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© 2006 Nature Publishing Group http://www.nature.com/natureimmunology REVIEW Host-pathogen interactions in drosophila: new tricks from an old friend Sara Cherry1 & Neal Silverman2 Insects rely solely on innate immune responses to combat a wide array of pathogens. With its powerful genetics, drosophila has proven especially powerful for the study of humoral innate immunity, characterized by the rapid induction of antimicrobial peptides. The two signaling pathways involved, Toll and Imd, have been studied intensely, but other aspects of the drosophila immune response are less well understood. A flurry of reports has focused on the mechanisms of phagocytosis, antiviral immunity and viral pathogenesis in drosophila. These studies have taken advantage of genome-wide RNA-mediated interference screening in drosophila cells, as well as more traditional genetic tools available in the fly. This review discusses advances in these exciting new areas of drosophila immunity. Insects rely entirely on innate immune responses for protection against pathogenic microorganisms. Humoral factors, especially antimicrobial peptides, are rapidly produced in the fat body (analogous to the mammalian liver) after infection to kill a wide array of microbes. In addition, circulating blood cells, known as hemocytes, efficiently phagocytose and destroy most microorganisms, whereas larger pathogens, such as parasitoid wasp eggs, are encapulated by blood cells and melanin. During the past decade, researchers working with drosophila, exploiting its unparalleled power as a model system, have elucidated many aspects of pattern recognition and innate immune signal transduction, especially in the production of antimicrobial peptides. Two pathways recognize different classes of pathogens to activate antimicrobial peptide gene expression. The Toll pathway responds to Gram-positive and fungal pathogens, activating the NF-κB transcription factors Dif and dorsal, resulting in the induction of various target genes, including the antifungal drosomycin. In contrast, the Imd pathway recognizes Gram-negative bacteria, activating a different NF-κB transcription factor (Relish) and the expression of a distinct, but overlapping set of genes encoding humoral factors, including the antibacterial diptericin1,2 (Fig. 1). Studies have suggested that similar paradigms also apply to other insects, including mosquitoes. Moreover, these studies have led directly to important findings in mammalian systems. In particular, the discovery of the function of drosophila Toll in the induction of antimicrobial peptide genes led to the identification of Toll-like receptors (TLRs) and their characterization as pattern-recognition receptors in mammals. The insect and mammalian signaling pathways are also highly conserved, with the drosophila Toll pathway sharing many similarities with adaptor protein MyD88– dependent interleukin 1 receptor–TLR pathway, whereas the Imd pathway is similar to the MyD88-independent (adaptor protein TRIF–dependent) branch3. More recently, drosophila immunologists have been exploring other aspects of innate immunity, taking advantage of the genetic tools available in flies: ‘forward genetic screening’ in adult flies, genome-wide RNA-mediated interference (RNAi) screening of drosophila cell lines, and examination of immune processes in pre-existing mutant strains. In this review, we will highlight important discoveries, and their relation to mammalian immunity, from genetic screens designed to examine phagocytosis and host-virus interactions. 1Department The CD36 homolog peste Mycobacterium fortuitum, an intracellular opportunistic pathogen of humans and insects with many similarities to Mycobacterium tuberculosis, readily infects S2 cells6. A genome-wide RNAi screen has identified a requirement for peste, CD36 homolog, in the phagocytosis of M. fortuitum and another intracellular bacterial pathogen, Listeria monocytogenes, but not for the engulfment of Escherichia coli or Staphylococcus aureus6,7. of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. 