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• W1978723280 abstract "The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gβγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gαo compared with rat Gαi1. In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species. The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gβγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gαo compared with rat Gαi1. In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species. IntroductionPolydnaviruses (PDVs) 2The abbreviations used are: PDV, polydnavirus; TR-FRET, time-resolved FRET; GTPγS, guanosine 5′-3-O-(thio)triphosphate; BV, bracovirus; Ni-NTA, nickel-nitrilotriacetic acid; GPCR, G-protein-coupled receptor; TRITC, tetramethylrhodamine isothiocyanate. are endogenous particles that are produced by some parasitoid wasps and injected, along with the wasp egg(s), into the hemocoel of host insects causing a range of developmental and immune effects that allow the parasitoid to successfully develop (1Webb B.A. Strand M.R. Gilbert L.I. Latrou K. Gill S.S. Comprehensive Molecular Insect Science. Elsevier, San Diego2005: 260-323Google Scholar, 2Beckage N.E. Gelman D.B. Annu. Rev. Entomol. 2004; 49: 299-330Crossref PubMed Scopus (328) Google Scholar, 3Kroemer J.A. Webb B.A. Annu. Rev. Entomol. 2004; 49: 431-456Crossref PubMed Scopus (148) Google Scholar, 4Glatz R.V. Asgari S. Schmidt O. Trends Microbiol. 2004; 12: 545-554Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Members of the Polydnaviridae are divided into two paraphyletic groups as follows: bracoviruses (BVs; genus Bracovirus) and ichnoviruses (genus Ichnovirus), which occur in some members of the wasp families, Braconidae and Ichneumonidae, respectively (5Webb B.A. Beckage N.E. Hayakawa Y. Lanzrein B. Stoltz D.B. Strand M.R. Summers M.D. Fauquet C.M. Mayo M.A. Maniloff J. Desselberger U. Ball L.A. 8th Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, San Diego2005: 255-267Google Scholar). Recent evidence suggests that these genera should perhaps be placed into separate virus families, as there is good evidence that Bracovirus and Ichnovirus derived from different ancestral viruses (6Bézier A. Annaheim M. Herbinière J. Wetterwald C. Gyapay G. Bernard-Samain S. Wincker P. Roditi I. Heller M. Belghazi M. Pfister-Wilhem R. Periquet G. Dupuy C. Huguet E. Volkoff A.N. Lanzrein B. Drezen J.M. Science. 2009; 323: 926-930Crossref PubMed Scopus (227) Google Scholar, 7Volkoff A.N. Jouan V. Urbach S. Samain S. Bergoin M. Wincker P. Demettre E. Cousserans F. Provost B. Coulibaly F. Legeai F. Béliveau C. Cusson M. Gyapay G. Drezen J.M. PLoS Pathog. 2010; 6: e1000923Crossref PubMed Scopus (106) Google Scholar). PDVs are replicated only in the ovaries of female wasps and are not known to affect the wasp (8Beckage N.E. Bioscience. 1997; 48: 305-311Crossref Scopus (36) Google Scholar). PDVs are highly unusual in that individual particles only contain a subset of the expressed genes and do not contain any genes for virus structure/replication. Therefore, the virus is only able to be transmitted vertically (between wasp generations) through the integration of the PDV genome into wasp chromosomes (8Beckage N.E. Bioscience. 1997; 48: 305-311Crossref Scopus (36) Google Scholar, 9Stoltz D.B. J. Gen. Virol. 1990; 71: 1051-1056Crossref PubMed Scopus (86) Google Scholar, 10Stoltz D.B. Beckage N.E. Thompson S.N. Federici B.A. Parasites and Pathogens of Insects. Academic Press, New York1993: 80-101Google Scholar). Indeed, it was the analysis of nonpackaged PDV genes (such as capsid proteins) that allowed the different ancestral viruses to be differentiated. Prior to this, a range of encapsidated PDV genes and/or gene families was postulated as being immune-suppressive, targeting cellular and humoral (cell-free) aspects of the innate insect immune system (4Glatz R.V. Asgari S. Schmidt O. Trends Microbiol. 