy et al., 2006; Pen-nacchio and Strand, 2006; Webb et al., 2006). In the waspgenomes, PDVs occur as integrated proviruses, whichundergo excision and replication in the ovary. PDV-carrying wasps parasitize larvae of lepidopteran hosts in aspecies-specific process involving an injection-mediateddeposition of fertilized parasitoid eggs into the abdominalcavity of host larvae and a concominant co-injection ofwasp venom, calyx fluid and PDVs.

Host immune suppression and suppression of normalphysiological processes, such as ecdysis, are criticalfactors contributing to the success of the parasitizationprocess (Strand and Pech, 1995; Schmidt et al., 2001;Webb and Strand, 2005; Pennacchio and Strand, 2006;Webb et al., 2006). Normally, parasitoid eggs should beidentified as non-self by the host immune surveillancemachinery and encapsulated by the host haemocytes(Lavine and Strand, 2002). However, several studies ondifferent parasitoid-host systems have shown that anencapsulation response is not mounted, a deficiency thatallows the development of the parasitoid’s progeny in thehost (Webb and Luckhart, 1994; Lavine and Beckage,1995; Strand and Pech, 1995; Asgari et al., 1996). Aprime suspect for the abolition of immune responses inthe parasitized host is the co-injected PDVs. AlthoughPDVs do not replicate inside the parasitized host larvae,they enter the cells of several tissues, predominantlyhaemocytes and fat body (Stoltz and Xu, 1990; Webb andLuckhart, 1994), and express in them multiple genes(Webb and Strand, 2005). Virus-encoded proteins arethought to be responsible for the physiological alterationsoccurring in the host, including the inhibition of immuneresponses that would normally preclude development ofparasitoid embryos.

The completion of several PDV genome sequencingprojects (Espagne et al., 2004; Webb et al., 2006; Tanaka

Received 8 January, 2008; revised 3 April, 2008; accepted 21 April,2008. *For correspondence. E-mail beslabro@bio.demokritos.gr; Tel.(+30) 210 6503621; Fax (+30) 210 6511767. †Present address: Insti-tute of Molecular Biology and Biotechnology, Foundation of Researchand Technology-Hellas, 71110 Heraklion, Crete, Greece.

Cellular Microbiology (2008) 10(10), 2118–2128 doi:10.1111/j.1462-5822.2008.01195.xFirst published online 10 July 2008

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et al., 2007) has contributed to the identification ofmultiple proteins that are likely to interfere with normalimmune function. Among them are included protein fami-lies with ankyrin repeats (Thoetkiattikul et al., 2005; Fala-bella et al., 2007), protein tyrosine phosphatases (Provostet al., 2004; Falabella et al., 2006; Gundersen-Rindal andPedroni, 2006; Pruijssers and Strand, 2007), cys-motifproteins (Fath-Goodin et al., 2006), cysteine proteaseinhibitors (Espagne et al., 2005) and proteins specific toeach parasitization system.

One of the better-studied proteins of the BV endo-symbiote of the wasp Cotesia rubecula (CrBV) is CrV1, asecreted glycoprotein expressed in the haemocytes andfat body cells of parasitized Pieris rapae larvae and impli-cated in the transient inactivation of host haemocyte func-tions (Asgari et al., 1996; 1997). Although the mechanismof inactivation is far from being resolved, it has beenshown that CrV1 is endocytosed by the host haemocytes.Evidence has also been presented that a specific coiled-coil domain of the protein is involved in binding anduptake (Asgari and Schmidt, 2002). Apparently, theprotein interacts, as a homodimer, with haemolymphfactors like lipophorin in order to gain access to thehaemocyte cytoplasm through a haemolymph clearancemechanism employing lipophorin or scavenger receptors(Asgari and Schmidt, 2002; Schmidt et al., 2005). In termsof functional consequences, it has been proposed thatCrV1 disrupts the haemocyte cytoskeletal structure by amechanism involving cytoskeletal actin destabilizationand disruption of cytoskeleton integrity, and that thesechanges may result in inhibition of haemocyte spreadingon foreign surfaces, aggregation and phagocytosis(Asgari and Schmidt, 2002; Schmidt et al., 2005).

CrV1 orthologues are found in PDV endosymbionts ofseveral other Cotesia species (Whitfield, 2000), includingthe BV of Cotesia congregata (CcBV), which parasitizesthe lepidopteran insect Manduca sexta. For the lattercase, a CrV1-like protein, termed CcV1, is expressed inparasitized M. sexta larvae (Le et al., 2003; Amaya et al.,2005). The CcV1 transcripts become evident in fat bodyand haemocyte cells of parasitized Manduca larvae within2 and 4 h post parasitization respectively, and theexpression lasts for at least 48 h (Le et al., 2003). TheCcV1 protein, a 477-amino-acid (aa)-long polypeptidethat includes a N-terminal hydrophobic signal peptidesequence (aa 1–23) cleaved in the process of secretion(Espagne et al., 2004), is visualized in the haemocytecytoplasm from 24 h post parasitization until the emer-gence of Cotesia pupae (Amaya et al., 2005). Given theconserved amino acid sequence and overall structuralsimilarity between CrV1 and CcV1, a conserved mode ofaction against M. sexta haemocyte targets has beenhypothesized, which has yet to be defined in molecularterms.