2Division of Infectious Disease, Department of Medicine, University of Massachusetts School of Medicine, Worcester, Massachusetts 01605, USA. Correspondence should be addressed to N.S. ([email protected]). Received 23 May; accepted 19 July; published online 21 August 2006; doi:10.1038/ni1388 NATURE IMMUNOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 2006 Phagocytosis Phagocytosis is a complex process that requires pathogen recognition followed by engulfment. Mammals have many (partly) redundant mechanisms for pathogen recognition. Phagocytosis by drosophila blood cells, or hemocytes, shares many similarities with the process in mammals, but is in a simpler system, with less genetic redundancy and readily amenable genetic tools4. For example, several groups have taken advantage of the fact that drosophila cell lines, such as S2 cells, are highly phagocytic and are easily used for whole-genome RNAi screens. In many screens with a wide variety of microbes, one common denominator has been the identification of actin and actin-related proteins, such as the Arp2/3 complex, as critical participants in phagocytosis, consistent with mammalian studies5–8. Also, various components of the endocytic pathway, including Rab5, have been identified in several screens, suggesting it has a more general function in phagocytosis. 911 REVIEW Toll Gram-positive bacteria Imd Fungi Gram-negative bacteria LYS-PGN DAP-PGN PGRP-SA PGRP-SD SPE Monomeric Polymeric port the engulfment of M. fortuitum when expressed in HEK293 cells. Mammalian CD36 is linked to the phagocytosis of apoptotic cells and the uptake of modified low-density lipoprotein and is necessary for the activation of TLR2 by a subset of ligands10,11. It will be useful to learn if other drosophila CD36 family members are involved in pathogen recognition, via a CD36 ‘code’ perhaps, and if these receptors are involved in the presentation of microbe-derived compounds to innate immune signaling receptors in the fly. L L C C PGRP-LCs x a Toll L L C C x x dMyD88 tube pelle dFADD imd bendless dUbc13 Dredd dUev1A dTAB2 dTAK1 P P P cactus P P P cactus Ub, proteas om Dif Dif γ kenny β ird5 γ e © 2006 Nature Publishing Group http://www.nature.com/natureimmunology Pro-spz spz DmIKK β P RelC C Relish Dif Dif drosomycin and other antimicrobials N RelN Cleavage diptericin and other antimicrobials Figure 1 The drosophila Toll and Imd pathways. The Toll pathway (left) responds to Gram-positive bacterial and fungal pathogens and culminates in activation of the NF-κB factors Dif and dorsal, whereas the Imd pathway (right) responds mainly to Gram-negative bacterial infection and leads to the activation of another NF-κB homolog, Relish. Both pathways rely on PGRPs to recognize bacterial pathogens. The Toll pathway is activated by lysine-type peptidoglycan (LYS-PGN), common to many Gram-positive bacteria, through the soluble receptors PGRP-SA and PGRP-SD, which in turn stimulate the proteolytic processing of spätzle (spz), the Toll ligand. In contrast, the Imd pathway is activated by diaminopimelic acid–containing peptidoglycan (DAPPGN), found on Gram-negative bacteria, that is recognized by the cell surface receptor PGRP-LC. Once these receptors are stimulated, signaling pathways very similar to mammalian NF-κB signaling pathways are activated. The Toll pathway is homologous to the MyD88-dependent pathway, which functions ‘downstream’ of most TLRs, whereas the Imd pathway has more homology with the MyD88-independent, TRIF-dependent pathway ‘downstream’ of TLR3 and TLR4. Activation of these drosophila immune response pathways leads to the induction of distinct but overlapping sets of target genes, including those encoding a battery of antimicrobial peptides. SPE, spätzle processing enzyme; Pro-spz, pro-spätzle; LCx and LCa, isoforms of PGRP-LC; dMyD88, drosophila MyD88; Ub, ubiquitin; dFADD, drosophila Fas-associated death domain; Dredd, Death-related ced-3–Nedd2-like protein; dUbc13, drosophila ubiquitinconjugating enzyme 13; dUev1A, drosophila ubiquitin-conjugating enzyme variant 1A; dTAB2, drosophila TAK1-associated protein 2; dTAK1, drosophila transforming growth factor-β-activated kinase 1; DmIKK, drosophila IκB kinase; ird5, immune response–deficient 5; RelC, Relish C terminus; RelN, Relish N terminus. In contrast, expression of peste in mammalian cells (HEK293) is sufficient for the uptake of M. fortuitum as well as E. coli. All of this work has been done in cell culture, and the in vivo function of peste in flies remains to be examined. Notably, peste is one of approximately thirteen CD36 family members expressed in drosophila (also called ‘scavenger receptor, class B’ (SR-B) proteins). Another family member, croquemort, is required for the phagocytosis of apoptotic cell ‘corpses’9. Of the four related molecules in mammals, SR-BI and SR-BII but not CD36 sup- 912 Eater: a critical phagocytic receptor It has been shown that another drosophila scavenger receptor, the class C homolog dSR-CI, contributes to the recognition and phagocytosis of both Gram-negative and Gram-positive bacteria in S2 cells. However, this receptor accounts for only a fraction of the phagocytic activity of these cells12. Expression profiling has been used to identify other phagocytic receptors, comparing phagocytic S2 cells to cells ‘reprogrammed’ into a nonphagocytic state13. That strategy identified eater, which