2004; 12: 545-554Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Proteins encoded by these genes include protein-tyrosine phosphatases (11Ibrahim A.M. Kim Y. Naturwissenschaften. 2008; 95: 25-32Crossref PubMed Scopus (52) Google Scholar), viral ankyrins (vankyrins) (12Kroemer J.A. Webb B.A. J. Virol. 2006; 80: 12219-12228Crossref PubMed Scopus (29) Google Scholar, 13Shi M. Chen Y.F. Huang F. Liu P.C. Zhou X.P. Chen X.X. Virology. 2008; 375: 374-382Crossref PubMed Scopus (17) Google Scholar), host translation inhibitory factors (14Barandoc K.P. Kim Y. Comp. Biochem. Physiol. Part D Genomics Proteomics. 2009; 4: 218-226Crossref PubMed Scopus (8) Google Scholar, 15Nalini M. Kim Y. J. Insect Physiol. 2007; 53: 1283-1292Crossref PubMed Scopus (37) Google Scholar), EGF-like proteins (16Beck M.H. Strand M.R. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19267-19272Crossref PubMed Scopus (118) Google Scholar), CrV1 homologs (17Asgari S. Schmidt O. Theopold U. J. Gen. Virol. 1997; 78: 3061-3070Crossref PubMed Scopus (104) Google Scholar, 18Gitau C.W. Gundersen-Rindal D. Pedroni M. Mbugi P.J. Dupas S. J. Insect Physiol. 2007; 53: 676-684Crossref PubMed Scopus (45) Google Scholar, 19Labropoulou V. Douris V. Stefanou D. Magrioti C. Swevers L. Iatrou K. Cell. Microbiol. 2008; 10: 2118-2128Crossref PubMed Scopus (32) Google Scholar), EP1-like proteins (20Kwon B. Kim Y. Dev. Comp. Immunol. 2008; 32: 932-942Crossref PubMed Scopus (52) Google Scholar), the H4 histone (21Gad W. Kim Y. J. Gen. Virol. 2008; 89: 931-938Crossref PubMed Scopus (40) Google Scholar), and C-type lectins (22Glatz R. Schmidt O. Asgari S. J. Biol. Chem. 2003; 278: 19743-19750Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 23Lee S. Nalini M. Kim Y. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2008; 149: 351-361Crossref PubMed Scopus (29) Google Scholar, 24Nalini M. Choi J.Y. Je Y.H. Hwang I. Kim Y. J. Insect Physiol. 2008; 54: 1125-1131Crossref PubMed Scopus (24) Google Scholar, 25Teramato T. Tanaka T. J. Insect Physiol. 2003; 49: 463-471Crossref PubMed Scopus (19) Google Scholar). Other PDV proteins are also thought to target insect immunity, but as yet their class and potential mode-of-action are yet to be elucidated. CrV2, expressed by the Cotesia rubecula bracovirus (CrBV), is an example of such a protein. CrV2 is expressed by CrBV only in the larvae of two closely related butterflies (Pieris spp.) and is secreted into the hemolymph. It was thought to be temporally associated with short term, subtle immune dysfunction in hemocyte cells that take up the secreted protein, although the molecular basis for its effects is not yet understood (26Glatz R. Schmidt O. Asgari S. J. Gen. Virol. 2004; 85: 2873-2882Crossref PubMed Scopus (13) Google Scholar). The protein does not cause toxic effects on the cells that recover their immune function once significant expression of CrV2 (and other early expressed proteins) is reduced. Here, we present data that infer a specific, high affinity interaction between CrV2 and invertebrate/mammalian Gα subunits of heterotrimeric G-proteins, which are important cell-signaling proteins, indirectly implicated in vertebrate and invertebrate immune function (see under “Discussion” and Refs. 27Cytryñska M. Zdybicka-Barabas A. Jakubowicz T. J. Insect Physiol. 2006; 52: 744-753Crossref PubMed Scopus (16) Google Scholar, 28Lentschat A. Karahashi H. Michelsen K.S. Thomas L.S. Zhang W. Vogel S.N. Arditi M. J. Immunol. 2005; 174: 4252-4261Crossref PubMed Scopus (42) Google Scholar, 29Marin D. Dunphy G.B. Mandato C.A. J. Insect Physiol. 2005; 51: 575-586Crossref PubMed Scopus (16) Google Scholar, 30Nakamura T. Furunaka H. Miyata T. Tokunaga F. Muta T. Iwanaga S. Niwa M. Takao T. Shimonishi Y. J. Biol. Chem. 1988; 263: 16709-16713Abstract Full Text PDF PubMed Google Scholar, 31Ozaki A. Ariki S. Kawabata S. FEBS J. 2005; 272: 3863-3871Crossref PubMed Scopus (21) Google Scholar, 32Solon E. Gupta A.P. Gaugler R. Dev. Comp. Immunol. 