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Short Communication |
School of Integrative Biology, University of Queensland, Brisbane, Australia
Correspondence
Karyn N. Johnson
karynj{at}uq.edu.au
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ABSTRACT |
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A supplementary table showing the primers used in this study is available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Dostert et al., 2005; Roxstrom-Lindquist et al., 2004; Sabatier et al., 2003), none have focused on the equally important contribution of DCV to these interactions. In the DCV replication cycle, DCV components have many points of interaction with the Drosophila cell and there are differences in the susceptibility between Drosophila strains and in the pathogenicity of the DCV isolates, suggesting that there is some specificity in the balance of the interaction between host and virus (Johnson & Christian, 1999; Thomas-Orillard et al., 1995).
DCV is a member of the genus Cripavirus of the family Dicistroviridae. DCV particles are non-enveloped and 30 nm in diameter with a T=3 icosahedral structure (Plus et al., 1975; Tate et al., 1999). The positive-sense RNA genome includes 9264 nt and contains two large open reading frames (ORFs) which encode the non-structural and structural polyproteins (Johnson & Christian, 1998). Non-structural proteins encoded by ORF1 include helicase, protease and RNA-dependent RNA polymerase (Johnson & Christian, 1998). The structural proteins encoded by ORF2 are VP0, VP1, VP2, VP3 and VP4, with VP0 a precursor of VP3 and VP4; these proteins assemble to form the DCV capsid (Reavy & Moore, 1983; Tate et al., 1999).
Relatively little is known about innate immune responses of Drosophila that are specific for viruses (Cherry & Silverman, 2006). RNA interference is the most studied and is a general antiviral defence. However, a number of viruses, including DCV, encode suppressors which inhibit this response (van Rij et al., 2006). Microarrays and proteomics have been used to identify genes and proteins that are upregulated in response to DCV infection in Drosophila (Dostert et al., 2005; Roxstrom-Lindquist et al., 2004; Sabatier et al., 2003). A number of genes have been found to be upregulated in response to DCV infection and one protein has been identified as being induced in the haemolymph of DCV-infected Drosophila (Sabatier et al., 2003).
Dostert et al. (2005) identified 140 genes that were upregulated in adult Drosophila 24 and 48 h post injection with DCV. Two thirds of these genes were not upregulated in response to bacterial or fungal infections (Dostert et al., 2005). Among the most upregulated of these virus-specific genes were CG12780, vir-1 and CG9080 (Dostert et al., 2005). Each of these three genes contain putative STAT-binding sequences in their promoter regions and loss of function hopM38/hopmsvl mutant Drosophila supported the implied role of the Jak-STAT regulation of these genes, with the mutant hop flies having a greatly reduced ability to induce these genes (Dostert et al., 2005). In addition, vir-1 was not upregulated in response to bacterial or fungal infection or stresses including heat shock, cold shock, mechanical pressure, dehydration and UV irradiation (Dostert et al., 2005). For the present study, CG12780, vir-1 and CG9080 were used as reporters of Drosophila response to DCV infection.
Throughout our experiments we used the Drosophila strain Champetieres (DGRC number 103403), which was obtained from the Drosophila genetic resource centre. The flies were maintained at 25 °C with a 12 h day/night cycle on standard Drosophila medium. DCV isolates EB, C and Z were originally purified from wild and laboratory stocks of Drosophila from Australia, France and Morocco, respectively (Christian, 1992; Jousset et al., 1972; Plus et al., 1975). DCVEB was plaque-purified and all three isolates were confirmed to be free of cricket paralysis virus contamination. DCV isolates were propagated in DL2 cells and purified by differential centrifugation through 10–40 % sucrose gradients, essentially as described previously (Johnson & Christian, 1998; Johnson et al., 2000). Each virus was resuspended in 50 mM Tris, aliquoted and stored at –20 °C.
The general approach for our experiments involved micro-injecting 4–5 days old, male Drosophila with either DCV, DCV component or buffer control into the upper lateral part of the fly abdomen, essentially as described previously (L'Héritier, 1952). The mortality of one group of Drosophila was monitored for survival curves. Forty-eight hours post injection, five Drosophila were collected and pooled from each treatment group and five Drosophila were collected from buffer-injected control flies (3–5 replicates per treatment for all experiments). The Drosophila samples were then homogenized with TRI reagent (Molecular Research Center) using a bead beater with 3 mm glass beads and total RNA was extracted as per manufacturer's instructions. Extracted RNA was DNase-treated to avoid genome contamination and reverse transcribed using random primers and SuperScript III reverse transcriptase (Invitrogen), following the manufacturer's instructions, with a 30 min incubation at 50 °C. To control for within-group variation all samples were reverse-transcribed in parallel.
Quantitative PCR was carried out using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), as per the manufacturer's instructions, using primers listed in Supplementary Table S1 (available with the online version of this paper). PCR was carried out using 2 µl cDNA template. The PCR temperature profile was 95 °C for 2 min, 50 °C for 2 min and 40 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s, and was performed with a Rotor-Gene 6000. A control without reverse transcriptase was used to confirm that all genomic DNA had been removed. RT-qPCR data were analysed using REST software (Pfaffl et al., 2002). Expression of CG12780, vir-1 and CG9080 was normalized using house-keeping genes actin 79B (GenBank accession no. NM_079486
[GenBank]
) and rpL32.