encodes a single-pass transmembrane receptor containing many extracellular ‘atypical’ epidermal growth factor–like repeats. ‘Knockdown’ of eater expression in S2 cells causes a reduction of more than 50% in phagocytosis of E. coli and S. aureus. Likewise, hemocytes (ex vivo) from eater mutant larvae show less engulfment of S. aureus and, notably, eater mutant flies show much less phagocytosis of S. aureus. Consistent with those findings, eater mutants are also hypersusceptible to infection with Serratia marcescens, a fly pathogen that is (partially) controlled by phagocytosis. Notably, it has been shown that the defect in the eater mutant flies is due mainly to a defect in hemocyte function. An N-terminal, 199–amino acid fragment of eater is able to bind several types of bacteria and has an activity profile similar to that of typical scavenger receptors in that it is inhibited by oxidized and acetylated low-density lipoprotein but not by unmodified low-density lipoprotein. Both eater and dSR-CI are also required for the efficient uptake of double-stranded RNA (dsRNA) by drosophila S2 cells14, a property of many drosophila cell lines that makes them particularly amenable to RNAi methodologies. Dscam: a diversity-generating system? Another receptor found to bind microbial pathogens and linked to phagocytosis in drosophila is Dscam (Down syndrome cell adhesion molecule). Dscam is single-pass transmembrane receptor with well established functions in axonal pathfinding15 that is also expressed in phagocytic hemocytes. Drosophila Dscam includes four alternatively spliced exons, each with multiple (2–33) mutually exclusive alternative exons, which potentially give rise to 38,016 distinct protein products (Fig. 2). Approximately 18,000 of these isoforms are probably expressed in hemocytes16. Most of the diversity in these Dscam isoforms is in their extracellular domain, which includes ten immunoglobulin-like domains and six fibronectin type III domains. In fact, this diversity occurs in three of the immunoglobulin-like domains, reminiscent of the diversity of human antigen receptors. For example, antibodies contain twelve immunoglobulin domains, of which four are highly diversified. Larval hemocytes, assayed ex vivo, and S2 cells require Dscam for efficient phagocytosis of heatkilled E. coli. Furthermore, some but not all isoforms of recombinant Dscam bind to E. coli. Based on those results, it has been suggested that the approximately 18,000 different Dscam isoforms expressed by drosophila hemocytes are a set of diverse immune receptors capable of recognizing a wide spectrum of microbial pathogens. In addition to its membrane-bound forms, Dscam is also found in insect sera, where it may function to bind microbes and thereby stimulate their phagocytosis (opsonization). Although those are intriguing findings, VOLUME 7 NUMBER 9 SEPTEMBER 2006 NATURE IMMUNOLOGY REVIEW a Exon 4 Exon 6 Exon 9 Exon 17 © 2006 Nature Publishing Group http://www.nature.com/natureimmunology b Figure 2 Dscam encodes a diverse set of immune receptors. Dscam contains multiple mutually exclusive alternative versions of exons 4, 6, 9 and 17 (a), creating 38,016 potential receptor isoforms. Much of that diversity occurs in second (orange), third (pink) and seventh (green) immunoglobulin-like domains (small circles; b). Longer ovals (b) represent the six fibronectin type III domains. These alternate Dscam isoforms may represent a diverse family of phagocytic receptors able to recognize a variety of microbial ligands. many important questions remain. Is Dscam important for phagocytosis in the intact organism? Are alternative Dscam isoforms capable of binding different microbes? Is Dscam required for phagocytosis of live bacteria or of other microbes beyond E. coli? And what cell surface receptors are required for the proposed opsonizing activity of soluble Dscam? Further supporting the hypothesis that Dscam is a hypervariable insect immune receptor, the anopheles mosquito Dscam undergoes infection-specific induction of alternative splicing that can produce about 31,000 possible splice isoforms. Moreover, depletion of a microbe-specific, induced isoform increases susceptibility to the same microbe but not to an unrelated bacteria17. Teps: complement like proteins Another family of insect proteins thought to function as opsonins are the Teps (thioester-containing proteins). The drosophila genome includes six potential Teps, all of which include several domains common to the α2-macroglobulin and complement (C3) family. These proteins form covalent adducts with their targets, such as microbial cell surfaces for complement C3, through their thioester motif. In mammals, complement attachment to microbial surfaces ‘marks’ these