In this study, we have examined the possible inter-actions of CcV1 with haemocyte proteins of immune-stimulated but non-parasitized M. sexta. We demonstratethat CcV1 interacts directly with hemolin and determinethe functional consequences of the interaction in terms ofinterference with the function of hemolin in the course of anormal immune response.

Results

CcV1 interacts with M. sexta hemolin

A library of cDNA clones generated from immune-stimulated haemocytes of non-parasitized M. sexta wasscreened initially for interactions with CcV1 using asscreening tool the yeast two-hybrid protein interactionassay. Following multiple rounds of qualitative and quan-titative screening assays in solid and liquid media forb-galactosidase activity, positive clones encoding CcV1interacting proteins were selected and analysed by PCR(data not shown). Clones with putative interacting prey-encoding inserts contained three cDNAs for M. sexta pro-teinase HP23 (Wang et al., 2006), two cDNAs for theserine protease homologue, SPH2, which lacks the serinemoiety of the active site (Yu et al., 2003), and threecDNAs of variable lengths (400–1100 bp) for hemolin(Wang et al., 1995).

To confirm the identified CcV1–hemolin interaction,CcV1 and hemolin were expressed as C-terminallytagged recombinant proteins, CcV1-myc.his and Hem-his.glu respectively, in stably transformed lepidopteraninsect cells. As expected, due to the presence of signalpeptide sequences at their N-termini, both proteins werequantitatively secreted into the cell culture media with onlyminor amounts of steady-state proteins being detectablein the cellular fractions (data not shown). Accordingly,the two proteins were purified to high purity (~90%)from the culture supernatants by single-step Ni2+-affinitychromatography. Upon PAGE, CcV1 (predicted mass of53 kDa) was found to migrate as an 85 kDa protein(Fig. 1A). Hemolin (predicted molecular mass of 47 kDafor the tagged protein), on the other hand, was found tomigrate with a mobility corresponding to a 50–52 kDaprotein (Fig. 1B). The increase in the apparent molecularweight of hemolin, was also observed previously (Suet al., 1998) and could be attributed, at least in part, to thepresence of a conserved N-glycosylation site (Bettencourtet al., 1999), which is found in all lepidopteran hemolins(Li et al., 2005).

Because based on its sequence (Espagne et al., 2004),CcV1 is also predicted to contain multiple glycosylationsites, we examined whether incubation of the producingcell cultures in the presence of tunicamycin or benzyl2-acetamido-2-deoxy-a-D-galactopyranoside, inhibitors of

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N- and O-glycosylation respectively, would result ina change in the protein’s migration properties. Theseexperiments, however, did not produce conclusive resultsas the presence of these inhibitors did not produce asignificant change in the mobility of the protein (data notshown). We note that a similar behaviour was observedwith recombinant CrV1 (Asgari and Schmidt, 2002). Thus,the abnormal mobility of these proteins in PAGE mayreflect intrinsic structural properties whose precise naturehas yet to be deduced.

To confirm the interaction between CcV1 and M. sextahemolin in an in vitro system, co-immunoprecipitationswere carried out in conjunction with Western blot assays.These assays were carried out using either a mixture ofaffinity-purified tagged proteins, CcV1-myc.his and Hem-his.glu (Fig. 1C) or culture media of cells coexpressingthese two proteins (Fig. 1D), in conjunction with antibod-ies recognizing the two antigenic epitopes, c-Myc andGlu-Glu respectively. Immunoprecipitation of either CcV1or hemolin using anti-myc or anti-glu antibodies respec-tively, resulted in co-immunoprecipitation of their interact-ing partner, hemolin and CcV1 respectively (Fig. 1C and

D). Moreover, results identical to those presented forhemolin were obtained when anti-hemolin rather thananti-glu antibody was employed for detection in theWestern blot assays (data not shown). Further evidencefor the in vitro interaction of the two proteins was obtainedby chemical cross-linking of culture supernatants with0.02% glutaraldehyde (data not shown).

