Download Host-pathogen interactions in drosophila: new tricks from an old friend

© 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.
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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
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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
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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-
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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
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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.
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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
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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.
Published online at http://www.nature.com/natureimmunology/
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