Characterization of a Novel Protein with Homology to C-type Lectins Expressed by the Cotesia rubecula Bracovirus in Larvae of the Lepidopteran Host, Pieris rapae

Abstract

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host (Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of ∼14 and ∼17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts. Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host (Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of ∼14 and ∼17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts. Polydnaviruses are particles specifically associated with the ovaries of certain Braconid and Ichneumonid endoparasitoids (1Stoltz D.B. Vinson S.B. Adv. Virus Res. 1979; 24: 125-171Crossref PubMed Scopus (233) Google Scholar). They are divided into genera, Ichnovirus and Bracovirus, based on differing host range and morphology (2Stoltz D.B. Whitfield J.B. J. Hym. Res. 1992; 1: 125-139Google Scholar). Polydnavirus genomes exist as a series of different circular DNA segments (3Krell P.J. Summers M.D. Vinson S.B. J. Virol. 1982; 43: 859-870Crossref PubMed Google Scholar), which are packaged singly or in groups into individual polydnavirus particles (1Stoltz D.B. Vinson S.B. Adv. Virus Res. 1979; 24: 125-171Crossref PubMed Scopus (233) Google Scholar). Particle-associated DNA segments are known to originate from wasp chromosomal DNA and are transferred in their integrated form to subsequent generations of wasps (4Stoltz D.B. Guzo D. Cook D. Virology. 1986; 155: 120-131Crossref PubMed Scopus (74) Google Scholar). Thus, polydnaviruses from different wasps are genetically isolated from each other and considered as separate “species” (5Webb B.A. Miller L.K. Ball L.A. The Insect Viruses. Plenum Publishing Corp., New York1998: 105-139Crossref Google Scholar). “Transmission” of particles is exclusively vertical (4Stoltz D.B. Guzo D. Cook D. Virology. 1986; 155: 120-131Crossref PubMed Scopus (74) Google Scholar, 6Stoltz D.B. J. Gen. Virol. 1990; 71: 1051-1056Crossref PubMed Scopus (86) Google Scholar), and particles are therefore not detected in males, although episomal polydnavirus DNA may exist (7Fleming J.G. Summers M.D. J. Virol. 1986; 57: 552-562Crossref PubMed Google Scholar). Production of particles is restricted to specialized ovarian calyx cells (1Stoltz D.B. Vinson S.B. Adv. Virus Res. 1979; 24: 125-171Crossref PubMed Scopus (233) Google Scholar) and is initiated in the pupal phase, soon after the onset of cuticular melanization, and continues in female adult wasps (8Norton W.N. Vinson S.B. Cell Tissue Res. 1983; 231: 387-398Crossref PubMed Scopus (53) Google Scholar, 9Albrecht U. Wyler T. Pfisterwilhelm R. Gruber A. Stettler P. Heiniger P. Kurt E. Schumperli D. Lanzrein B. J. Gen. Virol. 1994; 75: 3353-3363Crossref PubMed Scopus (93) Google Scholar, 10Pasquier-Barre F. Dupuy C. Huguet E. Monteiro F. Moreau A. Poirié M. Drezen J.-M. J. Gen. Virol. 2002; 83: 2035-2045Crossref PubMed Scopus (51) Google Scholar, 11Tanaka K. Matsumoto H. Hayakawa Y. Appl. Entomol. Zool. 2002; 37: 323-328Crossref Scopus (7) Google Scholar). Although the replication mechanism is not completely understood, recent evidence suggests that controlled localized chromosomal amplification occurs before excision of the particle segments (10Pasquier-Barre F. Dupuy C. Huguet E. Monteiro F. Moreau A. Poirié M. Drezen J.-M. J. Gen. Virol. 2002; 83: 2035-2045Crossref PubMed Scopus (51) Google Scholar). Larger chromosomal segments may have smaller segments nested within (12Cui L. Webb B.A. J. Virol. 1997; 71: 8504-8513Crossref PubMed Google Scholar). Particles accumulate in the oviduct and are injected into the host hemocoel, together with the parasitoid egg and various maternal secretions, at oviposition. The presence of polydnavirus particles is essential for survival of the egg and/or developing parasitoid larva (13Edson K.M. Vinson S.B. Stoltz D.B. Summers M.D. Science. 1981; 211: 582-583Crossref PubMed Scopus (242) Google Scholar, 14Stoltz D.B. Guzo D. J. Insect Physiol. 1986; 32: 377-388Crossref Scopus (107) Google Scholar, 15Guzo D. Stoltz D.B. J. Insect Physiol. 