CcV1-dependent interference with hemolin andhaemocyte function

Lipopolysaccharide binding inhibition. Upon demonstra-tion of the interaction between CcV1 and hemolin, weinvestigated whether this interaction interferes with the bio-logical activity of hemolin. First, enzyme-linked immuno-sorbent assay (ELISA) assays were carried out to find outwhether addition of CcV1 to purified hemolin (expressedwithout the tag) could inhibit hemolin’s binding tolipopolysaccharide (LPS). These experiments were carriedout in parallel with control experiments involving knownbinding competitors of hemolin, LPS from Escherichia coliserotypes 0111:B4 and 026:B6 and peptidoglycan. As is

Fig. 1. Purification of CcV1 and hemolin and in vitro interaction assays.A and B. Expressed proteins were purified from cell culture supernatants of stably transfected cells by affinity chromatography. Four fractions(lanes 1–4) were separated by 10% SDS-PAGE electrophoresis and stained with silver nitrate (left parts) or transferred to nitrocellulosemembranes and probed with antibodies against the tags (right parts), anti-myc for CcV1 (A) and anti-glu for hemolin (B).C and D. Western blots of immunoprecipitates from immunoprecipitation reactions carried out on mixtures of affinity-purified myc-tagged CcV1and glu-tagged hemolin (C) or media of cell cultures coexpressing myc-tagged CcV1 and glu-tagged hemolin (D). In the upper parts of eachpanel, the Western blots are showing the interaction of myc-tagged CcV1 with glu-tagged hemolin using either an anti-myc (C) or anti-gluantibody (D) to precipitate the relevant tag-bearing proteins. The lower parts of each panel show the results of re-probing of the samemembranes, after stripping, with the complementary antibodies to detect the immunoprecipitated partner. For the ‘IP (anti-myc)’ and ‘IP(anti-glu)’ indications, lanes with ‘-’ were loaded with input CcV1 or hemolin respectively; those indicated with ‘+(+)’ were loaded with theprecipitates of complete immunoprecipitation reactions, while lanes indicated with ‘ +(-)’ were loaded with the precipitates of incompleteimmunoprecipitation reactions lacking the indicated primary antibodies. The corresponding molecular weights of the proteins are shown onthe left.

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shown by the results presented in Fig. 2A, CcV1 inhibitshemolin binding to immobilized LPS at a level comparablewith that of LPS and peptidoglycan. When different con-centrations of CcV1 were used, a dose-dependentdecrease in hemolin binding was observed (Fig. 2B).

It should be noted that the degree of inhibition of hemolinbinding to LPS in the presence of the LPS or peptidoglycancompetitors were lower or higher respectively, than thosereported earlier by Yu and Kanost (2002). A possibleexplanation for the apparent discrepancy may be that theantigenic structure of hemolin produced by the cell linesmay differ somewhat from that of the native protein used inthe early experiments.

Inhibition of bacterial agglutination. Hemolin is alsoknown to bind to the surface of Gram+ and Gram- bacteriaand yeast causing their aggregation (Yu et al., 2002). To

examine whether CcV1 has the capacity to interferewith hemolin’s ability to bind to cellular surfaces, we incu-bated FITC-labelled bacteria either with purifiedC-terminally tagged hemolin or with a mixture of hemolinand CcV1 (also purified and C-terminally tagged). Asshown in Fig. 3, the addition of hemolin to the FITC-labelled bacteria caused their aggregation (Fig. 3B), whileneither BSA (Fig. 3A) nor CcV1 (Fig. 3C) were able toinduce an agglutination effect. Interestingly, the agglutina-tion effect caused by hemolin was abolished in the pres-ence of CcV1 (Fig. 3D) as well as by an anti-hemolinantiserum (data not shown).

Inhibition of haemocyte-mediated phagocytosis. A well-conserved mechanism of insect innate immunity is phago-cytosis (Beck and Strand, 2005; Mavrouli et al., 2005;Lamprou et al., 2007), which is initiated by the binding ofhaemocytes to non-self entities and is followed by theirinternalization. To find out whether CcV1 has the capacityto interfere with the host immune system also at the levelof phagocytosis, we examined the effect of its presenceon the ability of Bombyx mori haemocytes and a fewselected cultured cell lines, which included Trichoplusia niHighFive, Drosophila melanogaster S2 and Spodopteralittoralis Sl2b cells, to phagocytose bacteria. The rationalebehind the testing of these cell lines was based onprevious reports suggesting that HighFive and S2 cellsexhibit haemocyte-like properties and are able to phago-cytose FITC-labelled bacteria (Ramet et al., 2001; Beckand Strand, 2005). On the other hand, because theSpodoptera Sl2b cell line has been derived from

Fig. 2. Hemolin binding to plate-bound LPS in the presence ofcompetitors and purified CcV1.A. Hemolin (0.125 mg ml-1) was pre-incubated with knowncompetitors, such as LPS from E. coli strain 0111:B4, LPS, E. colistrain 026:B6 and peptidoglycan (PG), each at 0.7 mg ml-1 or with0.10 mg ml-1 of purified CcV1 for 60 min at room temperature.The mixtures were then added to LPS-coated plates and thebinding assay was performed as described in Experimentalprocedures. Results shown are the mean values of threeindependent measurements. Bars represent mean � standarddeviation.B. Hemolin binding to plate-bound LPS in the presence of differentconcentrations of purified CcV1.