microbes for opsonization. Drosophila TepI–TepIV contain functional thioester motifs, but none has yet been shown to form thioester adducts. However, several insect Teps have been linked to phagocytosis. For example, an S2 cell RNAi screen for genes involved in the phagocytosis of the yeast Candida albicans has shown that TepVI (also called Mcr, for ‘macroglobulin complement-related’) is required for the engulfment of C. albicans but not bacterial microbes (such as E. coli or S. aureus) or latex beads8. Although TepVI lacks an active thioester motif and presumably does not form covalent adducts, it binds C. albicans but not the nonpathogenic (and nonphagocytosed) yeast Saccharomyces cerevisiae. The function of the other four Teps in phagocytosis has also been assessed in cell culture. RNAi targeting of TepII causes a small but significant decrease in the engulfment of E. coli but not C. albicans or S. aureus, whereas targeting of TepIII specifically inhibits the uptake of S. aureus. All of this work has been done in S2 cells, and in vivo confirmation of the functions of the Teps in phagocytosis awaits further study. It will also be useful to learn if the presumed catalytic Teps (TepI–TepIV) form covalent adducts with the microbes whose phagocytosis they facilitate and to learn if drosophila phagocytes have cell surface receptors that specifically recognize these Tep-microbe adducts. Perhaps some of the receptors already linked to phagocytosis, outlined above, function as Tep receptors. NATURE IMMUNOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 2006 Notably, the mosquito Anopheles gambiae encodes approximately 15 Teps, and aTep1 covalently crosslinks E. coli to facilitate phagocytosis in mosquito cell lines18. The protein aTep1 is also found associated with the surface of the ookinete stage of the murine malarial parasite Plasmodium berghei. RNAi ‘knockdown’ of aTep1 in mosquitoes compromises clearance of this parasitic infection, although the antimalarial activity of aTep1 is thought not to involve phagocytosis19. Similar to mammalian complement, the insect Teps may therefore have evolved multiple mechanisms to combat infectious microbes. One issue that has not been examined in drosophila is whether other components of a complement-like system are involved in immunity. The classical pathway, which relies mainly on antibody recognition, is unlikely to be conserved, but are there other soluble pathogenbinding receptors involved in fixing the Teps to microbial surfaces? For example, could soluble Dscam be involved in fixing Teps? Are other proteases involved in activating the Teps? Drosophila encode many proteases with similarity to the MASPs, which are proteases that activate complement in the lectin pathway. Those questions and the in vivo analysis of Teps in phagocytosis await further study. We have summarized the present understanding of the function of Teps in phagocytosis as well as the other participants in the phagocytic processes discussed here (Fig. 3). A ‘picky’ participant Although the CD36 homologs, eater, and the Teps were first linked to phagocytosis in cell-based RNAi screens, the powerful ‘forward genetics’ available in drosophila have also been used to screen for mutant flies with defects in phagocytosis of some bacteria20. Because in adult flies hemocytes are mostly sessile and coalesce in the dorsal vessel, phagocytosed fluorescence-labeled microbes can be viewed directly through the cuticle of live animals. One mutant, picky eater (picky), was identified because it fails to engulf S. aureus particles but is able to phagocytose E. coli. Likewise, these mutants are hypersusceptible to infection with S. aureus but not E. coli. The mutation in the gene encoding picky has been mapped to a locus containing genes encoding three peptidoglycan-recognition receptors (PGRPs): PGRP-SC1a, PGRP-SC1b and PGRP-SC2. These PGRP genes encode peptidoglycan-digesting enzymes, in contrast to several other PGRP genes that encode noncatalytic peptidoglycan receptors necessary for bacterial recognition ‘upstream’ in the Toll and Imd pathways. Expression analysis has suggested that picky affects the expression of PGRP-SC1a and that a transgene expressing PGRP-SC1a ‘rescues’ the defects in the picky mutant. Notably, a mutant form of PGRP-SC1a 913 REVIEW helicase proteins, but may use other receptors to recognize viruses. Also, the PKR and RNaseL pathway is not conserved, but the RNAi pathway is highly conserved26. ? Soluble Dscam receptor Soluble Dscam Tep receptor E. coli PGRP-SC1a Tep II S. aureus Tep III Eater C.albicans Dscam Tep VI © 2006 Nature Publishing Group http://www.nature.com/natureimmunology Tep receptor eater Actin Tep receptor Arp2/3 Mycobacterium and listeria peste (CD36-like) Rab5 Actin Figure 3 Phagocytosis in drosophila. As in mammals, phagocytosis in drosophila uses a variety of recognition mechanisms for efficient engulfment of a broad range of microbes. The receptor ‘eater’ is important in the engulfment of both of Gram-negative and Gram-positive bacteria. Complement-like proteins, the Teps, are important for the phagocytosis of various of microbes, with different Teps (TepII, TepIII and TepVI) linked to the binding of different microbes. Dscam has been linked to the recognition and phagocytosis of E. coli and perhaps other microbes through its diverse isoforms. Dscam is both a cell surface receptor and a soluble receptor. Hypothetical receptors for soluble Dscam and for Tep-microbe adducts are in green. The CD36 homolog peste is specifically required for the phagocytosis of listeria as well as mycobacterium. Peste is sufficient but not necessary for the phagocytosis of E. coli. As in mammals, phagocytosis generally requires the actin network and the Arp2/3 complex. In addition, Rab5-dependent endocytic machinery has been linked to the phagocytosis of listeria and mycobacterium and is probably generally important for the engulfment of all microbes. In addition to these conventional receptor-microbe interactions, the generation of peptidoglycan fragments by the enzyme PGRP-SC1a is linked to efficient phagocytosis of S. aureus in flies (yellow). The mechanism whereby these peptidoglycan fragments stimulate phagocytosis is unclear. lacking catalytic activity is only mildly deficient in phagocytosing heatkilled S. aureus but completely fails to engulf live S. aureus. Moreover, injection of peptidoglycan into those flies restores their ability to phagocytose live S. aureus. That suggests that PGRP-SC1a functions at least in part by producing peptidoglycan fragments from S. aureus. Those peptidoglycan fragments may then function as signaling molecules to stimulate hemocyte function20. Antiviral immunity Innate immunity to all pathogens, including viruses, is the first and most ancient line of defense21. Mammals have developed a variety of mechanisms to detect and respond to viral pathogens22. For example, various structural components or viral nucleic acids are recognized by innate antiviral pattern-recognition receptors. For example, some TLRs recognize viral pathogens and their products either at the cell surface or in lysosomal or endosomal vesicles22. In addition, three well known systems detect the presence of intracellular dsRNA, which is produced as an intermediate in the replication cycle of single-stranded RNA viruses or as a byproduct of symmetrical transcription from DNA viruses23–25. Cytoplasmic pattern-recognition receptors (CARDhelicase proteins) activate many signaling pathways in common with the TLRs, inducing type I interferons through the NF-κB and IRF transcription factors. The dsRNA-activated protein kinase PKR and RNaseL pathway and RNAi pathways directly degrade viral RNA using host-encoded machinery. Drosophila do not seem to encode CARD- 914 RNAi pathway Although RNAi has been used as a methodology for depleting specific mRNA transcripts, leading to its use in genome-wide RNAi screening, RNAi probably first evolved as an innate immune mechanism. In that case, the RNAi pathway uses viral dsRNA to trigger host-mediated degradation of viral RNA. The dsRNA is processed by the endoribonuclease Dicer2 into small interfering RNA molecules that direct homologydriven RNA destruction through the Argonaute 2 (AGO2)–containing RNA-induced silencing complex27. In mammalian cells, induction of this pathway by the exogenous introduction of small interfering RNA directed against viral genomes can temporarily inhibit viral replication28,29. Moreover, this pathway has antiviral properties in plants, Caenorhabditis elegans and drosophila cells25,30. As further support for the idea of involvement of this system in antiviral immunity, many viruses, including influenza and flock house virus (FHV), encode accessory proteins (NS1 and B2, respectively) that inhibit RNAi in drosophila cells31. Moreover, FHV B2 is dispensable for infection only when components of the RNAi pathway are depleted in drosophila cells32. The essential antiviral function of RNAi in flies has now been demonstrated33–35. In transgenic drosophila expressing FHV RNA in embryos or adults, the virus-encoded RNAi inhibitor B2 is required for RNA replication, and loss of components of the cellular RNAi pathway allows for RNA replication of this viral RNA in the absence of B2 (refs. 33,34). Also, for adult flies, wild-type FHV is more pathogenic in Dicer-2 (Dcr-2) mutants than in wild-type flies, although RNA replication is not affected or is only slightly increased in this