1996; 20: 307-321Crossref PubMed Scopus (19) Google Scholar).Heterotrimeric G-proteins consist of three subunits, the Gα subunit and a dimer of Gβ and Gγ (Gβγ). There are a variety of subtypes for each G-protein class. The Gα subunit binds guanine nucleotides and exchanges GDP for GTP upon activation of the G-protein-coupled receptor (GPCR) with which the G-protein is usually associated. Nucleotide exchange causes conformation changes such that the Gα subunit and the Gβγ dimer interact with downstream effectors, including various enzymes and ion channels that alter cellular metabolism (33Oldham W.M. Hamm H.E. Nat. Rev. Mol. Cell Biol. 2008; 9: 60-71Crossref PubMed Scopus (793) Google Scholar). We previously developed a TR-FRET (time-resolved Förster resonance energy transfer) assay utilizing a terbium-chelate donor and Alexa546 acceptor to demonstrate interactions of the Gα subunit with the Gβγ dimer and the regulator of G-protein signaling 4 (RGS4) (34Leifert W.R. Bailey K. Cooper T.H. Aloia A.L. Glatz R.V. McMurchie E.J. Anal. Biochem. 2006; 355: 201-212Crossref PubMed Scopus (19) Google Scholar). The assay was recently adapted by others to monitor the effect of allosteric modulators of RGS4 in altering RGS4:Gα affinity and further developed into a high throughput screen for other such modulators (35Blazer L.L. Roman D.L. Chung A. Larsen M.J. Greedy B.M. Husbands S.M. Neubig R.R. Mol. Pharmacol. 2010; 78: 524-533Crossref PubMed Scopus (64) Google Scholar). Generally, TR-FRET uses a lanthanide donor fluorophore (such as terbium or europium) in combination with an appropriate acceptor (with an excitation spectrum overlapping the donor emission spectrum). When the fluorophores are in close proximity (<100 Å), energy transfer occurs between them, and acceptor emission is measured to determine binding kinetics (36Selvin P.R. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 275-302Crossref PubMed Scopus (471) Google Scholar). Using a lanthanide donor increases the signal:noise ratio when the long lived luminescence (characteristic of lanthanides) is exploited with time-gated measurements that eliminate short lived background signals. Lanthanides also exhibit other favorable properties, including multiple emission peaks, a large Stokes shift, and nonpolarized emission (37Nishioka T. Yuan J. Matsumoto K. Ferrari M. Ozkan M. Heller M.J. BioMEMS and Biomedical Nanotechnology. 3. Springer-Verlag Inc., New York2007: 437-446Google Scholar, 38Roda A. Guardigli M. Michelini E. Mirasoli M. Anal. Bioanal. Chem. 2009; 393: 109-123Crossref PubMed Scopus (53) Google Scholar).As part of validating the original TR-FRET assay, we determined that the CrV2 protein appeared to specifically interact with mammalian Gα subunits. Here, we adapt our TR-FRET assay to demonstrate a high affinity (nanomolar) interaction of acceptor-labeled CrV2 with mammalian and insect donor-labeled Gα. We also demonstrate that recombinant CrV2 is taken up by a specific hemocyte morphotype from larval Pieris rapae, which are key immune cells, where it could potentially interact with cellular proteins such as Gα. These results are intriguing as they suggest that insect hemocyte immune function could be regulated through G-protein signaling pathways and that some PDVs subvert host immunity by producing proteins that interact with Gα to alter immune signaling. This mode-of-action for immune suppression has not previously been reported, and its elucidation has the potential to facilitate the development of novel insect controls.EXPERIMENTAL PROCEDURESAll chemicals and reagents were of analytical grade and purchased from Sigma unless otherwise stated. All buffers were made in Milli-Q water.Production and Purification of Recombinant CrV2 and G-protein SubunitsTo produce CrV2 used