Fig. 3. Agglutination of FITC-labelled bacteria (E. coli) in thepresence of hemolin and CcV1 protein. Fluorescence microscopyof FITC-labelled E. coli bacteria incubated for 45 min with1 mg ml-1 BSA (A) as control, 0.45 mg ml-1 purified hemolin (B),0.45 mg ml-1 CcV1 (C) or a mixture of 0.5 mg ml-1 hemolin and0.45 mg ml-1 CcV1 (D) pre-incubated for 20 min at roomtemperature. The arrows point to examples of bacterialagglutination caused by the presence of purified hemolin.

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haemocytes (Volkoff et al., 2001), it was also expected tobe capable of phagocytosing bacteria. Indeed, all testedcell lines were found to be capable of phagocytosingFITC-labelled heat-killed bacteria in a similar fashion (see,for example, Fig. 4A for the Sl2b cells); however, thesilkmoth haemocytes (Fig. 4C, blue portion of fluores-cence graph and left inset image) were phagocytosingmore efficiently than any of the tissue culture cells. Whenthe same cells were pre-treated with purified CcV1protein, however, they showed a dramatic decrease intheir capability to phagocytose E. coli (Fig. 4B for Sl2bcells; and Fig. 4C, orange portion of fluorescence graphand right inset image for B. mori haemocytes).

CcV1 is uptaken by T. ni-cultured cells and B. morihaemocytes

In view of previous reports suggesting that CrV1 has theability to inactivate host haemocytes by a process that

involves uptaking of the protein by the cells (Asgari et al.,1996; 1997; Asgari and Schmidt, 2002), we testedwhether CcV1 behaves in a similar manner and is alsouptaken by HighFive cells, which are thought of as amodel of lepidopteran haemocytes due to their responsesto immune stimuli (Beck and Strand, 2005). As can beseen in Fig. 5A–D, upon incubation of the cells withpurified myc-tagged CcV1, the protein was found tobe present inside the cells. In contrast, another CcBVprotein, EP1, which contained the same C-terminal tag,could neither bind to nor be internalized by HighFive cellsupon incubation with the latter (data not shown).

To deduce whether hemolin and CcV1 interact on themembrane of recombinant hemolin-producing HighFivecells, a protein colocalization experiment was carried out.The results of this experiment (Fig. 5E–G) revealed thecolocalization of the two proteins in the periphery ofthe cells, thus providing strong supporting evidence forthe interaction between CcV1 and hemolin on the cellsurface. Furthermore, to investigate whether the interac-tion of CcV1 with hemolin may mediate hemolin entry intothe cells, control (hemolin-nonexpressing) HighFive cellswere incubated with hemolin in the presence or absenceof CcV1. These experiments did not yield any evidence forCcV1-mediated entry of hemolin into the cells (data notshown). On the other hand, the capacity of CcV1 for entryinto the cells in the presence of soluble hemolin remainedunaffected (data not shown), a finding suggesting that theinteraction between the two proteins does not result infunctional masking of the CcV1 determinants of cellularuptake (Asgari and Schmidt, 2002).

Finally, to examine whether the uptake of CcV1 mightbe subjected to species- or cell type-specific regulation,we examined its in vivo and in vitro association with B.mori larval haemocytes. Silkworm haemocytes isolatedfrom larvae injected with CcV1 displayed strong fluo-rescence indicative of CcV1 uptake (Fig. 6A and B).Furthermore, similar results were obtained when isolatedsilkmoth haemocytes were incubated in vitro with CcV1(Fig. 6C and D). The uptake of CcV1 by haemocytes wasalso confirmed by Western blot analysis using an antibodyspecific for the c-Myc epitope tag (Fig. 6G).

Discussion

The molecular mechanisms underlying PDV-assistedparasitization of lepidopteran larvae by hymenopteranparasitoids are far from being resolved. One majorcommon element of the different parasitization systems,however, is the ability of the parasitoids to avoid encap-sulation by interfering with the host immune responses.

Previous work has established that certain PDV pro-teins may be uptaken by and act as destabilizers of hosthaemocytes. The prototype protein for this type of behav-

Fig. 4. Phagocytosis of FITC-labelled E. coli in the presence ofCcV1. Top: fluorescence of S. littoralis Sl2b cells incubated withFITC-labelled E. coli in the absence (A) or presence (B) ofrecombinant CcV1, after trypan blue quenching. Bottom:phagocytosis of FITC-labelled E. coli by primary B. morihaemocytes. Compilation of FACS analyses of B. mori haemocytesshowing endogenous fluorescence (yellow), fluorescence afterphagocytosis of FITC-labelled E. coli (blue) and fluorescence fromCcV1-incubated haemocytes after phagocytosis of FITC-labelled E.coli (orange). Inset images: photomicrographs of untreated (left) orCcV1-treated (right) B. mori haemocytes used in FACS analysisafter in vivo phagocytosis of FITC-labelled E. coli.