mutant33,34. However, neither of those reports determined if loss of Dcr-2 is sufficient to restore lethality to B2-deficient virus-infected flies, as would be predicted. In embryos, mutations in genes encoding components of the RNAi pathway do not fully ‘rescue’ the B2 mutant FHV defects, suggesting that RNAi inhibition is only one of the functions of B2 in viral replication34. The disconnection between RNA replication and survival in the Dcr-2 strain may relate to the observation that in embryos, B2 has functions beyond RNAi inhibition. Unexpectedly, viral replication in embryos and larvae is not lethal, and only adult flies succumb to infection33,34. That result may be due to the presence of additional antiviral mechanisms during development or to differential sensitivity of adults to replicating virus. Because FHV is more pathogenic in flies lacking Dcr-2, even in the presence of B2 and without considerable changes in viral replication, the antiviral mechanism of the RNAi machinery remains unclear. A better understanding of the mechanism of virus-induced death might resolve some of those issues. Notably, three other RNA viruses, drosophila C virus (DCV), cricket paralysis virus (CrPV) and Sindbis virus, are also more pathogenic in Dcr-2 mutants33,34. Moreover, with these viruses, loss of RNAi causes a more noticeable increase in viral RNA replication. Those data emphasize the generality of RNAi as an antiviral response. Another notable aspect of the antiviral function of the RNAi pathway is that although Dcr-2 is required for protection against infection with FHV, DCV, CrPV and Sindbis, replication of drosophila X virus (DXV, a dsRNA virus) is insensitive to loss of this gene33–35. In contrast, DXV is more pathogenic in other mutants effecting RNAi. In particular, mutations affecting components ‘downstream’ of Dcr-2, including mutations in AGO2, r2d2 and piwi (which encodes an argonaute family member linked to RNAi-mediated silencing), cause greater sensitivity to DXV36. Also, expression of piwi is required for the repression of retroelements, including the VOLUME 7 NUMBER 9 SEPTEMBER 2006 NATURE IMMUNOLOGY © 2006 Nature Publishing Group http://www.nature.com/natureimmunology REVIEW endogenous retrovirus gypsy, in the germline of flies, suggesting a general antiviral function for this factor37,38. The repression of endogenous elements by RNAi has also been noted in diverse species, including C. elegans and plants, demonstrating the ancient and conserved nature of this repression39. The antiviral function of RNAi is further emphasized by the finding that CrPV (a close relative of DCV) encodes a product that can suppress RNAi in tissue culture34. Whereas FHV B2 is thought to repress the RNAi pathway by sequestering dsRNA, the mechanism of action of the CrPV product has not yet been studied. Many other mammalian, insect and plant viral inhibitors also inhibit RNAi in drosophila cells in the absence of viral infection, although which step in the pathway each targets is unknown31,40. Those data have established RNAi as an antiviral mechanism in animals. However, it is unclear whether the only function of these viral factors is to block RNAi or if they have additional functions during viral infection, as suggested for FHV B2. Bioinformatic analysis of drosophilids has demonstrated that Dcr-2, AGO2 and r2d2 are among the most rapidly evolving genes in their genomes41. Viruses are an important class of pathogens for drosophila (40% of wild drosophila are infected with viruses42), and this interaction drives cycles of adaptation between the virus and host43. Moreover, it is apparent that the RNAi machinery also controls the activation and movement of endogenous retrotransposons, thereby controlling the mutation frequency in flies. The rapid evolution of these genes, with strong directional selection, demonstrates the selective pressure to modulate this pathway, further supporting the proposition that RNAi is a principal part of the continual ‘arms race’ between virus and host. Whether the driving force for these population-wide changes is external pressures, such as horizontally transmitted viruses, or internal forces, such as vertically transmitted viruses or transposons, is yet to be determined. Global transcriptional profiling has been used to characterize the response to infection by DCV46. Two different infection routes, oral inoculation and intrathoracic injection, have been used in these studies. Both routes have been investigated because DCV is a natural pathogen of flies, but the natural route of infection is unknown. After oral administration of DCV to flies, there is little mortality and only very few genes are induced47. In contrast, intrathoracic injection leads to mortality and a robust