The Epigenetic Regulator G9a Mediates Tolerance to RNA Virus Infection in <i>Drosophila</i>

<div><p>Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. Here, we demonstrate that the histone H3 lysine 9 methyltransferase <i>G9a</i> regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in <i>Drosophila</i>. <i>G9a</i>-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. Our results identify an essential epigenetic mechanism underlying tolerance to virus infection.</p></div

Genetic interaction between <i>G9a</i> and the Jak-Stat pathway.

<p>(<b>A,B</b>) Survival upon DCV infection (1,000 TCID₅₀ units) of wild-type or <i>G9a</i> mutant and wild-type flies overexpressing (<b>A</b>) a dominant negative version of the <i>domeless</i> receptor (dome^ΔCyt), or (<b>B</b>) the negative regulator of Jak-Stat signaling <i>Socs36E</i>. The UAS/Gal4 system was used to drive transgene expression. Gal4 is expressed under control of the actin promoter (<i>Act-Gal4</i>) to drive ubiquitous expression of the <i>UAS-dome</i>^ΔCyt and <i>UAS-Socs36E</i> transgenes. Control flies expressing only the <i>Act-Gal4</i>, the <i>UAS-dome</i>^ΔCyt, or the <i>UAS-Socs36E</i> transgenes were included as controls (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s012" target="_blank">S5A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s012" target="_blank">S5B</a> Dataset). Mock infections where performed along the experiments and are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s006" target="_blank">S6A and S6B Fig</a>. (<b>C,D</b>) Expression of <i>TotA</i> and <i>vir-1</i> upon DCV infection of wild-type or <i>G9a</i> mutant flies, expressing (<b>C</b>) dome^ΔCyt, or (<b>D</b>) <i>Socs36E</i>. Expression of the gene of interest (by RT-qPCR) was normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and expressed as fold change relative to mock infection (Tris buffer). Data are means and s.d. of three independent pools of at least 15 male flies for each genotype. (<b>A</b>,<b>B</b>) A representative experiment of two independent experiments is shown. Differences in expression of <i>TotA</i> and <i>vir-1</i> were evaluated with a Student’s t-test (*<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

Hyperactivation of the Jak-Stat pathway renders flies hypersensitive to virus infection.

<p>(<b>A</b>) Experimental set-up. Expression of the <i>Upd</i> transgene was induced specifically in adult flies using the <i>Gal4/Gal80ts</i> system. <i>Gal80ts</i> is a temperature-sensitive allele of the Gal80 inhibitor that binds Gal4 to prevent activation of gene expression at 20°C. At 29°C, Gal80ts is degraded, allowing Gal4 to bind to the Upstream Activating Sequences (UAS) to induce gene expression. Flies were reared at 20°C, and 0 to 3-day-old adults were conditioned at 29°C for 3 days prior to viral challenge. (<b>B</b>) Expression levels by RT-qPCR of <i>Upd</i> and <i>TotA</i> in flies carrying the temperature-dependent <i>Upd</i> overexpression system (<i>UAS-Upd; tubulin-Gal4/Gal80ts</i>) after 3 days conditioning at 29°C. The <i>Gal4</i> and <i>Gal80ts</i> transgenes were combined with the <i>UAS-Upd</i> by standard genetic crosses at 20°C and 0 to 3-day-old adult offspring was cultured for 3 days at 20°C or at 29°C before RNA levels were analyzed by RT-qPCR. Transcript levels of <i>Upd</i> and <i>TotA</i> were normalized to RNA levels of the housekeeping gene <i>Ribosomal Protein 49</i>, and expressed as fold change relative to control flies carrying only the <i>UAS-Upd</i> transgene. (<b>C</b>) Survival of flies carrying the temperature-dependent <i>Upd</i> overexpression system (<i>UAS-Upd; tubulin-Gal4/Gal80ts</i>) and genetic control flies upon DCV infection (1,000 TCID₅₀ = units) at 29°C. Data are means and s.d. of three independent pools of at least 10 male flies for each genotype. Data in (<b>C</b>) are from one experiment representative of 2 independent experiments. Differences in expression of <i>Upd</i> and <i>TotA</i> were evaluated with a Student’s t-test (*<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

Hyperactivation of the Jak-Stat pathway by virus infection of <i>G9a</i> mutants.