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iour has been CrV1, a secreted protein of C. rubecula BV(Asgari et al., 1997; Le et al., 2003; Amaya et al., 2005).Besides containing a signal peptide, CrV1 possessescharacteristic structural elements, including a coiled-coildomain, which is probably involved in protein–proteininteractions and a C-terminal region comprised of a vari-able number of repeat sequence motifs. CrV1 orthologuesin other Cotesia species (Whitfield, 2000), including CcV1of the BV of C. congregata, share such structural featuresand have been hypothesized to act in a CrV1-like fashion(Le et al., 2003; Amaya et al., 2005).

In this study, we attempted to deduce CcV1’s possiblerole(s) in the process of immunosuppression of the para-sitized host through the identification of proteins ofimmune-stimulated host haemocytes that may interactwith it. Our results, using initially a yeast two-hybrid inter-action screen as a diagnostic test and subsequently, invitro co-immunoprecipitation tests involving recombinantproteins expressed in lepidopteran insect cells (Fig. 1),uncovered a previously unsuspected property of CcV1, itscapacity for direct interaction with hemolin. Hemolin, amember of the immunoglobulin superfamily containingfour immunoglobulin domains (Sun et al., 1990; Su et al.,1998), is one of the most significant molecules of the

humoral and cellular immune defence systems of theLepidoptera (Yu et al., 2002; Kanost et al., 2004). It isproduced by fat body cells and haemocytes and occursboth as a soluble protein in the haemolymph and as amembrane-bound haemocyte protein (Bettencourt et al.,1997). Although its biological functions are not yet fullydeduced, it is considered to be a pattern recognition mol-ecule (Kanost et al., 2004) acting as lectin by binding toLPS of Gram- bacteria and lipoteichoid acid of Gram+

microorganisms (Yu and Kanost, 2002).Our functional experiments have clearly shown that the

interaction of CcV1 with hemolin interferes with the capac-ity of the latter to bind LPS (Fig. 2) and causebacterial agglutination (Fig. 3). Both effects are attributedto the interaction-mediated masking of hemolin’simmunoglobulin-like repeats, the domains responsible forhemolin’s function as a pattern recognition molecule. Byconjecture, we hypothesize that CcV1 has a similar role inthe context of parasitized larvae by mediating a humoralcounter-action against the host immune system, whichshould normally be activated in order to combat the para-sitic infection.

The second level of CcV1’s counter-action against thehost immune response detected through our studies is

Fig. 5. The uptake of myc-tagged CcV1 by T. ni BTI-TN-5B1-4 (HighFive; Invitrogen) cells. The upper panels show HighFive cells before(A and B) and after (C and D) treatment with purified myc-tagged CcV1, with A and C showing fluorescence micrographs and B and Dshowing phase contrast micrographs of the cells. The presence of CcV1 in the cells was detected by indirect immunofluorescence usinganti-myc antibody and FITC-labelled secondary antibody as described in Experimental procedures. The lower panels show confocalmicroscopy of M. sexta hemolin-producing HighFive cells after treatment with CcV1. Immunostaining was performed using a double stainingtechnique. The uptaken myc-tagged CcV1 was detected (E) using an anti-myc antibody (dilution 1:500) and an anti-mouse FITC-labelledsecondary antibody (1:100), while hemolin was detected (F) using an anti-hemolin serum (dilution 1:500) and anti-rabbit Rhodamine X-labelledsecondary antibody (dilution 1:200). G shows the merging of the photographs from E and F.

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clearly exercised at a cellular level. This is manifested asthe suppression of the haemocytes’ capacity to phagocy-tose bacteria in vitro and in vivo (Fig. 4). The CcV1-mediated in vitro inhibition of phagocytosis was examinedand found to occur with several cell types examined,leading to the conclusion that the endocytosis inhibition-related function of CcV1 is not species-specific. Thiseffect could be mediated by the interaction of CcV1 witheither plasma membrane-anchored or soluble hemolin,which is capable of binding to haemocytes in a calcium-