Experiments were undertaken to delineate the DCV components or processes that induce upregulation of the Drosophila reporter genes. The DCV isolates are estimated to differ by up to 10 % at the nucleotide level (Johnson & Christian, 1999), so the Drosophila response to three DCV isolates, DCVC, DCVEB and DCVZ, was compared in order to determine whether virus genetic variation affected upregulation of the reporter genes. Drosophila were injected with approximately 100 nl of either 1.2x108 IU DCVEB ml–1, an equivalent particle concentration of DCVC or DCVZ or buffer, and the relative expression of CG12780, vir-1 and CG9080 was determined as described above.
Compared with the negative control Drosophila, Drosophila infected with each DCV isolate showed statistically significant upregulation of all three genes (Fig. 1, P<0.05 in each case). This confirmed that the response was not restricted to the DCV–Drosophila strain combinations used in the previous study (Dostert et al., 2005). However, direct comparison between isolates found no difference in the level of upregulation of these genes, except for the statistically significant but small (1.5-fold, P=0.03) difference in the upregulation of vir-1 between DCVEB and DCVZ. Therefore, there was no correlation between the genetic differences of the virus isolates and the level of gene upregulation of the reporter genes. This is consistent with the previous finding that vir-1 is upregulated in response to infection by flock house virus (FHV), which like DCV is a non-enveloped, single-stranded, positive-sense RNA virus (Dostert et al., 2005), suggesting that infection with positive-sense RNA viruses may stimulate a common response pathway in Drosophila. This being the case, it is possible that the reporter genes would be stimulated by processes in the replication cycle that are common across virus groups.
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). To test whether non-infectious virus induced the reporter genes, flies were challenged with DCVEB that had been inactivated with UV light. It is anticipated that the UV-inactivated virus attaches to the cell surface and is taken up into the host cell, though uptake by the cells of the UV-inactivated virus is technically difficult to prove and was not assayed.
DCV was UV-inactivated by exposure to a total of 12 000 mJ UV light (4x3 min bursts). Inactivation was confirmed by an infectivity assay in DL2 cells (Scotti, 1977). Untreated DCV was prepared from the same virus stock and processed as for inactivated virus, except there was no exposure to UV light. Drosophila were injected with approximately 100 nl of either 1.2x108 IU ml–1 of DCV, UV-inactivated DCV of the same dilution or buffer control. The relative abundance of CG12780, vir-1 and CG9080 transcripts in response to UV-inactivated DCV was compared with buffer-injected Drosophila, with DCV-injected Drosophila as a positive control.
UV-inactivated DCV did not induce any significant differences in the levels of reporter gene transcript expression compared with the control Drosophila (P>0.05 in each case), whereas the DCV-infected Drosophila showed significant (P<0.05) upregulation of all three genes (Fig. 2a). Mortality of DCV-infected flies was similar to previous experiments at 100 % mortality by 4 days post infection, whereas 0 % mortality was observed in the UV-inactivated DCV-injected group (Fig. 2b). This suggests that the reporter genes are not induced in flies challenged with non-infectious virus.
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; van Rij et al., 2006; Wang et al., 2006). While the DCV reporter genes are not involved in this pathway, it is possible that the DCV dsRNA is inducing Drosophila responses that are independent of RNA silencing. It has previously been shown that dsRNA is taken up into cells of adult Drosophila following injection of dsRNA into the abdominal cavity (Dzitoyeva et al., 2001; Goto et al., 2003); here, we introduced dsRNA into adult Drosophila using a similar approach. Briefly, a region of the DCV genome was amplified using the primers DCV-ORF2-Fw and DCV-ORF2-Rv (Supplementary Table S1) and purified using a MinElute PCR purification kit (Qiagen). RNA was transcribed using a MEGAscript kit (Ambion) and purified using the MEGAclear protocol (Ambion). Drosophila were injected with 400 ng µl–1 DCV-ORF2 dsRNA, 1.2x108 IU ml–1 DCVEB, buffer control or 200 ng µl–1 non-specific dsRNA (kindly provided by Darren Underwood, University of Queensland, Australia) and the relative expression of CG12780, vir-1 and CG9080 was determined.
The DCV-ORF2 dsRNA did not induce vir-1 transcription, although CG12780 and CG9080 were induced 1.9 and 1.7-fold, respectively (P=0.04, Fig. 3a). The Drosophila response to non-specific dsRNA was not significantly different from the buffer-injected controls (P>0.05 in each case), and positive control DCV infection caused significant upregulation of each of the three reporter genes (P<0.05 in each case). DCV-infected Drosophila died by day four, whereas the mortality of flies injected with DCV dsRNA or non-specific dsRNA was similar to the buffer-injected control Drosophila (Fig. 3b). Thus, while induction was observed following injection of DCV-dsRNA for two of the reporter genes, CG12780 and CG9080, the biological significance is not clear as it was below twofold difference, and no induction was observed following injection of non-specific dsRNA. Taken together, this suggests that dsRNA is not sufficient to stimulate upregulation of those three Drosophila genes.
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Studies of the complex interactions underlying the Drosophila–virus system have largely ignored the contribution of the virus to these interactions. The replication cycle of DCV involves a number of interactions with Drosophila host cells which could be responsible for the upregulation of the DCV-infection-induced genes CG12780, vir-1 and CG9080. This study found that neither dsRNA nor non-infectious DCV particles were sufficient to stimulate upregulation of the three reporter genes. The upregulation of these genes seems to be a general response, with little or no difference observed in the Drosophila response to three different DCV isolates.
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ACKNOWLEDGEMENTS |
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Received 23 December 2007;
accepted 19 February 2008.
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