Fig 1.
Turandot genes are expressed upon IIV-6 Infection A) Heatmap of mRNA levels for selected immune-related genes following IIV-6 infection of adult w1118 flies for 12, 24 and 48 hours assayed in duplicate by NanoString nCounter. RNA was isolated from PBS-injected flies at the same time points. Each data point is a biologically independent sample. A’) Detailed comparison of mRNA levels for Tot A or Tot M from nCounter data. B) Fold induction of all eight Tots from w1118 flies infected with IIV-6, relative to PBS injected controls, at 6, 12, or 24 hours, quantified by qRT-PCR. n = 3, error bars represent SEM and statistical significance determined by Multiple t-tests with correction for multiple comparisons using the Holm-Sidak method. By this analysis, all Turandots were significantly induced with p values between 0.05 and 0.0003 at all time-points with the exception of TotF (ns) and TotE, which was undetectable (nd). C) S2* cells were infected with IIV-6 and TotA expression was assayed by qRT-PCR at the indicated time points. Significance was determined by two-way ANOVA and Sidak’s multiple comparisons test, comparing the infected sample to its time-matched uninfected control. * p < 0.05; *** p < 0.001; and **** p < 0.0001 Error bars indicate standard deviation and black bars indicate the mean. A.U., Arbitrary Units.
Fig 2.
Viral replication is required for IIV-6 induced turandot expression.
A) S2* cells were infected with heat- or UV-inactivated IIV-6 or infected with VACV or IIV-6 in serum-free (SF) media, and TotA induction was assayed by qRT-PCR. B, C) S2* cells were treated with the viral polymerase inhibitors B) phosphonoacetic acid (PAA) or C) cidofovir at the indicated concentrations for one hour prior to IIV-6 infection. For all panels, TotA induction was assayed at 24 hours post-infection by qRT-PCR. Significance, compared to IIV-6 infected samples without treatment or drugs, was determined by one-way ANOVA and Sidak’s multiple comparisons test (* p < 0.05; *** p < 0.001; and **** p < 0.0001). Error bars indicate standard deviation and black bars indicate the mean. A.U., Arbitrary Units.
Fig 3.
JAK-STAT signaling is required for IIV-6-induced turandot expression and survival from virus infection A) S2* cells were transfected with dsRNA targeting hopscotch, domeless, or Stat92E, and 48 hours later were infected with IIV-6 for 24 hours. RNA was then isolated and TotA induction was quantified by qRT-PCR. Data shown are from three biologically independent assays. Two non-overlapping dsRNAs were used to target each gene. Error bars indicate standard deviation and black bars indicate mean. Statistical analysis was performed comparing control dsRNA (GFP or mCherry) transfected cells to target gene knockdowns by two-way ANOVA with corrections for multiple comparisons using the Holm-Sidak method (**** p <0.0001). A.U., Arbitrary Units. B) Kaplan-Meier curves showing survival of Stat92ERNAi expressing (UAS-Stat92ERNAi (VDRC)x tubulin-Gal4) flies (green lines) or control (w1118 x tubulin-Gal4) flies (black lines) following infection with IIV-6 (solid lines) or injection with PBS (dotted lines). C) Kaplan-Meier curves showing survival of Stat92ERNAi expressing (UAS-Stat92ERNAi (TRiP-1)x c564-Gal4) flies (green lines) or control (w1118) flies (black lines) following infection with IIV-6 (solid lines) or injection with PBS (dotted lines). Survival assays utilized at least 50 animals per treatment in each trial and statistical significance was determined by Log-rank (Mantel-Cox) test, comparing IIV-6 infected RNAi lines to IIV-6 infected control animals, or PBS-injected RNAi lines to PBS-injected controls. ****p <0.0001.
Fig 4.