1987; 33: 19-31Crossref Scopus (78) Google Scholar). Polydnavirus DNA segments do not contain genes for particle replication, so no particles are produced in the lepidopteran host (1Stoltz D.B. Vinson S.B. Adv. Virus Res. 1979; 24: 125-171Crossref PubMed Scopus (233) Google Scholar, 16Webb B.A. Cui L.W. J. Insect Physiol. 1998; 44: 785-793Crossref PubMed Scopus (46) Google Scholar). Particles enter most host cell types (17Strand M.R. Mckenzie V. Grassl B.A. Aiken J.M. J. Gen. Virol. 1992; 73: 1627-1635Crossref PubMed Scopus (114) Google Scholar, 18Harwood S.H. Beckage N.E. Insect Biochem. Mol. Biol. 1994; 24: 685-698Crossref Scopus (64) Google Scholar), and viral transcripts are produced in the first few hours after parasitization. Transcripts are generated either transiently (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar) or persistently (17Strand M.R. Mckenzie V. Grassl B.A. Aiken J.M. J. Gen. Virol. 1992; 73: 1627-1635Crossref PubMed Scopus (114) Google Scholar) during parasitism. Relative levels of Campoletis sonorensis ichnovirus gene expression in Helicoverpa virescens larvae depend largely on gene copy number (16Webb B.A. Cui L.W. J. Insect Physiol. 1998; 44: 785-793Crossref PubMed Scopus (46) Google Scholar); therefore, segment nesting could conceivably function to increase the copy number of genes essential for parasitoid survival. Such genes presumably would encode abundantly expressed, secreted proteins rather than intracellular proteins (16Webb B.A. Cui L.W. J. Insect Physiol. 1998; 44: 785-793Crossref PubMed Scopus (46) Google Scholar). Cotesia rubecula bracovirus (CrBV) 1The abbreviations used are: CrBV, Cotesia rubecula bracovirus; CTL, C-type lectin; CRD, carbohydrate recognition domain; PBS, phosphate-buffered saline; RT, reverse transcription; ORBC, ovine red blood cell. genes are expressed in the host larvae, Pieris rapae, over a relatively short time period, from 4 to 12 h after parasitization (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar). CrBV appears to express only 4 major genes, which differs from other systems, such as C. sonorensis ichnovirus, which is suspected of expressing over 35 genes comprising several gene families (20Turnbull M. Webb B. Adv. Virus Res. 2002; 58: 203-254Crossref PubMed Google Scholar). The products of particle-associated genes act to suppress the host immune response (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar, 21Strand M.R. Noda T. J. Insect Physiol. 1991; 37: 839-850Crossref Scopus (132) Google Scholar, 22Li X. Webb B.A. J. Virol. 1994; 68: 7482-7489Crossref PubMed Google Scholar, 23Lavine M.D. Beckage N.E. J. Insect Physiol. 1996; 42: 41-51Crossref Scopus (98) Google Scholar, 24Luckhart S. Webb B.A. Dev. Com. Immunol. 1996; 20: 1-21Crossref PubMed Scopus (75) Google Scholar, 25Cui L. Soldevila A. Webb B.A. Arch. Insect Biochem. Physiol. 1997; 36: 251-271Crossref PubMed Scopus (58) Google Scholar), most often by targeting hemocytes. Gene products may also lead to physiological disorders (e.g. arrested development) by interfering with the host endocrine system (26Vinson S.B. Iwantsch G.F. Q. Rev. Biol. 1980; 55: 143-165Crossref Google Scholar, 27Webb B.A. Dahlman D.L. J. Insect Physiol. 1986; 32: 339-345Crossref Scopus (21) Google Scholar, 28Lawrence P.O. Lanzrein B. Beckage N.E. Thompson S.N. Federici B.A. Parasites and Pathogens of Insects. 1. Academic Press, CA1993: 59-86Google Scholar, 29Balgopal M.M. Dover B.A. Goodman W.G. Strand M.R. J. Insect Physiol. 1996; 42: 337-345Crossref Scopus (36) Google Scholar). Suppression of the host immune response appears to be the primary function of most polydnavirus genes expressed in lepidopteran larvae and is considered an important evolutionary adaptation for an organism directly exposed to the immune system of its host. One of the four major CrBV genes, CrV1, encodes a glycoprotein that is abundantly expressed in host tissues and inactivates hemocytes by destabilizing the cytoskeleton (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar, 30Asgari S. Schmidt O. Theopold U. J. Gen. Virol. 