<p>(<b>A</b>,<b>B</b>) Expression of inducible immune genes at 24 hours after DCV infection (TCID₅₀ = 10,000) determined by RT-qPCR in (<b>A</b>) whole flies, and (<b>B</b>) fat bodies of wild-type or <i>G9a</i> mutant flies. Expression of the gene of interest was normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and expressed as fold change relative to mock infection (Tris buffer). Data are means and s.d. of three independent pools of (<b>A</b>) 30 female flies and (<b>B</b>) 30 fat bodies for each genotype. (<b>C,D</b>) Basal expression levels of the indicated genes measured by RT-qPCR on 3 to 5-day-old unchallenged wild-type and <i>G9a</i> mutant female flies (<b>C</b>) or fat bodies <b>(D</b>). Basal expression levels are expressed as dCt values (difference between Ct of the gene of interest and the Ct of <i>Ribosomal Protein 49)</i>. (<b>E</b>-<b>I</b>) Expression of inducible Jak-Stat dependent immune genes at (<b>E</b>-<b>H</b>) 24 hpi or (<b>I</b>) 7 dpi with 10,000 TCID₅₀ units of (<b>E</b>) CrPV, (<b>F</b>) DXV, (<b>G</b>) FHV or (<b>H</b>,<b>I</b>) 14,000 TCID₅₀ units of IIV-6. Data are means and s.d. of three independent pools of at least 15 female flies (<b>C,E-I</b>) or at least 10 fat bodies (<b>D</b>) per genotype. Data are from one experiment representative of 3 (<b>A,B,E</b>), and 2 (<b>C</b>,<b>D</b>) independent experiments. *<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001 (Student’s t-test).</p

Loss of <i>G9a</i> does not affect viral loads upon DCV infection.

<p>(<b>A,B</b>) Wild-type or <i>G9a</i> mutant flies were inoculated with DCV and viral titers were determined over time in (<b>A</b>) whole flies, and (<b>B</b>) dissected fat bodies. Data represent means and s.d of three independent experiments. Each experiment contained three biological replicates of 5 female flies (<b>A</b>), or 10 fat bodies (<b>B</b>) per replicate for each genotype. (<b>C</b>,<b>D</b>) DCV RNA levels over the course of 3 days post-infection analyzed by RT-qPCR in (<b>C</b>) whole flies or (<b>D</b>) fat bodies of wild-type and <i>G9a</i> mutant flies. DCV RNA levels were normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and are calculated relative to the viral RNA levels in flies harvested immediately after inoculation (t₀). Data represent means and s.d. of three biological replicates of 5 female flies (<b>C</b>) or 10 fat bodies (<b>D</b>) per replicate for each genotype. Data in panel <b>C</b> and <b>D</b> are from one experiment representative of 2 independent experiments. *<i>P</i> < 0.05 (Student’s t-test).</p

<i>G9a</i> targets genes of the Jak-Stat pathway.