transcriptional response distinct from the imd- and Toll-dependent responses noted with bacterial or fungal infections. That further emphasizes the finding that the canonical NF-κB signaling pathways are dispensable for antiviral responses to DCV48. In the group of DCV-inducible genes, a subset requires the Janus kinase–signal transducer and activator of transcription (JakSTAT) signaling pathway for expression. That pathway was first characterized in mammals for its function in interferon signaling, a well known antiviral cascade that induces a plethora of antiviral mediators49. Consistent with involvement of that pathway in fly antiviral immunity, susceptibility to infection is modestly increased after loss of that pathway in adults and viral loads are increased. Therefore, it seems likely that some of those virally induced Jak-STAT responsive genes have antiviral properties. However, the best characterized of the DCV-induced, Jak-STAT–dependent genes, vir-1, is dispensable for resistance to DCV infection, and overproduction has no protective effect. Also, activation of the pathway taking advantage of a hyperactive allele of the Jak homolog hopscotch is insufficient to induce the Jak-STAT–dependent DCV-inducible gene program, suggesting that Viral products Cellular debris Antiviral factors? ? Toll ? ? Toll Dom Entry Antiviral signaling responses in drosophila In addition to RNAi, mammals use innate immune receptors (TLRs and CARD-helicase receptors) to recognize viral pathogens and activate signaling cascades that culminate in the production of antiviral products, such as interferons. Drosophila seem to have analogous innate antiviral mechanisms. Studies using DXV have found that both the Toll and Imd pathways are activated during infection44. However, only the Toll pathway seems to provide a protective antiviral response, as Dif mutants, which lack the NF-κB component of this pathway, are more susceptible to viral challenge and allow increased viral replication. Counterintuitively, flies carrying an activated allele of Toll are also hypersusceptible to infection, yet show slightly lower viral replication. Also, the known components that connect the Toll receptor to the transcription factor are dispensable, suggesting an alternate signaling pathway ‘downstream’ of the Toll receptor that converges on the NF-κB factor Dif. Whether those factors are required in the infected tissues and how they affect viral replication have yet to be elucidated. Further support for the idea of NF-κB as antiviral factor comes from the fact that many viruses, including insect-specific polydnaviruses, inhibit this signaling pathway by encoding inhibitors structurally homologous to the IκB proteins known to sequester NF-κB in the cytoplasm. These viral IκBs probably function in an analogous way in infected tissues and repress the ability of the host to mount an NF-κB-dependent antiviral response45. In contrast to the robust activation of those NF-κB pathways by DXV, infection by DCV does not activate those pathways, nor are they required for antiviral activity46. The pathogenesis and tropisms of these viruses differ, as do their replication cycles, suggesting that specific aspects of their life cycle may be important in defining how the host responds to a given invader. NATURE IMMUNOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 2006 Jak Uncoating ? ? Translation IκB -lik STAT e in hib ito r RNA replication B2 CrPV Nterm Dif (NFκB) Dif (NFκB) Antiviral factors Dcr-2 r2d2 AGO2 RISC Antiviral factors? Antiviral factors? Antiviral factors?, vir-1 Infected cell Uninfected cell Viral replication Virus-encoded inhibitors Innate antiviral immunity Figure 4 Virus-host interactions in drosophila. Viral infection requires cellular factors for each step in the life cycle, including entry, uncoating, translation and RNA replication (blue). Recognition of intracellular dsRNA produced during RNA replication by the host-encoded RNAi machinery in the infected cell leads to the targeted destruction of viral RNA (green). To combat that host-encoded antiviral process, viruses encode factors that inhibit the RNAi antiviral activity (red). Infection also leads to the activation of signaling pathways, including the Toll pathway for DXV infection and the Jak-STAT pathway for DCV and FHV infection (green). Activation of the JakSTAT pathway occurs in uninfected cells in response to unknown mediators and leads to the activation of many factors, some of which probably have antiviral properties. It is unknown where the NF-κB pathway is activated during DXV infection; however, polydnaviruses encode inhibitors of this pathway, which suggests that this pathway is activated in the infected cell (red). Nterm, N terminus; Dom, Domeless. 