1997; 78: 3061-3070Crossref PubMed Scopus (106) Google Scholar). As a result, infected hemocytes are unable to encapsulate the parasitoid egg. A 32-kDa wasp-specific protein (Crp32) produced in calyx cells is associated with particles and also covers the parasitoid's eggs, providing passive immune protection for the developing embryo (31Asgari S. Theopold U. Wellby C. Schmidt O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3690-3695Crossref PubMed Scopus (94) Google Scholar). Whereas Crp32 appears to provide passive protection for the parasitoid, polydnavirus genes provide protection by actively suppressing host immune function. Both elements are required for survival and development of the C. rubecula parasitoid (31Asgari S. Theopold U. Wellby C. Schmidt O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3690-3695Crossref PubMed Scopus (94) Google Scholar). C-type lectins (CTLs) are proteins that bind to specific glycodeterminants and require the presence of divalent metal ions, most commonly Ca2+, to exhibit 2002; PubMed Scopus Google Scholar). are by a series of conserved residues in their carbohydrate recognition K. Rev. Cell Biol. Scopus Google Scholar). in various a range of carbohydrate are and have into groups based on gene and of 2002; PubMed Scopus Google Scholar). One has isolated from and appears to function as of immune 2002; PubMed Scopus Google Scholar), presumably to on the of or lectins are with each monomer CRD, and most often bind as the primary K. Rev. Cell Biol. Scopus Google Scholar). Here we on a novel CrBV the of which shows divalent ion-dependent lectin and has a conserved to those isolated from and Although have isolated from a range of is the first of a associated with invertebrate Insect rubecula wasps were on P. rapae as S. Schmidt O. J. Insect Physiol. 1994; Scopus (46) Google Scholar). Virus and DNA from female wasps was in and by of The was a and at in a for N.E. Arch. Insect Physiol. Biochem. 1994; Scopus Google Scholar). particles were in of PBS, and DNA was isolated from as (4Stoltz D.B. Guzo D. Cook D. Virology. 1986; 155: 120-131Crossref PubMed Scopus (74) Google Scholar). DNA was isolated from ovaries and female and wasps by in a of and was to a of and the were at were with A at for and with DNA was by of and of and at for was with at and in and were on a and transferred to a as J. T. A Scholar). was isolated from h parasitized P. rapae to and P. Biochem. 1987; PubMed Scopus Google Scholar). were on and transferred to as J. T. A Scholar). and of a h P. rapae was from P. rapae larvae at h after parasitization by C. rubecula wasps was isolated from The isolated was used for of the packaged in and The was and to the before with CrBV DNA with and and with were as DNA were resulting in of the CrV3 CrV3 was and reverse directly from the produced by the and subsequent to the CrV3 and were and to for of the into the expression were as are and of DNA from female C. rubecula wasps or of DNA was used as in A was by of of of of of and of DNA and at amplification were at for at for and at for was for at products were on at and and were used in of isolated from h parasitized P. rapae larvae, reverse of and of in a of were to for to before on was by of of of reverse and of before at for h and at for The were used in a by of of of of of DNA and of and were as for of of and rapae larvae were into with of a and the was at for at was and the was in and were and the fat body was and in before for and of body were at and on as by PubMed Scopus Google Scholar). were not before the of were in with protein to subsequent of protein were either within the transferred to a as J. T. A Scholar). were with a of an was used at a of of CrV3 in were and to the of the CrV3 a to the first amino of the protein were used in of produced during to the required for into the expression The was and with and as was before of the DNA of was with the were identified by of cells Production of CrV3 was by the of to before for h at The protein was identified by and in the of with only a of CrV3 of was at for at 4 were in a and and for The was at for at 4 before of the with of in and proteins were with with and proteins were with with were with of before in and at 4 to to the was by CrV3 was on by with CrV3 protein were from the with and One was used to by an of the CrV3 with by two with CrV3 with at and 4 after the was after the and used to at a of was by of