<p>(<b>A</b>) Expression levels of <i>domeless</i>, <i>dPIAS</i>, and <i>Socs36E</i> at 24 hpi in fat bodies of 3 to 5-day-old female wild-type or <i>G9a</i> mutant flies challenged with DCV (10,000 TCID₅₀ units). Data are expressed as fold change relative to mock infection (Tris buffer). (<b>B</b>) Basal expression levels of Jak-Stat genes measured by RT-qPCR on fat bodies of 3 to 5-day-old unchallenged female wild-type and <i>G9a</i> mutant flies. Basal expression is presented as dCt (difference between Ct of the gene of interest and the Ct of <i>Ribosomal Protein 49)</i>. (<b>C</b>) Representative example of a <i>G9a</i> target locus within the <i>domeless</i> gene, defined as a genomic region in which the H3K9me2 mark is present in wild-type flies, but not in <i>G9a</i> mutants, in a previous study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.ref020" target="_blank">20</a>]. Blue and red plots represent sequence reads in H3K9me2 ChIP-seq analyses of wild-type and <i>G9a</i> mutants, respectively [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.ref020" target="_blank">20</a>]. Gene structure is indicated with boxes for exons, lines for introns, and gray boxes for untranslated regions. The arrow represents the position of the amplicon generated by qPCR after Chromatin-Immunoprecipitation (ChIP-qPCR). (<b>D</b>) H3K9me2 ChIP-qPCR on fat bodies of wild-type or <i>G9a</i> mutant flies. Fold enrichment is the percentage of input of the gene of interest normalized to that of a reference gene with very low H3K9me2 marks (<i>moca</i>). Specificity control experiments for ChIP-qPCR experiments are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s005" target="_blank">S5E–S5J Fig</a>. Data are means and s.d. of (<b>A,B</b>) three independent pools of at least 10 fat bodies, or (<b>D</b>) three independent pools of 80 female fat bodies, for each genotype. Data are from one experiment representative of 2 (<b>A</b>,<b>B</b>) or 6 (<b>D</b>) independent experiments. *<i>P</i> < 0.05 (Student’s t-test).</p

VP1 suppressor activity is species-specific.

<p>(<b>A</b>) Western blot analysis of S2 cells expressing V5 epitope-tagged VP1 from <i>D. melanogaster</i> Nora virus (DmelNV) and <i>D. immigrans</i> Nora-like virus (DimmNV). S2 cells were transfected with plasmids encoding full-length VP1 (FL) and C-terminal (ΔC) or N-terminal (ΔN) deletions thereof. Expression of the VP1 constructs was analyzed by western blot using an anti-V5 (α-V5) antibody. Detection of tubulin with anti-tubulin (α-tub) antibody was used as a loading control. Molecular mass (in kDa) is indicated on the left. For DmelNV VP1^ΔN284, bands of lower mobility were observed in addition to the expected 26 kDa protein, the nature of which remains unknown. Note that these additional bands are not consistently observed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256.s002" target="_blank">Figure S2A</a>, lane 5, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256-vanMierlo2" target="_blank">[14]</a>). (<b>B</b>) RNAi sensor assay in S2 cells. Firefly luciferase (Fluc) and <i>Renilla</i> luciferase (Rluc) reporter plasmids were transfected into S2 cells, together with plasmids encoding the indicated VP1 constructs. Two days after transfection, S2 cells were soaked in either control (Ctrl) dsRNA or Fluc dsRNA, and luciferase activities were measured the next day. Fluc counts were normalized to Rluc counts, and presented as fold silencing relative to the corresponding control dsRNA treatment. (<b>C</b>) Hairpin-based RNAi sensor assay in S2 cells. S2 cells were transfected with plasmids coding for Fluc, Rluc, and an Rluc-hairpin RNA together with a control vector (Vector) or plasmids encoding the N-terminal deletion mutants of DmelNV VP1^ΔN284 or DimmNV VP1^ΔN295. Rluc counts were normalized to Fluc counts, and presented as fold silencing over non-hairpin control transfections. Bars in Panels B and C represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's <i>post hoc</i> test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). ** <i>P</i><0.01; *** <i>P</i><0.001; ns, not significant.</p

VP1 suppressor activity is species-specific.