915 © 2006 Nature Publishing Group http://www.nature.com/natureimmunology REVIEW Jak-STAT must work in conjunction with other pathways to induce the antiviral response. Notably, those Jak-STAT–responsive genes are not induced in well established immune tissues or in virus-infected tissues but instead in uninfected, nonimmune tissues48. Because that pathway has been shown to respond to various stressors in drosophila, that might suggest that those nonimmune cells are responding either to toxic byproducts of the infection (such as products of lysed cells) or to cytokines released by the infected cells50,51. These data collectively demonstrate the existence of a previously unknown antiviral immune response in insects that is partly dependent on the Jak-STAT pathway. However, they raise many more questions than they answer. What are the mechanisms of viral recognition that lead to DCV-induced gene activation, including Jak-STAT activation? What is the nature of the Jak-STAT– independent pathway? What are the effector mechanisms activated by these pathways that combat viral pathogens? And what factors or pathways act in conjunction with the Jak-STAT pathway to activate the Jak-STAT–dependent genes? RNAi screen has identified conserved negative and positive regulators of the NFAT pathway, including the long-sought calcium release– activated calcium channel57,58. The advent of genome-wide RNAi screening has only added to the power of drosophila as a model system for innate immunity. This new technology, coupled with traditional genetic approaches and the blossoming interest in several new areas of host-pathogen interactions, will undoubtedly lead to many important and exciting discoveries. Virus-host interactions Another important facet of virus-host biology is the cellular factors ‘hijacked’ by viruses to complete their life cycle; drosophila has proven very powerful for dissecting these interactions. Screening mutant flies for susceptibility to infection has shown that viral entry of DCV requires clathrin-mediated endocytosis. In fact, this process is limiting for infection: heterozygous mutant flies that are otherwise healthy, viable and fertile are attenuated in their susceptibility to DCV52. A cell-based genome-wide RNAi screen has identified about 100 cellular factors required for efficient DCV replication, including many genes involved in translation. Moreover, it has been found that translation is limiting for viral replication both in cells and animals53. DCV and mammalian picornaviruses (such as poliovirus) are translated by an internal ribosome entry site–dependent, cap-independent mechanism that is dependent on abundant ribosomal machinery for function, as poliovirus replication is also more sensitive to depletion of host ribosomal machinery than to cellular messages. Thus, the host may be under strong selective pressure to downregulate those processes (endocytosis and translation), which are essential for viral pathogenesis but are limiting, as a means of viral evasion. In humans, there is a similar phenomenon in the increasing frequency of mutated CCR5 alleles among people who despite high exposure to human immunodeficiency virus (HIV) remain uninfected and in people who are slow progressors, due to the fact that CCR5 is needed by most HIV strains to enter cells; that finding has spurred the development of a new class of anti-HIV therapeutics54–56. Those data demonstrate that the cellular factors used by viruses in insect cells are often used by human viral pathogens and that the further study of these interactions in insect systems, especially those identifying the limiting factors for viral replication, may identify new targets for antiviral drug development. We have summarized the present understanding of virus-host interactions in drosophila (Fig. 4). 1. Royet, J., Reichhart, J.M. & Hoffmann, J.A. Sensing and signaling during infection in Drosophila. Curr. Opin. Immunol. 17, 11–17 (2005). 2. Kaneko, T. & Silverman, N. 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NFAT, a critical target of T cell receptor signaling, is activated by calcium entry. Although drosophila do not encode a calcium-activated NFAT homolog, the pathways that regulate NFAT shuttling between the cytoplasm and nucleus are highly conserved; when expressed in S2 cells, mammalian NFAT translocates to the nucleus in a calcium-dependent way. With that assay, a genome-wide 916 ACKNOWLEDGMENTS We thank R. Doms, M. Tudor, E. Lien and members of the Silverman lab for comments and insights; and B. Graveley for the Dscam figure (Fig. 3). Supported by the National Institutes of Health (AI060025 to N.S.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. 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