proteins from of h parasitized P. rapae larvae were with SDS-PAGE was to a of before of of and for h at of was by of CrV3 with of and ovine red blood cells in were in before at for to form a over the of the cells a at the of the was as the of the for of in was before in of the of used to in the and were as for the other to a of of concentrations of lectin was for tested. for of lectin on divalent of CrV3 was in divalent and or EDTA and with of as concentrations of divalent were also to CrV3 to lectin and of rubecula parasitoid wasps polydnavirus particles into the of P. rapae larvae at to of host tissues by the particles and expression of particle-associated genes (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar). was isolated by a from h parasitized CrBV DNA as a (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar). The was used to a the of a CrBV gene and a the as the was and used as a in a of CrBV DNA and a of from and h parasitized larvae to a CrBV of and to a of and the that the to DNA from female wasps but not to that from P. rapae not indicate that the from particles to the larvae at oviposition. of the to only in the that CrV3 shows no homology with other The was subsequently with an of A at the of the was identified as the only with a for Res. 1991; PubMed Scopus Google Scholar). The of CrV3 is with a of of the amino a the first amino of the with a at the of the indicating that CrV3 protein is probably secreted from cells of A was produced J. J. Mol. Biol. 1982; PubMed Scopus Google Scholar). residues the of composed of amino were found in the as as a of the were used to specific to the CrV3 and of and DNA these the presence of a in the CrV3 The was within the conserved lectin The CrV3 the was into and used to cells in which the CrV3 protein was subsequently of proteins from and cells the presence of a protein that was in cells and in the of the proteins not were used to the of of the protein was by with as a protein from the was used for into and of from injected was used to from and h parasitized P. rapae larvae. The to a protein that was not by not of two which are in the and monomers were and kDa in size and were in a of as by the of and of from h parasitized larvae with a in of the monomer and an increase in the smaller that the monomer is an form of the smaller monomer A and were by lectin monomers by have identified in S. T. S. A. S. J. Biol. 1996; PubMed Scopus (53) Google Scholar), although the biological of of the lectin is not from the CrV3 was used to for of CrV3 in fat body and hemocytes from h parasitized larvae indicate that CrV3 is produced by hemocytes and fat body was on proteins from fat and at h after parasitization The presence of a of CrV3 in the with fat body or hemocytes that the protein is secreted and with appears that the of each CrV3 monomer with its within parasitized larvae hemolymph, the of 17-kDa monomer to 14-kDa monomer is and in fat the is are consistent with CrV3 secreted from fat body into the is most of the 17-kDa monomer is The smaller monomer detected in fat body and hemocytes is probably only monomers are secreted from infected cells before wasp or host elements to the 14-kDa monomer. A has for the which has residues by Pieris S. Schmidt O. Theopold U. J. Gen. Virol. 1997; 78: 3061-3070Crossref PubMed Scopus (106) Google Scholar). CrV3 was at a at h parasitization but was in by at h parasitization not an consistent with the expression of CrV3 (19Asgari S. Hellers M. Schmidt O. J. Gen. Virol. 1996; 77: 2653-2662Crossref PubMed Scopus (129) Google Scholar). and CrV3 were detected in in parasitized larvae and were to contain monomers The of different to with individual larvae, and often only was detected and A and The of is not of proteins from h parasitized larvae in an increase in CrV3 and a in that the and the from a with a or CrV3 CrV3 and smaller were detected in CrV3 CrV3 to in a of smaller into their in an increase of indicating that the CrV3 is that are at The that CrV3 appears to only form that are of suggests that CrV3 are the required for The that CrV3 that residues are not required for were probably not the or were not transferred to the of is of several of the invertebrate characterized J. J. Mol. Biol. 32: PubMed Scopus Google Scholar, Y. K. A. Scholar, T. S. J. Biochem. 