<p>(<b>A</b>) Western blot analysis of S2 cells expressing V5 epitope-tagged VP1 from <i>D. melanogaster</i> Nora virus (DmelNV) and <i>D. immigrans</i> Nora-like virus (DimmNV). S2 cells were transfected with plasmids encoding full-length VP1 (FL) and C-terminal (ΔC) or N-terminal (ΔN) deletions thereof. Expression of the VP1 constructs was analyzed by western blot using an anti-V5 (α-V5) antibody. Detection of tubulin with anti-tubulin (α-tub) antibody was used as a loading control. Molecular mass (in kDa) is indicated on the left. For DmelNV VP1^ΔN284, bands of lower mobility were observed in addition to the expected 26 kDa protein, the nature of which remains unknown. Note that these additional bands are not consistently observed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256.s002" target="_blank">Figure S2A</a>, lane 5, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256-vanMierlo2" target="_blank">[14]</a>). (<b>B</b>) RNAi sensor assay in S2 cells. Firefly luciferase (Fluc) and <i>Renilla</i> luciferase (Rluc) reporter plasmids were transfected into S2 cells, together with plasmids encoding the indicated VP1 constructs. Two days after transfection, S2 cells were soaked in either control (Ctrl) dsRNA or Fluc dsRNA, and luciferase activities were measured the next day. Fluc counts were normalized to Rluc counts, and presented as fold silencing relative to the corresponding control dsRNA treatment. (<b>C</b>) Hairpin-based RNAi sensor assay in S2 cells. S2 cells were transfected with plasmids coding for Fluc, Rluc, and an Rluc-hairpin RNA together with a control vector (Vector) or plasmids encoding the N-terminal deletion mutants of DmelNV VP1^ΔN284 or DimmNV VP1^ΔN295. Rluc counts were normalized to Fluc counts, and presented as fold silencing over non-hairpin control transfections. Bars in Panels B and C represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's <i>post hoc</i> test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). ** <i>P</i><0.01; *** <i>P</i><0.001; ns, not significant.</p

Species-specific inhibition of AGO2 slicer activity.

<p>(<b>A</b>) <i>In vitro</i> RNA cleavage (slicer) assays in lysates from <i>D. melanogaster</i> embryos (left panel) or <i>D. immigrans</i> embryos (right panel). Radioactively cap-labelled target RNA was incubated in embryo lysate together with a non-specific control siRNA (lanes 1 and 6) or a target specific siRNA (lanes 2–5, 7–10). Target cleavage was determined either in the absence of recombinant protein (lanes 2 and 7) or in the presence of 0.3 µM of MBP (lanes 3 and 8), MBP-DmelNV VP1 (lanes 4 and 9), or DimmNV VP1 (lanes 5 and 10). (<b>B</b>) Quantification of target cleavage in <i>D. melanogaster</i> and <i>D. immigrans</i> embryo lysate in the presence of MBP, DmelNV VP1, or DimmNV VP1 protein. The fraction of cleaved RNA was determined by dividing the intensity of the cleavage product by the total intensity of cleavage product and non-cleaved target. Data are normalized to MBP. Bars represent means and standard deviations of two independent experiments.</p

VP1 interferes with the effector phase of the RNAi pathway.

<p>(<b>A</b>) Mobility shift assays for binding of viral RNAi suppressor proteins to siRNAs. Radiolabeled siRNAs were incubated in buffer (lane 1) or with decreasing amounts of recombinant MBP-VP1^ΔN284 (lanes 2–5), MBP (lanes 6–9), and MBP-NS3 (lane 10–13). Ten-fold dilutions were used, starting at 2 µM for MBP-VP1^ΔN284 (lane 2) and 2.6 µM for MBP (lane 6). MBP-NS3 was tested in two-fold dilutions (highest concentration of 8 µM, lane 10). RNA mobility shifts were analyzed on an 8% native polyacrylamide gel. (<b>B</b>) RISC Slicer assay in <i>Drosophila</i> embryo lysate. Lysates were incubated with non-targeting control siRNA (Ctrl, lane 1) or with Fluc siRNA (lanes 2–4) in the absence (lane 2) or presence of recombinant MBP-VP1^ΔN284 (lane 3) or MBP (lane 4). RISC cleavage products were analyzed on an 8% denaturing polyacrylamide gel. Slicer assay is representative for two independent experiments.</p