in cell and far Western blot experiments, PCR was used to generate full CrV2 constructs containing the N-terminal signal peptide with a His tag incorporated at the 3′ end and NotI and KpnI restriction enzyme sites at the 5′ and 3′ ends, respectively. The forward primer sequence was 5′-gcg gcc gca tgt tgt cta caa agc-3′, and the reverse primer sequence was 5′-ggt acc tta gtg atg gtg atg gtg atg ggg atg atc tcg agc cct-3′. PCR products were produced with proofreading polymerase and cloned into pCR®-Blunt (Invitrogen) to allow subsequent restriction of the cloned products for insertion into pFastBac1 (Invitrogen). Recombinant baculovirus was then generated using the Bac-to-Bac system (Invitrogen) as per the manufacturer's instructions. Sf9 cells at 2 × 106 cells/ml were infected with amplified virus at a multiplicity of infection of ∼2 for 48–72 h in suspension at 27 °C with shaking. Cells were harvested by centrifugation, and expression was confirmed by Western blot. The media containing secreted CrV2 were stored at −20 °C or −80 °C for longer term storage.To produce purified recombinant CrV2 for TR-FRET experiments, the coding sequence without the first 21 N-terminal amino acids, which contain the secretory signal peptide, was cloned into pQE30 (Qiagen) and transformed into M15[pREP4] Escherichia coli as per Glatz et al. (26Glatz R. Schmidt O. Asgari S. J. Gen. Virol. 2004; 85: 2873-2882Crossref PubMed Scopus (13) Google Scholar). Expression of recombinant CrV2 was induced in 400-ml bacterial cultures in YT broth (8 g/liter tryptone, 5 g/liter yeast extract, 2.5 g/liter NaCl, pH 7.0) as per the manufacturer's instructions. Induced bacterial cells were pelleted by centrifugation and stored at −80 °C. Cells were later resuspended in 10 ml of TBP buffer (50 mm Tris, pH 8, 10 mm β-mercaptoethanol, 0.02 mg/ml phenylmethanesulfonyl fluoride, 0.03 mg/ml benzamidine). Lysozyme was added to a final concentration of 0.2 mg/ml and gently mixed at 4 °C for 30 min. MgCl2 was then added to a final concentration of 5 mm followed by DNase I to a concentration of 0.01 mg/ml. Mixing was continued for a further 30 min. Twenty percent (w/v) cholate solution (50 mm NaHEPES, pH 8.0, 3 mm MgCl2, 50 mm NaCl, and 200 g/liter cholic acid (Na+)) was added to give a final cholate concentration of 1% (v/v). The preparation was then gently stirred (1 h at 4 °C) before ultracentrifugation in a Beckman Coulter OptimaTM LE-80K at 100,000 × g for 40 min. Ni-NTA-agarose beads (800 μl; Qiagen) in TBP (50%) were added to the supernatant and incubated on ice for 30 min with occasional stirring. Supernatants were then applied to gravity-fed columns. Columns were then washed with 20 ml of TBP containing 100 mm NaCl followed by washing with 5 ml of TBP containing 100 mm NaCl and 10 mm imidazole. All washing procedures were carried out at 4 °C. His-tagged CrV2 was eluted from the column in 400-μl fractions using TBP containing 100 mm NaCl and 250 mm imidazole. Elution fractions were run on a polyacrylamide gel and stained using Coomassie Blue. Elutions containing CrV2 were identified and fractions pooled. Protein concentration was determined according to Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213288) Google Scholar) or by laser densitometry, before aliquoting and storage at −80 °C.G-protein subunits were produced from 1 to 2 liters of Sf9 cells infected with the desired recombinant baculovirus. Recombinant full-length Drosophila Gαo (isoform I)-expressing baculovirus was produced using the Bac-to-Bac expression system as described by the manufacturer (Invitrogen). Cell culture, virus amplifications, and infections, as well as the purification of G-protein subunits using Ni-NTA-agarose beads and fluorescent labeling of G-proteins or CrV2 with cysteine-reactive Alexa Fluor 546 C5-maleimide (Invitrogen) or CS124-DTPA-EMCH-Tb (Invitrogen), were performed as described by Leifert et al. (34Leifert W.R. Bailey K. Cooper T.H. Aloia A.L. Glatz R.V. McMurchie E.J. Anal. Biochem. 