A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response

Abstract

Alphaviruses are RNA viruses transmitted between vertebrate hosts by arthropod vectors, primarily mosquitoes. How arthropods counteract alphaviruses or viruses per se is not very well understood. Drosophila melanogaster is a powerful model system for studying innate immunity against bacterial and fungal infections. In this study we report the use of a novel system to analyze replication of Sindbis virus (type species of the alphavirus genus) RNA following expression of a Sindbis virus replicon RNA from the fly genome. We demonstrate deficits in the immune deficiency (Imd) pathway enhance viral replication while mutations in the Toll pathway fail to affect replication. Similar results were observed with intrathoracic injections of whole virus and confirmed in cultured mosquito cells. These findings show that the Imd pathway mediates an antiviral response to Sindbis virus replication. To our knowledge, this is the first demonstration of an antiviral role for the Imd pathway in insects.

Figure 1. UAS-GAL4 controlled launch of SIN genome replication in Drosophila.

(A) Schematic of the SINrep:GFP and SINΔrep:GFP constructs used to generate the transgenic flies. SINrep:GFP replicon RNA encodes the 5′cap, the nonstructural proteins the subgenomic promoter, GFP and 3′poly A tail. The SINΔrep:GFP RNA has a large deletion region encoding the nonstructural proteins. The hypothesized launch of alphavirus genome replication under the control of UAS/GAL4 (B) compared to a natural virus infection (C). Introduction of viral genomic plus sense RNA into cytoplasm differs for each system (steps1 and 2). In (B) the viral RNA is introduced after transcription by host RNA pol II where as in infection (C) the RNA is introduced by receptor mediated viral entry and endocytosis. The process of genome replication and subgenomic mRNA expression is the same for each system (steps 3 to 8). In cytoplasm, genomic RNA is translated into nsPs (Step 3). The nsPs copy genomic RNA into a minus-strand RNA (step 4 and 5). The viral replicase complex recognizes the minus-strand RNA and copies it into genomic (step 6) and subgenomic RNA (step 7). The subgenomic RNA encodes for the GFP (B) or structural proteins (C) (step 8). The structural proteins assemble with a copy of the genome into infectious virus particles (C, step 9).

Figure 2. Alphavirus genome replication in Drosophila.

Bright field and fluorescence images showing pattern of GFP expression in (A) control GFP Act5C-GAL4,UAS-GFP flies, (B) SIN replicon Act5C-GAL4,UAS-SIN:GFP flies and (C) mutant SIN replicon Act5C-GAL4/UAS-SINΔrep:GFP flies during development. (D) Real-time qRT-PCR analysis of GFP mRNA in SIN replicon flies without GAL4 driver (UAS- SINrep:GFP) and with GAL4 driver (Act5C-GAL4,UAS-SIN:GFP) and in (E) mutant SIN replicon flies without (UAS-SINΔrep:GFP) and with GAL4 driver (Act5C-GAL4,UAS-SINΔrep:GFP). The results were normalized to actin, and the value obtained for control flies was considered as one fold. Data shown is representative of three independent experiments. Error bars represent SD.

Figure 3. Dicer-2 mutant flies have enhanced alphaviral replication.

Bright field and fluorescence images of (A) wild type SIN replicon flies and SIN replicon flies heterozygous for dcr-2 mutation (dcr-2^L811FXS) and (B) control GFP and GFP flies heterozygous for dcr-2 mutation. (C and D) GFP expression levels in SIN replicon and control GFP flies measured by fluorometry. Adult flies were homogenized and GFP in homogenates was detected by fluorescence measurements. The value obtained for control flies was considered as one. (E and F) Real-time qRT-PCR analysis of GFP mRNA in SIN replicon and control GFP flies. The results were normalized to actin, and the value obtained for control flies was considered as one. Data shown is representative of three independent experiments. Error bars represent SD.

Figure 4. Alphaviral replication is higher in Relish mutant flies.

(A) Bright field and fluorescence images of wild type SIN replicon flies and flies heterozygous for SIN replicon and relish (rel^e20), dif (Df(2L)Exel8036) dorsal (dorsal^KG06652), and stat (stat92E^HJ) mutations. (B) GFP expression levels in the above flies measured by fluorometry. The value obtained for control SIN replicon flies was considered as one. Data shown is representative of three independent experiments. (C) SIN replication was measured by real-time qRT-PCR analysis of nsP1 mRNA in control SIN replicon flies and flies heterozygous for SIN replicon and relish (rel^e20), dif (Df(2L)Exel8036) dorsal (dorsal^KG06652), and stat (stat92E^HJ) mutations. The value obtained for control SIN replicon flies was considered as one. Error bars represent SD.

Figure 5. The Imd pathway is involved in controlling alphaviral replication.

SIN replication was measured by real-time qRT-PCR analysis of nsP1 (A) and GFP (B) mRNAs in control SIN replicon flies and flies heterozygous for SIN replicon and relish (rel^e20), imd (imd ^ey08573), dfadd (BG4^EY10870), dredd (dredd^EY08404), tab2 (tab2 ^201y), ird5 (ird5^EY02434) and kenny (key^c02831) mutations. The value obtained for control SIN replicon flies was considered as one. (C) The expression of Diptericin, Metchnikowin and Drosomycin AMPs was measured in w¹¹¹⁸ flies, GFP expressing flies, mutant SIN replicon expressing flies, and SIN replicon expressing flies by real-time qRT-PCR. The value obtained for w¹¹¹⁸ flies was considered as one. Data shown is representative of three independent experiments. Error bars represent SD.

Figure 6. Alphaviral replication in Relish flies.

(A) SIN virus replication was measured by real-time qRT-PCR analysis of nsP1 mRNA in control w¹¹¹⁸ flies, dif (dif¹) and relish (rel^e20) mutant flies. (B) Viral titers of SIN in w¹¹¹⁸, Dif and Relish mutant flies 5 days post infection. (C) SIN virus replication in control w¹¹¹⁸, UAS-Relish.his6 flies and flies over-expressing Relish (cg-GAL4,UAS-Relish.His6). Replication was measured by qRT-PCR analysis of nsP1 mRNA. Flies were infected with 200pfu of SIN:GFP virus. Viral replication and viral titers were measured five days post infection. Data shown is representative of three independent experiments. Error bars represent SD.

Figure 7. Relish activation in mosquito cells.

Western blot with anti-Relish antibody on cytoplasmic and nuclear extracts of c6/36 cells infected with SIN. C6/36 cells were not infected or infected with SIN for 6 and 48 h and lystes obtained and fractionated and probed for Relish.