dependent manner (Bettencourt et al., 1999), and actingas an opsonin linking haemocytes to microorganisms andfacilitating haemocyte aggregation, nodule formationand phagocytosis (Lanz-Mendoza et al., 1996; Daffre andFaye, 1997; Eleftherianos et al., 2007). In fact, thedemonstration of CcV1 colocalization with hemolin inrecombinant hemolin-expressing HighFive cells (Fig. 5)supports the contention that CcV1 acts at the level ofhaemocyte cell surface. This hypothesis is also in com-plete agreement with the results of recent studies, whichhave demonstrated that RNAi-mediated downregulationof hemolin gene expression in M. sexta larvae results in anumber of phenotypes, which include a reduction in: (i)the rate of naive haemocyte aggregation induced by addi-tion of E. coli to them, (ii) the numbers of bacteria (E. coli)endocytosed by haemocytes following bacterial (E. coli)infection and (iii) the numbers of melanotic tubules formedupon E. coli infection (Eleftherianos et al., 2007). Fur-thermore, RNAi-mediated knock-down of hemolin geneexpression in M. sexta resulted in increased susceptibilityto the insect pathogen Photorhabdus (Eleftherianos et al.,2006) and reduced levels of phenoloxidase activity (Tere-nius et al., 2007). Finally, based mostly on observationsrelated to events occurring during lepidopteran insectinfection by baculovirus pathogens, hemolin has alsobeen proposed to represent an important antiviral defencefactor (Hirai et al., 2004; Roxstrom-Lindquist et al., 2005;Terenius, 2008). Although it is unknown at presentwhether the tissue culture cells examined in our studiesexpress hemolin or not, for the case of haemocytes, our invitro experiments were carried out using haemocytes iso-lated from BSA-injected, therefore immune-stimulatedlarvae, which should be expressing plasma membrane-anchored hemolin.

The uptake of CcV1 by phagocytosis-competentcells (Figs 5 and 6) and the observed inhibition of thecapacity of silkmoth haemocytes isolated from CcV1-injected pupae to phagocytose bacteria in vivo (Fig. 4)clearly suggest a CcV1-mediated disruption of normalhaemocyte function. In this regard, we have establishedthat CcV1 uptake may occur in the absence ofhaemolymph factors, a finding that is not in agreementwith the conclusions of previous studies on CrV1 entryinto C. rubecula haemocytes. The latter suggested that: (i)CrV1 interacts with haemolymph factors and gains entryinto the haemocytes by a mechanism involving endocyto-sis of lipophorin and scavenger receptors and (ii) as aresult, the uptake cannot occur in the absence ofhaemolymph factors (Asgari and Schmidt, 2002; Schmidtet al., 2005). In contrast, our results have clearly demon-strated that haemocytes and other established cell lineswith haemocyte-like features, which are cultured in simplebuffers devoid of any macromolecular factors, are: (i)capable of in vitro CcV1 uptake and (ii) inhibited in their

Fig. 6. CcV1 protein is uptaken by B. mori haemocytes in vivo andin vitro.A and B. Isolated B. mori haemocytes from larvae injected withmyc-tagged CcV1 were stained with anti-myc antibody andFITC-labelled secondary antibody as described in the Experimentalprocedures. For the study of the in vitro uptake of CcV1, isolatedB. mori haemocytes were incubated with 2 mg of myc-tagged CcV1in PBS (in C and D) or with PBS alone (in E and F) and stained asabove.A, C and E. Phase contrast micrographs.B, D and F. Fluorescence micrographs.G. Western blot analysis showing the presence of myc-taggedCcV1 in B. mori haemocytes. Cell lysates were prepared fromhaemocytes isolated from B. mori larvae 24 h after injection withheat-killed E. coli (lane 1) or CcV1 protein (lane 2). Purifiedhemolin (lane 3) was included as a negative control, while celllysates from CcV1-producing HighFive cells (lane 4) and purifiedmyc-tagged CcV1 (lane 5) served as positive controls.

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capacity to phagocytose E. coli by the presence of CcV1.Thus, CcV1 may also be endocytosed upon binding tomembrane-anchored hemolin.

Irrespective of the mechanistic details of CcV1 internal-ization, our studies indicate that the immune function ofhaemocytes and haemocyte-like cell lines, which displayhemolin on their surface, is compromised by the interac-tion between membrane-bound hemolin and CcV1. Con-sequently, we predict that the inhibition in the capacity ofhaemocytes to phagocytose invading microorganisms byCcV1 is effected both at the level of the cell surfacethrough the interactions of CcV1 with the microorganism-binding hemolin and at an intracellular level through inter-ference with the integrity of the cytoskeletal structure. Thecorrectness of this hypothesis should be validatedthrough future studies.

In conclusion, this study provides novel evidence forthe direct interaction of a virally encoded protein with thehost immune protein hemolin and the functional conse-quences of the interaction. This as well as other, as yetunidentified interactions between virally expressed geneproducts and host proteins associated with the inductionof insect immune responses, may provide valuable toolsfor gaining in-depth insights into the mechanisms ofimmunosuppression of parasitized hosts.