2006; 355: 201-212Crossref PubMed Scopus (19) Google Scholar).Antibody Labeling of CrV2 in Hemocytes in VitroLarval P. rapae were surface-sterilized in ice-cold 70% ethanol for a few minutes and then kept on ice until they were bled from a severed proleg directly into an ice-chilled 1.5-ml microcentrifuge tube containing ∼200 μl of Sf900II medium (Invitrogen) saturated with phenylthiourea (which inhibits melanization reactions). Hemocytes were aliquoted into wells of a printed glass slide and incubated at room temperature until cells attached, and spreading was observed. Obviously, spread cells were considered to be a plasmatocyte morphotype and attached cells that retained a rounded, apparently unspread, conformation were considered as a granulocyte morphotype. Insect cell culture medium containing secreted baculovirus-expressed CrV2 was applied to the hemocyte-containing slides and incubated for the desired time. Medium was then removed, and cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 4 min, and washed in TBST (8.8 g/liter NaCl, 0.2 g/liter KCl, 3 g/liter Trizma (Tris base), 500 μl/liter Tween 20 detergent). Anti-CrV2 polyclonal rabbit antibodies (26Glatz R. Schmidt O. Asgari S. J. Gen. Virol. 2004; 85: 2873-2882Crossref PubMed Scopus (13) Google Scholar) in TBST (1:500) were added to the glass slide and incubated for 1.5–2 h at room temperature. Cells were washed with TBST, and fluorphore-conjugated anti-rabbit antibody (1:200–250) with 1:50 dilution of FITC-phalloidin (0.1 mg/ml) in TBST were applied to slides for 1 h at room temperature in the dark. Cells were then washed with TBST and stained with 1:10,000 dilution (1 μg/ml) of DAPI for 5 min. Cells were then washed with TBST and PBS; a coverslip was applied, and the coverslip sealed with nail varnish.TR-FRET AssaysThe interaction between Alexa546 (Alexa)- and CS124-DTPA-EMCH-Tb (Tb)-labeled proteins was measured using TR-FRET as described in Leifert et al. (34Leifert W.R. Bailey K. Cooper T.H. Aloia A.L. Glatz R.V. McMurchie E.J. Anal. Biochem. 2006; 355: 201-212Crossref PubMed Scopus (19) Google Scholar). Briefly, these experiments were conducted in black 96-well plates. 20× working solutions of proteins were made in TMN buffer (50 mm Tris, pH 7.6, 100 mm NaCl, 10 mm MgCl2). Five microliters of each was then applied to opposite sides of the well such that mixing did not occur. Other indicated components such as proteinase K could also be added in this manner where required. TMN buffer was then added to give a final assay volume of 100 μl, and the reaction was initiated by mixing of all components. TR-FRET was then measured using a Victor3 multilabel plate reader (PerkinElmer Life Sciences) using an excitation wavelength of 340 nm and a delay of 50 μs, before measuring the emission at 572 nm for 900 μs. Where appropriate, measurements were ceased so that unlabeled proteins or buffer could be added and then measurements resumed.[35S]GTPγS Binding Assay40 nm purified Gα subunit or CrV2 was mixed with 1 nm [35S]GTPγS in a final volume of 100 μl of TMN buffer and incubated in a shaking water bath for 90 min at 27 °C. Twenty five microliters in triplicate were then filtered through glass microfiber 1-μm filter papers (GFCs; Filtech), and unbound [35S]GTPγS was removed by washing with three times with 4 ml of TMN buffer. The filters were then dried, and the amount of bound [35S]GTPγS was measured by scintillation counting for 60 s in Pico Pro VialsTM with 4 ml of Ultima GoldTM scintillation mixture (PerkinElmer Life Sciences) using a Wallac 1410 liquid scintillation counter.Far Western AssayPurified Drosophila Gαo and bovine serum albumin were subjected to SDS-PAGE on an “any kDa” TGX gel (Bio-Rad) using a nonreducing sample buffer. Proteins were transferred onto