Inducible response to virus infection

A hallmark of the immune antiviral response in mammals is the production of interferons, which are induced upon recognition of viral pathogens by innate immunity receptors (Toll-like receptors (TLRs) and RIG-like receptors). Interferons then trigger the production of antiviral molecules and establishment of an antiviral state in the infected cells (Beutler et al, 2007). There is now also evidence for an inducible innate antiviral defence system in Drosophila. Imd and Toll pathways One major characteristic of the response to bacterial or fungal infections is the inducible secretion into the haemolymph of a cocktail of antimicrobial peptides (Lemaitre and Hoffmann, 2007). In a first step to characterize the response of Drosophila to virus infection, an attempt was made to identify molecules induced by DCV infection that could serve as markers of the antiviral response. Proteomic analysis of the haemolymph of DCV-infected flies revealed that antimicrobial pep-tides regulated by the Toll or Imd pathways are not induced upon infection by this virus (Sabatier et al., 2003). An independent proteomic analysis more recently identified some 150 proteins upreg-ulated in FHV-infected Drosophila tissue-culture cells, but, again, the known Drosophila antimicrobial peptides were not induced (Go et al., 2006). Genome-wide microarray analysis of the tran-scriptome of flies infected with DCV, either by injection or by ingestion, suggest that some genes encoding antimicrobial peptides regulated by the Toll or the Imd pathway are induced (Roxström-Lindquist et al., 2004; Dostert et al., 2005). However, at least in the case of the injection model, quantitative analysis by RNA blot hybridization or quantitative real-time PCR showed that these genes are only weakly upregulated compared to bacterial or fungal challenges (Dostert et al., 2005). Thus, neither DCV nor FHV seem to activate the Toll or the Imd pathways.

Different results were obtained with DXV, which appears to induce several genes encoding antimicrobial peptides to the same levels as infection with the Gram-negative bacterium Escherichia coli. However, genetic analysis revealed that flies mutant for the NF-KB-related transcription factor Relish are not more susceptible to DXV infection than wild-type flies, ruling out a role for the Imd pathway in the resistance to this virus. By contrast, flies mutant for Dif, the NF-KB-like transcription factor regulated by the Toll pathway, are more susceptible to DXV infection than wild-type flies. Surprisingly, loss-of-function mutants for other components of the Toll pathway (Toll, Spätzle, Tube, and Pelle) succumb to DXV infection at a similar rate as wildtype controls (Zambon et al., 2005), suggesting that Dif mediates resistance to DXV infection independently of the classical Toll pathway. Of note, the gene ref(2)P, which mediates refractoriness to SIGMAV infection (see above), has been proposed to encode a component of the Toll pathway, suggesting a connection between SIGMAV infection and the activity of Dif and Dorsal (Avila et al., 2002). However, flies containing a permissive allele of ref(2)P are more susceptible to SIGMAV infection than flies that are deficient for the gene, indicating that SIGMAV uses the permissive allele to infect flies. This recent finding implies that the control of SIGMAV by ref(2) P may not result from a host defence mechanism (Carre-Mlouka et al., 2007).

SIGMAV was also shown to induce expression of antimicrobial peptides, at least at the mRNA level. Of note, SIGMAV infection leads to upregulation of the genes encoding the antibacterial peptides dip-tericin and drosocin, regulated by the Imd pathway, but not the antifungal peptide drosomycin, suggesting that the Imd pathway, rather than the Toll pathway, is involved (Tsai et al, 2008). It should be noted, however, that induction of antimicrobial peptides was not shown to confer protection against SIGMAV infection. Finally, a last indication for the possible participation of NF-KB pathways in the control of viral infections in Drosophila comes from the fact that some insect DNA viruses, such as the insect-specific polydnaviruses, inhibit this signalling pathway by encoding inhibitors structurally homologous to the inhibitory kB (IkB) proteins known to inhibit Dif and Relish expression in the cytoplasm (e.g. vankyrins). These viral proteins probably function in a way analogous to IkB in infected tissues and compromise the insect immune response by repressing NF-KB-like transcription factors (Thoetkiattikul et al., 2005; Kroemer and Webb, 2006).