Experimental procedures

Plasmid constructions

The coding sequence of CcV1 was amplified from a cDNA clonein pTriplEX2 vector (gift of J.-M. Drezen, University of Tours,France) using Pfu DNA Polymerase (Promega) with specificprimers carrying EcoRI and BamHI restriction sites (primer F:5′-agtcgaattcATGTCACTCGTCAAAAGTAC-3′ and primer R:5′-agtcggatccGAGAAATTGATGAGAAATGAG-3′) and intro-duced into the EcoRI and BamHI sites of vector pGBT9(Clontech) encoding the Gal4 DNA-binding domain (Gal4BD).The yeast vector pGAD424 encoding the Gal4 activation domain(Gal4AD) was engineered to include a XhoI site by replacing aBamHI site in order to allow directional cloning of a library ofcDNA clones generated from immune stimulated haemocytes ofnon-parasitized M. sexta larvae (obtained from T. Trenzeck, Uni-versity of Giessen, Germany). Yeast two-hybrid system screen-ing using the MATCHMAKER Gal4-based yeast two-hybrid screenfrom Clontech and b-galactosidase assays were carried out aspreviously described (Andronopoulou et al., 2006).

For generation of expression plasmids, the original CcV1cDNA clone was amplified with Pfu DNA polymerase (Promega)using the following primers, which contained a BamHI site(underlined) and a consensus Kozak-type sequence (italics)upstream of the initiation codon: CcV1F, 5′-tataggatcccaccATGTCACTCGTCAAAAAGT-3′ and CcV1R, 5′-gcgcggatccAGAAATTGATGAGAAATG-3′. The corresponding BamHI fragmentwas subcloned into vector pEIA-myc.his (c-Myc epitope andhexahistidine tag; Douris et al., 2006) in frame with the C-terminalmyc.his tag to generate the pEIA.CcV1-myc.his construct. Thecomplete ORF of M. sexta hemolin, including the signal peptide,

was subcloned from the original pBluescript SK+ clone (a giftfrom M. Kanost, Kansas State University) into vector pEIA-glu.his(Douris et al., 2006) using the same strategy and primers HemF:5′-atatggatcccaccATGGTTTCCAAAAGTATCG-3′ and Hem2R:5′-ctctggatccAGCAACAATCACGAGCGTCTCAGC-3′. All ex-pression constructs were verified by sequencing.

Protein expression and purification

BTI-TN-5B1-4 (HighFive; Invitrogen) cells derived from T. ni werecultured at 28°C in serum-free medium ESF921 (ExpressionSystems). Transfection with Lipofectin reagent (Invitrogen) wasperformed as previously described (Keith et al., 2000). Forthe generation of stably transformed cell lines, cells wereco-transfected with relevant expression plasmids and pBmA.pac,a plasmid conferring resistance to puromycin (P.J. Farrell and K.Iatrou, unpublished), at molar ratios of expression to selectionconstructs of 500:1, using 2.5 mg of total plasmid DNA per 106

cells. Stably transformed cell lines were selected as previouslydescribed (Douris et al., 2006) and maintained in ESF921medium supplemented with 50 mg ml-1 gentamicin (Invitrogen)and 50 mg ml-1 puromycin (Sigma). Cell culture supernatantscontaining secreted CcV1 and/or hemolin were used for proteinpurification by affinity chromatography on Ni-NTA agarose matrix(Qiagen) as described by the manufacturer. Purified proteinswere analysed on 10% SDS-PAGE gels, stained with silvernitrate or electroblotted to PVDF (Bio-Rad) membranes and visu-alized by Western analysis using commercial antibodies againstthe c-Myc (mouse monoclonal 9E10, Santa Cruz or 9B11, CellSignalling) and glu-glu epitopes (QED Bioscience) or specificanti-hemolin antisera (a gift from M. Kanost), at dilutions of1:1000. Detection was done by enhanced chemiluminescence(Amersham Pharmacia Biotech). Purified proteins were dialysedagainst TBS (Tris-buffered saline; 25 mM Tris–HCl, 137 mMNaCl and 3 mM KCl, pH 7.0) or PBS (phosphate-buffered saline,137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4,pH 7.4) and protein concentrations were determined using theBradford assay (Sigma).

To determine whether the increase in the apparent molecularmass of CcV1 was due to glycosylation, stably transformedinsect cells expressing recombinant CcV1 were cultured in thepresence of inhibitors for either N-glycosylation (tunicamycin,1–10 mg ml-1) or O-glycosylation (benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside, 2, 10 and 30 mM). Cell culture superna-tants as well as total cell extracts were then subjected to Westernblot analysis.

Immunoprecipitations

Mixtures of affinity-purified proteins or culture supernatants dialy-sed against TBS were incubated with 5 mg of the primary anti-body for 16 h at 4°C with gentle rotation. Then, 40 ml of a 50%slurry protein A/G-sepharose were added and incubated 3–4 h.After washing six times for 5 min each at 4°C with TBS, boundproteins were released from the beads by boiling in SDS loadingbuffer. Following gel electrophoresis and electrotransfer, the blotswere probed with anti-myc (9B11) or anti-glu primary antibodies.