a Hybond-C nitrocellulose membrane (GE Healthcare) at 100 V for 1 h. Membranes were blocked with 5% skim milk powder in TBS buffer (8.8 g/liter NaCl, 0.2g/liter KCl, 3 g/liter Trizma) for >2 h. Membranes were then probed with CrV2 in Sf900II medium or conditioned medium overnight. Membranes were then washed with TBST buffer (see above) and probed with rabbit anti-CrV2 (1:10,000) in blocking solution for 2 h. Following washing with TBST, membranes were probed with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:20,000) in blocking solution for 2 h. Membranes were washed with TBST and developed using 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and nitro blue tetrazolium chloride to detect any CrV2 bound to Gαo.TR-FRET Data AnalysesData were analyzed using PrismTM 4.00 (GraphPad software Inc., San Diego). Data are presented as mean ± S.E., where n is equal or greater than 3. Where n = 2, data are presented as the mean, and error bars represent the range of the duplicates. If error bars are not visible, they are small and therefore hidden by the symbols.Amino Acid Sequence AnalysesThe CrV2 amino acid sequence was subjected to protein Blast (blast.ncbi.nlm.nih.gov) to detect any similar protein sequences. Three similar sequences were detected. ClustalW2 software was then used to align the amino acid sequences of the four similar proteins and to produce a simple phylogenetic tree of the four protein sequences. Protein BLAST was also used to determine the amino acid identity between the two experimental Gα subunits used here, i.e. rat Gαi1 (GenBankTM accession number NP_037277) and Drosophila melanogaster Gαo isoform I (GenBankTM accession number AAS64873). IntroductionPolydnaviruses (PDVs) 2The abbreviations used are: PDV, polydnavirus; TR-FRET, time-resolved FRET; GTPγS, guanosine 5′-3-O-(thio)triphosphate; BV, bracovirus; Ni-NTA, nickel-nitrilotriacetic acid; GPCR, G-protein-coupled receptor; TRITC, tetramethylrhodamine isothiocyanate. are endogenous particles that are produced by some parasitoid wasps and injected, along with the wasp egg(s), into the hemocoel of host insects causing a range of developmental and immune effects that allow the parasitoid to successfully develop (1Webb B.A. Strand M.R. Gilbert L.I. Latrou K. Gill S.S. Comprehensive Molecular Insect Science. Elsevier, San Diego2005: 260-323Google Scholar, 2Beckage N.E. Gelman D.B. Annu. Rev. Entomol. 2004; 49: 299-330Crossref PubMed Scopus (328) Google Scholar, 3Kroemer J.A. Webb B.A. Annu. Rev. Entomol. 2004; 49: 431-456Crossref PubMed Scopus (148) Google Scholar, 4Glatz R.V. Asgari S. Schmidt O. Trends Microbiol. 2004; 12: 545-554Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Members of the Polydnaviridae are divided into two paraphyletic groups as follows: bracoviruses (BVs; genus Bracovirus) and ichnoviruses (genus Ichnovirus), which occur in some members of the wasp families, Braconidae and Ichneumonidae, respectively (5Webb B.A. Beckage N.E. Hayakawa Y. Lanzrein B. Stoltz D.B. Strand M.R. Summers M.D. Fauquet C.M. Mayo M.A. Maniloff J. Desselberger U. Ball L.A. 8th Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, San Diego2005: 255-267Google Scholar). Recent evidence suggests that these genera should perhaps be placed into separate virus families, as there is good evidence that Bracovirus and Ichnovirus derived from different ancestral viruses (6Bézier A. Annaheim M. Herbinière J. Wetterwald C. Gyapay G. Bernard-Samain S. Wincker P. Roditi I. Heller M. Belghazi M. Pfister-Wilhem R. Periquet G. Dupuy C. Huguet E. Volkoff A.N. Lanzrein B. Drezen J.M. Science. 2009; 323: 926-930Crossref PubMed Scopus (227) Google Scholar, 7Volkoff A.N. Jouan V. Urbach S. Samain S. Bergoin M. Wincker P. Demettre E. Cousserans F. Provost B. Coulibaly F. Legeai F. Béliveau C. Cusson M. Gyapay G. Drezen J.M. PLoS Pathog. 