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Comparative Study
, 102 (20), 7257-62

The Toll Pathway Is Important for an Antiviral Response in Drosophila

Affiliations
Comparative Study

The Toll Pathway Is Important for an Antiviral Response in Drosophila

Robert A Zambon et al. Proc Natl Acad Sci U S A.

Abstract

The innate immune response of Drosophila melanogaster is governed by a complex set of signaling pathways that trigger antimicrobial peptide (AMP) production, phagocytosis, melanization, and encapsulation. Although immune responses against both bacteria and fungi have been demonstrated in Drosophila, identification of an antiviral response has yet to be found. To investigate what responses Drosophila mounts against a viral infection, we have developed an in vivo Drosophila X virus (DXV)-based screening system that identifies altered sensitivity to viral infection by using DXV's anoxia-induced death pathology. Using this system to screen flies with mutations in genes with known or suggested immune activity, we identified the Toll pathway as a vital part of the Drosophila antiviral response. Inactivation of this pathway instigated a rapid onset of anoxia induced death in infected flies and increases in viral titers compared to those in WT flies. Although constitutive activation of the pathway resulted in similar rapid onset of anoxia sensitivity, it also resulted in decreased viral titer. Additionally, AMP genes were induced in response to viral infection similar to levels observed during Escherichia coli infection. However, enhanced expression of single AMPs did not alter resistance to viral infection or viral titer levels, suggesting that the main antiviral response is cellular rather than humoral. Our results show that the Toll pathway is required for efficient inhibition of DXV replication in Drosophila. Additionally, our results demonstrate the validity of using a genetic approach to identify genes and pathways used in viral innate immune responses in Drosophila.

Figures

Fig. 1.
Fig. 1.
DXV pathology in Drosophila WT adults. (A) The anoxia sensitivity phenotype correlates with injection of DXV into WT flies, with a dramatic drop in survival at 6 dpi. More than 200 flies were used for each survival curve. Error bars show one standard deviation. (B) Viral titer, as measured by quantitative RT-PCR, shows a rapid increase in DXV. Each point represents a reaction from a pooled sample of 10 flies. Error bars show one standard deviation. (C) Immunostaining of DXV in sagittal sections of WT whole flies after injection with water or DXV. The head is oriented to the right. Immunostaining of H2O injection control shows no significant background. DXV infected samples show a steady spread of virus through entire WT organism over time. (D) DXV immunostaining and TUNEL staining of sagittal sections of a 7 dpi Drosophila showing cell death in the tissue where DXV antigens are detected. No TUNEL staining was observed in H2O-injected flies. Sections are ≈25 μm apart in the same organism.
Fig. 2.
Fig. 2.
AMP expression levels in DXV-infected flies measured by quantitative RT-PCR. (A) Two hours after DXV injection. (B) Twenty-four hours after DXV injection. No significant difference between the DXV- and E. coli-infected groups exists, with even the greatest difference being under a 1-fold alteration. Data are representative of three experiments.
Fig. 3.
Fig. 3.
Increased susceptibility of Toll and Dif mutant fly lines. Dif1 lacks the ability to activate the Toll pathway. Tl10b is a constitutively active mutant of the Toll pathway. relE20 is a mutant in the IMD pathway. All measurements normalized to H2O-injected samples of the same mutant Drosophila line. Only Dif1 and Tl10b lines have significant alteration in survival, defined as two (or more) time points being outside of one standard deviation of the screened lines average survival (gray region). relE20 displays average survival. The 7-day time point increase in relE20 survival is due to base line effects from the wounding controls.
Fig. 4.
Fig. 4.
Model for Drosophila antiviral response. 1, Virus enters system and infects the cell. 2, Virus replication. 3, Lysis of infected cell, releasing internal cellular compounds and virus. 4, Released materials activate the IMD and Toll humoral immune pathways and cause local activation of the cellular response. 5, Global activation of hemocytes via Toll pathway signaling. 6, Activated hemocytes signal to the fat body enhancing Toll and IMD pathways activation. 7, Hemocytes recognize aberrant infected cells and engulf and eliminate these cells. Activated hemocytes then enforce stringent recognition of aberrant tissue and destroy infected cells more efficiently. Green arrows indicate signaling to activate the humoral response. Blue arrows indicate signaling to activate the cellular response.

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