In summary, there are at present no clear experimental data connecting the Toll and Imd NF-kB pathways to antiviral immunity. One should, however, remain open-minded until a larger number of viruses have been tested, and additional features other than antimicrobial peptides have been examined. The Imd pathway, for example, regulates apoptosis, a well-known antiviral response, and can be suppressed by expression in transgenic Drosophila of the baculovirus anti-apoptotic protein p35 (Georgel et al., 2001). Clearly, the involvement of the Toll and Imd pathways in the resistance to viral infections deserves further investigation. vir-1 and the JAK/STAT pathway Genome-wide microarray analysis of the transcrip-tome of flies 24 or 48 h after injection of DCV identified some 140 genes induced by a factor of at least two. Only one-third of these genes are also upregu-lated following bacterial or fungal infections, confirming that pathways different from Toll and Imd are activated in response to DCV infection. Since the list of genes induced did not provide any hints pointing to the pathway activated, the regulation of the gene vir-1 (virus-induced RNA 1) was studied. This gene is not expressed in adult flies, and is strongly induced by DCV and FHV infection, but not by bacteria or fungi (Dostert et al., 2005; Hedges and Johnson, 2008). vir-1 is a previously unrecognized transcript of the gene CG31764, which is produced from an inducible promoter. Promoter truncation experiments in transgenic flies led to the demonstration that the virus-response element maps to a 190 bp fragment, which contains a consensus binding site for the transcription factor STAT92E. Introduction of point mutations in this STAT-binding site strongly reduces induction of the vir-1 promoter, and DCV infection triggers induction of STAT DNA-binding activity in fly nuclear extracts (Dostert et al, 2005; C. Dostert and J.L. Imler, unpublished results). Altogether, these data suggest that the Janus kinase/signal transduction and activators of transcription (JAK/STAT) pathway is involved in the induction of vir-1. In Drosophila, this pathway is composed of a single JAK kinase, encoded by the gene hopscotch, and a single STAT factor (known as STAT92E) encoded by the gene marelle. The kinase is regulated by the cytokine receptor Domeless, which bears some similarity with the gp130 subunit of the interleukin-6 receptor in mammals (Figure 4.4). The Drosophila JAK/ STAT pathway controls cell multiplication and differentiation in multiple tissues and developmental stages (Arbouzova and Zeidler, 2006). Most mutants of the pathway therefore exhibit developmental phenotypes, making genetic experiments in adult flies a difficult task. However, analysis of the flies carrying a viable combination of a null and a hypomorph allele of hopscotch (Agaisse et al., 2003) revealed that the JAK kinase is required for induction of vir-1. This result was confirmed in flies over-expressing either a dominant-negative version of the Domeless receptor, or the negative regulator of the pathway dPIAS (Dostert et al., 2005). These data are consistent with a model in which one cytokine of the Unpaired (Upd) family (Upd-1, -2, and -3) is induced by DCV infection, and triggers an antiviral state in cells through activation of the JAK/ STAT pathway.

Of note, several other genes induced by DCV were found to contain consensus binding sites for STAT92E in their proximal promoter, and to be dependent on Hopscotch for full induction in virus-infected flies. Importantly, this response is associated with protection against infection, as shown by the fact that hopscotch mutant flies contain higher viral load than wild-type controls, and succumb more rapidly. Thus, at least some of the genes induced by DCV participate in the control of the viral amplification, by mechanisms that remain to be identified. Some of these genes may encode antiviral molecules targeting the virions or interfering with one step of the viral replication cycle. These antiviral mechanisms are probably distinct from RNA interference, since the list of DCV-induced genes does not contain any genes of the RNAi pathway. Interestingly, there is some


Type I interferon IFNAR1 ^2^IFNAR2



Drosophila Upd?

Drosophila Upd?

I Virus-induced genes STAT92E consensus: TTTCNNNGAAA



I Virus-induced genes STAT92E consensus: TTTCNNNGAAA

Figure 4.4 The JAK/STAT signalling pathway and antiviral immunity in mammals and flies. In mice (and humans) stimulation of the interferon receptor (IFNAR) by type I interferon produced in virus-infected cells leads to activation of the JAKs TYK2 and JAK1. These tyrosine kinases then phosphorylate (P) the transcription factors STAT1 and STAT2, triggering SH2-domain-mediated dimerization and nuclear translocation. Once in the nucleus, STAT transcription factors induce expression of interferon-stimulated genes (ISGs), such as those encoding the Mx proteins. In Drosophila, the JAK encoded by the gene hopscotch activates the STAT transcription factor STAT92E and induces expression of several virus-induced genes, such as vir-1. The Domeless cytokine receptor is required for the induction of the pathway in response to viral infection, but the cytokine involved, which is most likely one of the three members of the Unpaired (Upd) family, has not been identified yet. The consensus recognition DNA sequences for STAT1 and STAT3 in mammals and STAT92E in Drosophila are indicated.

