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Retrotransposon activation during Drosophila metamorphosis conditions adult antiviral responses

Nature Genetics volume 54, pages 1933–1945 (2022)Cite this article

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Abstract

Retrotransposons are one type of mobile genetic element that abundantly reside in the genomes of nearly all animals. Their uncontrolled activation is linked to sterility, cancer and other pathologies, thereby being largely considered detrimental. Here we report that, within a specific time window of development, retrotransposon activation can license the host’s immune system for future antiviral responses. We found that the mdg4 (also known as Gypsy) retrotransposon selectively becomes active during metamorphosis at the Drosophila pupal stage. At this stage, mdg4 activation educates the host’s innate immune system by inducing the systemic antiviral function of the nuclear factor-κB protein Relish in a dSTING-dependent manner. Consequently, adult flies with mdg4, Relish or dSTING silenced at the pupal stage are unable to clear exogenous viruses and succumb to viral infection. Altogether, our data reveal that hosts can establish a protective antiviral response that endows a long-term benefit in pathogen warfare due to the developmental activation of mobile genetic elements.

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Fig. 1: Monitoring mdg4 mobilization in somatic cells via a transposition reporter.
Fig. 2: mdg4 selectively mobilizes in the regenerating tissues during metamorphosis.
Fig. 3: Nanopore sequencing detected full-length copies of mdg4 from both wild and laboratory strains.
Fig. 4: mdg4 activation at the pupal stage safeguards adult flies upon persistent viral infection.
Fig. 5: mdg4 activation at the pupal stage safeguards adult flies upon one-time viral infection.
Fig. 6: Relish activation at the pupal stage protects adult flies from viral infections.
Fig. 7: mdg4 triggers Relish activation in both the hindgut and fat body at the pupal stage.
Fig. 8: dSTING triggers Relish activation at the pupal stage for adult antiviral responses.

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Data availability

The sequencing data from this study were deposited in the National Center for Biotechnology Information BioProject database under accession number PRJNA7843705. The published datasets that were used in this manuscript can be found in the Sequence Read Archive under accession numbers SRR1197325, SRR1197324, SRR1197326, SRR8627922, SRR8627923 and SRR8643355. Source data are provided with this paper.

Code availability

Code from this manuscript is available at https://github.com/ZhaoZhangZZlab/2021_fly_mdg4.

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Acknowledgements

We thank P. Zamore for providing DCV and M.-C. Saleh for providing FHV and CrPV. We thank D. Fox for critical suggestions, B. Kegeris for assistance on cloning and K. Poss for reading the manuscript (all members of the Z.Z. laboratory). This work was supported by grants from the Pew Biomedical Scholars Program and National Institutes of Health (DP5 OD021355 and R01 GM141018) to Z.Z. and a grant from the National Institutes of Health (AI060025) to N.S.

Author information

Author notes

  1. These authors contributed equally: Lu Wang, Lauren Tracy.

Authors and Affiliations

  1. Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA

    Lu Wang, Lauren Tracy, Weijia Su, Fu Yang & Z. Z. Zhao Zhang

  2. State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China

    Lu Wang & Yu Feng

  3. Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Neal Silverman

  4. Duke Regeneration Center, Duke University School of Medicine, Durham, NC, USA

    Z. Z. Zhao Zhang

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Contributions

Z.Z.Z.Z. and L.W. conceived of the project. All authors designed the experiments. L.T. performed the experiments for Fig. 3 and Extended Data Figs. 3c, 4, 8f and 9c, as well as all of the statistical analysis for survival assays and Relish signal quantification in the nucleus. Y.F. performed the experiments of RT-qPCR for dcr-2, ago-2 and pelle expression in Fig. 6a (right panel). F.Y. contributed to Fig. 3. W.S. performed computational analysis L.W. performed the rest of the experiments and data analysis. N.S. designed the experiments and contributed to data interpretation. Z.Z. and L.W. wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Lu Wang or Z. Z. Zhao Zhang.