1987; PubMed Scopus Google Scholar, K. H. 1990; PubMed Scopus Google Scholar, T. M. S. S. J. Biol. 1997; PubMed Scopus (93) Google Scholar). of CrV3 and of the amino with those from the that CrV3 shows with various C-type lectins. amino are conserved in from and that are also found in CrV3 Thus, that CrV3 is a lectin is on the presence of divalent metal the levels of similarity are with hypothetical proteins from C. and C. and indicating that these proteins may also function as The lectins are proteins from and although similarity with these lectins is that of the bracovirus lectins. CrV3 was also found to be to a of P. lectins not is of that the Cotesia-associated polydnavirus lectins similarity with invertebrate lectins with those of other Virus lectins are proteins that are in of the to specific on cells S. Google Scholar). polydnavirus particles enter host cells before lectin expression and probably express lectins as of immune a function to that of invertebrate lectins. CrV3 has a of only a and CRD, with several invertebrate lectins. other appears to be The few known or parasitoid lectins to homology with host proteins that are important for immune the A. R. PubMed Scopus Google Scholar). is and to host lectins, CrV3 with host lectins for that are in recognition or of the immune lectins and invertebrate lectins and in lectins a that some genes originate from host CrV3 and on CrV3 agglutinated and ovine red blood was to be in the presence of and but was of was completely in the presence of EDTA and was by the of or but not by concentrations to is in to other which are The of a CrV3 the of as CrV3 levels were not and the of by may indicate that may be important for of CrV3 in is that metal of CrV3 differs from that of of of various divalent or EDTA on in a was not inhibited by any of over at would be that the completely at concentrations of the and were a that was not the relatively of the lectins the lectin from was used to cells and was completely inhibited by its S. E. PubMed Scopus Google Scholar). is that CrV3 has with CrV3 to in is that lectin is by red blood cell and on divalent that CrV3 a and/or amino residues for its or is in to which is or elements are to the monomer. residues on each of the conserved in are known to be important of carbohydrate K. Rev. Cell Biol. Scopus Google Scholar). The lectins to CrV3 exhibit and are characterized by the the CrV3 is Whereas other lectins have a the conserved K. Rev. Cell Biol. Scopus Google Scholar), as do the hypothetical bracovirus lectins, the of the in CrV3 is among such lectins. Thus, the CrV3 may its and do not as and may indicate that CrV3 is is that CrV3 may have specific presumably an individual associated with host from E. at also to of various on in a in that CrV3 may the of host hemocytes to on a and may of these cells at high concentrations in the but not to to these is that CrV3 with a that is required for of of CrV3 of CrV3 to from parasitized P. rapae larvae the CrV3 gene from CrBV has conserved amino residues consistent with known from and and the protein shows divalent ion-dependent lectin is unique in that not require for its lectin lectin was not inhibited by that may be specific to or may require an CrV3 monomers are composed of a C-type and to into Thus, we propose that CrV3 is a novel expressed as of CrBV of host The most function of CrV3 is to with host components to immune the developing the characterized CrV3 shows similarity with proteins from CrV3 appears to hypothetical proteins isolated from associated with two other Cotesia that these hypothetical proteins also function as lectins and may have metal to appears that express a novel polydnavirus gene family of lectins. polydnavirus gene families have identified (20Turnbull M. Webb B. Adv. Virus Res. 2002; 58: 203-254Crossref PubMed Google Scholar), but no invertebrate protein has characterized as a exists as to the form of The bracovirus lectins may be important for evolutionary and to a that a bracovirus was in a Cotesia and that some bracovirus genes from their hosts. to the CrV3 and its of We of for in during in