LPS binding assays

Experiments were carried out according to Yu and Kanost (2002).Hemolin, in binding buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl,

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0.1 mg ml-1 BSA), was incubated for 3 h at room temperaturewith 0.7 mg of LPS [from E. coli serotypes 0111:B4 and 026:B6(Sigma)] or peptidoglycan and different concentrations of CcV1.The mixtures were then added to the wells of a 96-well plate,which had been coated with LPS (2 mg well-1) and pre-incubatedwith 3 mg ml-1 BSA for 1 h, and incubated for 6 h at roomtemperature. Following removal of the mixture, the plates werewashed three times with PBS-0.1% Triton X-100 and incubatedovernight with 1:1000 anti-hemolin at 4°C. Detection was carriedout with anti-rabbit HRP (1:3000 dilution) following incubation at37°C for 1 h. The TMB substrate kit (Pierce) was used for devel-opment and the reaction was stopped by the addition of 2 Msulphuric acid.

FITC labelling of bacteria and agglutination assays

Saturated bacterial cultures were centrifuged and the pellet waswashed three times with PBS, re-suspended in 0.1 M sodiumcarbonate buffer pH 9.6 containing 0.1 mg of FITC (Sigma), andincubated for 30 min at 37°C. The bacteria were extensivelywashed with PBS, re-suspended in TBS, and kept at -20°C priorto use in agglutination assays as previously described (Yu et al.,1999). Briefly, 10 ml of bacteria (2.5 ¥ 109 cells ml-1) were mixedwith the indicated concentrations of purified hemolin, CcV1 or amixture of both. As negative agglutination control, bacteria weremixed with 1 mg ml-1 BSA. After 45 min incubation at roomtemperature, the cells were observed under a fluorescencemicroscope.

Immunofluorescence

Cells were incubated with recombinant CcV1-myc.his protein for2 h at room temperature. After extensive washes with PBS, thecells were fixed with 4% paraformaldehyde (PFA) in PBS for20 min. Fixed cells were permeabilized with PBS-T for 10 minand stained overnight at 4°C with anti-myc antibody (9B11; CellSignalling). Following four to five washes in PBS, a FITC-conjugated goat anti-mouse secondary antibody was added for1 h at room temperature. Cells were again extensively washedwith PBS, mounted and examined under a fluorescence micro-scope (Zeiss Axiovert 25 inverted microscope). For confocalmicroscopy, the cells were treated as described above andobserved in a Bio-Rad confocal microscope (MRC 1024 ES)equipped with Lasersharp software (Bio-Rad) and a krypton-argon laser.

Isolation and treatment of B. mori haemocytes

The prolegs of stage days 5–6 fifth instar larvae of B. mori werecut off and the haemolymph was collected on ice, in tubescontaining saturated phenyl thiourea (Sigma) in PBS. Thehaemocytes were collected by centrifugation at 1000 g for 10 minat 4°C and washed three to four times with PBS. For in vitrouptake experiments, collected haemocytes were incubated withpurified recombinant CcV1 and then fixed on glass slides using4% PFA in PBS. Haemocytes from larvae injected with CcV1were collected in the same way and, after washing in PBS, theywere fixed on glass slides. Indirect immunostaining was done asdescribed above.

Phagocytosis assays and FACS analysis

FITC-labelled, heat-killed bacteria (E. coli) were added to cul-tured cells and incubated at 28°C for 45 min. The fluorescence ofFITC-labelled particles attached to the haemocytes extracellu-larly was quenched by adding 0.2% Trypan blue and cells wereobserved under a fluorescence microscope. For FACS analysis,B. mori haemocytes were incubated with FITC-labelled bacteriaas described above and subsequently fixed with 2% PFA in PBS,washed, re-suspended in PBS and analysed by FACS (Calibur,Becton Dickinson) (Ramet et al., 2001; Ohta et al., 2006;Lamprou et al., 2007). Collected data were analysed using theBecton Dickinson CELL Quest program. Untreated haemocyteswere also analysed by FACS to deduce the levels ofautofluorescence.

Acknowledgements

This work has been supported by a research grant to K.I. (03ED-122) implemented within the framework of the ‘ReinforcementProgramme of Human Research Manpower’ (PENED) andco-financed by National (Greek Ministry of Development, GeneralSecretariat of Research and Technology) and the EuropeanCommission (E.U., European Social Fund); and by funds of theEU FP5 Grant (QLK3-CT-2001-01586, BIP – Bioinsecticides frominsect parasitoids) to K.I. We thank Jean-Michel Drezen, Univer-sity of Tours, France for providing the CcV1 clone; Tina Trenczek,University of Giessen, Germany for providing the M. sextahaemocyte cDNA library; Michael Kanost, Kansas State Uni-versity, USA for clones and antisera for M. sexta hemolin;Anastasia Apostolidou, Athens Academy Institute of BiomedicalResearch, Greece for the initial subcloning of the haemocytecDNA library into a yeast expression vector; and SotirisTsakas, University of Patras, Greece for guidance in the FACSstudies.

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