p38-dependent IIV-6 induced unpaired expression A) S2* cells were infected with IIV-6 for the indicated times and induction of upd1, upd2, or upd3 was monitored by qRT-PCR, compared to PBS injected controls. Three biologically independent replicates are shown and statistical analysis was performed by Multiple t-tests with the Holm-Sidak correction for multiple comparisons. Error bars indicate standard deviation and black bars indicate the mean. B) TotA or TotM expression was measured by qRT-PCR from control w1118 flies as well as outstretcheds (oss), upd2Δ, upd3Δ, or upd2Δ,3Δ mutant flies 24 hours after IIV-6 infection or PBS injection. The results of 3–5 biologically independent assays are displayed. Error bars represent the standard deviation and black bars represent the mean. Statistical significance compared to the IIV-6 infected control strain (w1118) was determined by two-way ANOVA with Holm-Sidak correction. C) S2* cells were treated with inhibitors for the three MAPKs: JNK (SP600125, 25μΜ), ERK (U0125, 10μΜ), and p38 (SB203580, 10μΜ), or treated with a vehicle control (DMSO), for one hour prior to IIV-6 infection or mock treatment. After 24 hours of infection, upd3 expression was assayed by qRT-PCR. The results of 3 biologically independent assays are shown. Statistical significance, compared to the DMSO treated control, was determined by two-way ANOVA with Holm-Sidak correction. D) S2* cells were treated with the JAK inhibitor Tofacitinib (CP690,550 10 μM), or treated with a vehicle control (DMSO), for one hour prior to IIV-6 infection or mock treatment. After 24 hours of infection, TotA and upd3 expression were assayed by qRT-PCR. 3 biologically independent assays with statistical significance, comparing virus infected vehicle to inhibitor treatments, determined by two-way ANOVA with Holm-Sidak's multiple comparisons test. Significance is indicated in A-D as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; and ns, not significant. A.U., Arbitrary Units.
Fig 5.
p38b is required for turandot induction and survival A) Mutant strains for the three p38 homologs, p38aMPK1, p38bex9, or p38c1A1, were infected with IIV-6 or injected with PBS, and TotA expression was measured by qRT-PCR 24 hours post-infection. Control genotypes included w1118 and p38bex9/CyO heterozygotes. The results of 3–5 biologically independent replicates are shown. Black bars indicate the mean and error bars represents standard deviation. Statistical analysis, comparing TotA levels in the virus infected control to mutant strains, was determined by two-way ANOVA with Sidak’s correction for multiple comparisons (***p = 0.0004, ** p = 0.0045) B) Kaplan-Meier curves showing survival of homozygous (p38bex9, red lines) or heterozygous (p38bex9/CyO, orange lines) p38b mutant flies following IIV-6 infection (solid lines) or PBS-injected controls (dotted lines) compared to control (w1118, black lines) flies. C) Kaplan-Meier curves showing survival of p38a mutant flies (red lines) following IIV-6 infection (solid lines) or PBS-injected controls (dotted lines) compared to control (w1118, black lines) flies. D) Kaplan-Meier curves showing survival of p38bRNAi expressing (blue lines) or E) p38aRNAi expressing flies (purple lines) following IIV-6 infection (solid lines) or PBS-injected controls (dotted lines). UAS-p38aRNAi and UAS-p38bRNAi flies were crossed to tubulin-GAL4 for ubiquitous knock-down, while the control was generated by w1118 crossed to tubulin-GAL4 (black lines). Statistical significance was determined by Log-rank (Mantel-Cox) test (**** p <0.0001; ns, not significant). A.U., Arbitrary Units. F) Viral loads as determined by QPCR for p38bex9 and p38bex9/CyO heterozygous siblings. Each data point represents 5 flies. Error bars indicate standard error, and black lines indicate the mean. A.U., arbitrary units. Statistics were determined using two-way ANOVA. ns, not significant.
Fig 6.
Nox is required for IIV-6 induced upd3 and turandot expression S2* cells were treated with the indicated concentrations of DPI (an NADPH oxidase inhibitor) or vehicle control (DMSO) for one hour, and then infected with IIV-6 or mock treated. A) TotA expression and B) unpaired3 expression were quantified by qRT-PCR 24 hours post-infection. For A) and B), 3 biologically independent replicates are shown, and statistics performed comparing virus infected vehicle treated to DPI treated conditions. C) UAS-NoxRNAi and UAS-DuoxRNAi flies were crossed to c564-GAL4 for fat body specific knock-down, while the control was generated by w1118 crossed to c564-GAL4. Progeny were infected with IIV-6 or injected with PBS, and TotA expression was measured in whole flies by qRT-PCR 24 hours post-infection. 5 biologically independent replicates are shown. For all panels, error bars represent standard deviation, black lines indicate the mean, and statistical significance was determined using two-way ANOVA with Sidak’s correction.(***p = 0.0004, ****p<0.0001). A.U., Arbitrary Units.
Fig 7.
IIV-6 infection activates a protective response through p38b and JAK-STAT signaling IIV-6 infection activates p38b through the NADPH oxidase Nox and presumably ROS production. p38b triggers the induction of the unpaireds, which encode for secreted ligands which activate the JAK-STAT pathway via the receptor, Domeless. JAK-STAT signaling leads to induction of the Turandots (Tots), a family of small, secreted proteins with no known function. JAK-STAT as well as p38b signaling protects Drosophila from IIV-6 infection, and p38b in particular increases tolerance to this virus.