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

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

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

<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

<i>Aedes aegypti</i> Aag2 cells produce transposon-derived piRNAs with a ping-pong signature.

<p><b>A.</b> Size distribution of the small RNA reads that match with 0 mismatches against an <i>Aedes aegypti</i> transposon dataset that contain full-length non-composite transposons sequences (TEfam: <a href="http://tefam.biochem.vt.edu/tefam/index.php" target="_blank">http://tefam.biochem.vt.edu/tefam/index.php</a>). <b>B.</b> Heat map for 25–29 nt small RNAs that mapped to individual retrotransposons with more than 1000 reads. Read count and log-transformed ratios of antisense/sense small RNAs are presented. <b>C</b>. Profile of 25–29 nt reads that mapped to the transposon Copia Ele56 (TF000691) allowing 0 mismatch during alignment. Transposon-derived piRNAs that mapped to the sense and antisense strand of the transposon sequence are shown in red and blue, respectively. <b>D.</b> Conservation and relative nucleotide frequency per position of 25–29 nt reads that mapped to the sense (top) and the antisense (bottom) strands of the entire transposon dataset. n indicates the number of reads used to generate each logo.</p

U4.4 cells produce vsiRNAs and vpiRNAs through a ping-pong mechanism upon (+) ssRNA arbovirus infection.

<p>Profile of 21 nt vsiRNAs (<b>A</b>) and 25–29 nt (<b>B</b>) SINV-GFP-derived small RNAs allowing 0 mismatch during alignment. Viral small RNAs that mapped to the sense and antisense strand of the SINV-GFP genome are shown in red and blue, respectively. <b>C.</b> Conservation and relative nucleotide frequency per position of 25–29 nt SINV-GFP-derived reads that mapped to the sense (top) and the antisense (bottom) strands of the SINV-GFP genome. The overall height of the nucleotide stack indicates the sequence conservation; the height of the nucleotides within each stack represents their relative frequency at that position. n indicates the number of reads used to generate each logo. <b>D.</b> Frequency map of the distance between 25–29 nt small RNAs that mapped to opposite strands of the SINV-GFP genome. The peak at position 9 on the sequence (the first nucleotide being position 0) indicates the position of maximal probability of finding the 5′ end of a complementary small RNA.</p

<i>Aedes albopictus</i> U4.4 cells are Dcr-2 competent and produce two populations of viral small RNAs.

<p><b>A.</b> Dicer assay in uninfected U4.4 cells. Lane 3 shows processing of a 113-bp dsRNA substrate into 21-nt siRNAs after incubation in a U4.4 cell extract. Synthetic siRNA (21-nt) and input dsRNA (113-nt) are used as size markers in lanes 1 and 2, respectively. <b>B.</b> RNAi reporter assay. Co-transfection of firefly luciferase specific dsRNA with reporter plasmids encoding firefly and <i>Renilla</i> luciferase into U4.4 cells results in silencing of the firefly luciferase reporter. GFP dsRNA was used as non-specific dsRNA control. <i>Renilla</i> luciferase activity was used as internal control to normalize the firefly luciferase activity. Error bars represent the standard deviations of three individual samples. <b>C.</b> Size distribution of the small RNA reads that match the genome of SINV-GFP with 0 mismatches.</p

The C-terminus of Nora virus VP1 is essential for RNAi suppressor activity.

<p>(<b>A</b>) Schematic presentation of full-length (FL) and N- and C-terminal deletion mutants (ΔN and ΔC) of VP1. (<b>B</b>) Western blot analysis of VP1 expression constructs. V5 epitope tagged expression constructs were transfected into <i>Drosophila</i> S2 cells and expression of VP1^FL and the deletion mutants was analyzed by Western blot using a V5 antibody (αV5). (<b>C</b>) RNAi reporter assay in S2 cells. The experiment was performed as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002872#ppat-1002872-g002" target="_blank">Figure 2D</a>, using plasmids encoding either CrPV 1A, VP1^FL or the VP1 deletion mutants. Bars represent averages and standard deviations of three independent samples. The graph is representative for two independent experiments.</p