2010; 6: e1000923Crossref PubMed Scopus (106) Google Scholar). PDVs are replicated only in the ovaries of female wasps and are not known to affect the wasp (8Beckage N.E. Bioscience. 1997; 48: 305-311Crossref Scopus (36) Google Scholar). PDVs are highly unusual in that individual particles only contain a subset of the expressed genes and do not contain any genes for virus structure/replication. Therefore, the virus is only able to be transmitted vertically (between wasp generations) through the integration of the PDV genome into wasp chromosomes (8Beckage N.E. Bioscience. 1997; 48: 305-311Crossref Scopus (36) Google Scholar, 9Stoltz D.B. J. Gen. Virol. 1990; 71: 1051-1056Crossref PubMed Scopus (86) Google Scholar, 10Stoltz D.B. Beckage N.E. Thompson S.N. Federici B.A. Parasites and Pathogens of Insects. Academic Press, New York1993: 80-101Google Scholar). Indeed, it was the analysis of nonpackaged PDV genes (such as capsid proteins) that allowed the different ancestral viruses to be differentiated. Prior to this, a range of encapsidated PDV genes and/or gene families was postulated as being immune-suppressive, targeting cellular and humoral (cell-free) aspects of the innate insect immune system (4Glatz R.V. Asgari S. Schmidt O. Trends Microbiol. 2004; 12: 545-554Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Proteins encoded by these genes include protein-tyrosine phosphatases (11Ibrahim A.M. Kim Y. Naturwissenschaften. 2008; 95: 25-32Crossref PubMed Scopus (52) Google Scholar), viral ankyrins (vankyrins) (12Kroemer J.A. Webb B.A. J. Virol. 2006; 80: 12219-12228Crossref PubMed Scopus (29) Google Scholar, 13Shi M. Chen Y.F. Huang F. Liu P.C. Zhou X.P. Chen X.X. Virology. 2008; 375: 374-382Crossref PubMed Scopus (17) Google Scholar), host translation inhibitory factors (14Barandoc K.P. Kim Y. Comp. Biochem. Physiol. Part D Genomics Proteomics. 2009; 4: 218-226Crossref PubMed Scopus (8) Google Scholar, 15Nalini M. Kim Y. J. Insect Physiol. 2007; 53: 1283-1292Crossref PubMed Scopus (37) Google Scholar), EGF-like proteins (16Beck M.H. Strand M.R. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19267-19272Crossref PubMed Scopus (118) Google Scholar), CrV1 homologs (17Asgari S. Schmidt O. Theopold U. J. Gen. Virol. 1997; 78: 3061-3070Crossref PubMed Scopus (104) Google Scholar, 18Gitau C.W. Gundersen-Rindal D. Pedroni M. Mbugi P.J. Dupas S. J. Insect Physiol. 2007; 53: 676-684Crossref PubMed Scopus (45) Google Scholar, 19Labropoulou V. Douris V. Stefanou D. Magrioti C. Swevers L. Iatrou K. Cell. Microbiol. 2008; 10: 2118-2128Crossref PubMed Scopus (32) Google Scholar), EP1-like proteins (20Kwon B. Kim Y. Dev. Comp. Immunol. 2008; 32: 932-942Crossref PubMed Scopus (52) Google Scholar), the H4 histone (21Gad W. Kim Y. J. Gen. Virol. 2008; 89: 931-938Crossref PubMed Scopus (40) Google Scholar), and C-type lectins (22Glatz R. Schmidt O. Asgari S. J. Biol. Chem. 2003; 278: 19743-19750Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 23Lee S. Nalini M. Kim Y. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2008; 149: 351-361Crossref PubMed Scopus (29) Google Scholar, 24Nalini M. Choi J.Y. Je Y.H. Hwang I. Kim Y. J. Insect Physiol. 2008; 54: 1125-1131Crossref PubMed Scopus (24) Google Scholar, 25Teramato T. Tanaka T. J. Insect Physiol. 2003; 49: 463-471Crossref PubMed Scopus (19) Google Scholar). Other PDV proteins are also thought to target insect immunity, but as yet their class and potential mode-of-action are yet to be elucidated. CrV2, expressed by the Cotesia rubecula bracovirus (CrBV), is an example of such a protein. CrV2 is expressed by CrBV only in the larvae of two closely related butterflies (Pieris spp.) and is secreted into the hemolymph. It was thought to" @default.
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• W1978723280 title "Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins" @default.
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