evidence that the inducible response contributes to the pathogenesis of DCV-infected flies, as hopscotch mutant flies succumb more rapidly to DCV infection than wild-type flies when they are challenged with a low dose of virus, but not when they are challenged with a high dose of virus (Dostert et al., 2005). One interpretation for this result is that hopscotch mutant flies, which make an attenuated inducible response, can cope with a higher viral load. A similar type of observation was made when the sensitivity of Dicer-2 mutant flies to FHV infection was studied: Dicer-2 mutant flies resist infection only poorly when infected with a high dose of FHV, even though they do not contain more viral RNA than wild-type flies (because of the expression of B2, which suppresses RNAi; see above). This strong susceptibility to viral infection correlates with an increased induction of vir-1 in Dicer-2 mutant flies (Galiana-Arnoux et al, 2006). Altogether, these data indicate that the inducible response to virus infection is more complex than the inducible response to bacteria or fungi, where detection of the infection primarily triggers the production of effector molecules. It appears that some of the virus-induced genes alter the physiology of the host, a situation reminiscent of inflammation in mammals, which is well known to be associated with adverse effects in severe cases of sepsis. The JAK/STAT pathway is necessary, but not sufficient for the inducible antiviral response Although playing an important role in the control of at least some viral infections, the inducible antiviral response is not limited to the JAK/STAT pathway. Indeed, genes from the Turandot (Tot) family, which are regulated by the JAK/STAT pathway, are not induced by DCV infection, at least in the first 2-3 days of infection. Tot genes are, however, strongly induced following FHV infection. The TotA gene was initially identified in a screen for genes differentially expressed in bacteria-infected flies (Ekengren et al., 2001). TotA belongs to a family of eight genes in D. melanogaster that are upregu-lated by bacterial challenge, but also by a number of other stresses, such as extreme heat shock (temperatures above 37°C), mechanical pressure, or ultraviolet irradiation in larvae. The induction by the Gram-negative bacterium E. coli was later shown to involve the JAK/STAT pathway, since (1) TotA and TotM induction is abolished in flies carrying loss-of-function mutations in the hopscotch gene and (2) Tot genes are constitutively expressed in flies carrying the constitutively active allele of hopscotch, hopTum-l (Agaisse et al., 2003). These data suggest that the JAK/STAT pathway is both necessary and sufficient for the regulation of Tot genes, implying that if the pathway is activated during DCV infection, Tot genes should be induced. Experimental data demonstrate that things are more complex, however, and the regulation of vir-1, and probably also that of Tot genes, cannot be narrowed down to a single pathway. The fact that vir-1 is not con-stitutively expressed in flies containing the hopTum-l allele clearly points out that activation of the JAK/ STAT pathway is not sufficient to trigger expression of this gene. In mammals, STAT transcription factors have long been known to function with cofactors. For example, the antiviral effects of type I interferon are not mediated by STAT-1 only, but by the association of STAT-1 with the IRF9 transcription factor. Both STAT-1 and IRF9 bind DNA, and the juxtaposition of STAT-1- and IRF9-binding sites defines the subset of STAT-1-regulated promoters that are induced by type I interferon. In Drosophila, the Bcl6-related factor encoded by the gene Ken & Barbie (Ken) provides an example of a transcription factor that selectively modulates the activity of STAT92E on some promoters in vivo (Arbouzova et al., 2006). Induction of Tot genes also requires the Relish transcription factor (Agaisse et al., 2003), and the MEKK-1 (mitogen-activated protein kin-ase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase kinase 1) pathway (Brun et al, 2006), providing a further possible explanation for the differential regulation of vir-1 and Tot genes.

The fact that some virus-response genes remain fully inducible in hopscotch mutant flies provides an independent line of evidence that other signalling pathways contribute to the inducible antiviral response. The gene Vago (CG2081), which remains inducible in hopscotch mutant flies, provides a good model to identify these alternative pathways (Dostert et al., 2005). Unlike vir-1, Vago is induced in DCV-infected cells of the fat body, suggesting that it may be induced directly upon sensing the presence of virus in the cells, rather than by a cyto-kine produced from virus-infected cells. Vago is induced following infection by DCV and SINV, but not by FHV. The protein B2 accounts for this difference, and acts as a suppressor of Vago induction (S. Deddouche and J.L. Imler, unpublished results). Because this viral protein is a dsRNA-binding protein, these findings strongly suggest that Drosophila cells, like their mammalian counterparts, sense dsRNA as a molecular pattern betraying the presence of virus in the cell. It will be particularly interesting to understand how the expression of Vago is regulated, and compare this signalling mechanism to the one leading to interferon production in mammalian cells.

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