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Extended data

Extended Data Fig. 1 Monitoring retrotransposon mobilization in somatic cells via a transposition reporter.

a, Detailed schematic design of eGFP transposition reporter to monitor mdg4 mobilization. b, Summary of mobilization events from different somatic tissues for 9 retrotransposon families, as assayed by corresponding eGFP reporter. No: no eGFP positive cells are detected; Yes: eGFP positive cells can be detected. c, Detecting eGFP signals in somatic tissues from positive control, negative control, and mdg4 transposition reporter in 2-4-day-old adult flies. Note: Positive control construct gives low number of eGFP positive cells in brain and malpighian tubules, indicating that transcription of mdg4 is suppressed in these tissues. Three independent biological replicates were performed. d, Zoom-in display of the box region in Fig. 1b. In DAPI channels, green arrows point to the nuclei that have GFP expression.

Source data

Extended Data Fig. 2 mdg4 selectively mobilizes in the regenerating tissues during metamorphosis.

a, Schematic of Drosophila hindgut. Both larval and adult hindgut include the pylorus, ileum and rectum. During pupal stage metamorphosis, the pylorus and ileum from larval stage degenerate; the anterior part of pylorus (ring) regenerates to produce adult pylorus and ileum. b, Detecting eGFP positive cells from mdg4 transposition reporter in midgut, salivary gland and proventriculus at different stages. c, The box plot shows the number of eGFP positive cells from mdg4 transposition reporter in midgut, salivary gland and proventriculus.

Source data

Extended Data Fig. 3 Probing transposition events by PCR.

a, Transposition events generate intron-removed DNA, which produces a short PCR product. b, Probing mobilization events at different developmental stages. Only the DNA from 48 hour pupal hindguts can harbor enough mobilization events to be detected by this PCR assay. c, Probing mobilization events from different adult tissues. These tissues either have no––or too few––mobilization events to be detected by this method. Three independent biological replicates were performed for b and c.

Source data

Extended Data Fig. 4 RNA-Seq to measure transcripts from mdg4.

a, Bar graph to display the abundance of full-length and Env mRNAs from mdg4. Full-length mdg4 transcripts are constantly expressed at all stages. Env mRNAs can be detected from early stage embryos and pupal stage, but not adult stage. b, IGV browser screenshot to display the representative sequencing reads that support the expression of Env mRNAs.

Extended Data Fig. 5 Multiple RNAi constructs were designed to silence mdg4.

a, RT-qPCR to quantify the expression of mdg4 upon suppression by using one of the 7 RNAi constructs based on the two-tailed t-test. Each sh-RNA construct was driven by ac-Gal4. Flies were raised at 25˚C and newly eclosed flies were used to extract RNA. Data are normalized to rp49 (RpL32) expression; the bars report mean ± standard deviation for three biological replicates (apply to all RT-qPCR data from this manuscript). b, Visualizing mdg4 transposition events (GFP positive cells) in hindgut from either white or mdg4 suppressed 2-4-day-old adults. Flies carrying sh-mdg4-5 construct were used for Fig. 6. sh-mdg4-1 was used in the rest of the experiments. The findings from it were validated by using other constructs (shown in Extended Data Fig. 11). c, RT-qPCR to measure the amount of DCV from fly bodies after feeding animals virus for 8 hours based on the two-tailed t-test. Data are normalized to rp49 (RpL32) expression; the bars report mean ± standard deviation for three biological replicates. d, By activating mdg4 RNAi from embryonic to adult stage, RT-PCR experiments were performed to monitor the amount of DCV in fly bodies after one-time infection. Together with Extended Data Fig. 5a, these data indicate RNAi efficiency negatively correlates with the robustness of antiviral response. e, By using sh-mdg4-2 to silence mdg4 at the stage specific manner, similar findings were made as Fig. 5. Three independent biological replicates were performed for sh-mdg4-2 in d and e, one time experiment was performed for sh-mdg4-3, 4, 5, 6 and 7 in d.

Source data

Extended Data Fig. 6 Schematic design of the virus-feeding assays used in this study.

a, Top panel: Schematic design to achieve RNAi from embryonic to adult stage. Middle panel: Schematic design to achieve RNAi only at the pupal stage. At lower temperature, Gal80 inhibits Gal4 activity. At 29˚C, Gal80 becomes inactive and cannot suppress Gal4. Bottom panel: Schematic design to achieve RNAi only at the adult stage. b, Validating ac-Gal4+tub-Gal80ts system by driving UAS-GFP expression at high temperature (29 °C) and low temperature (18 °C) in larval midgut and proventriculus. This experiment was only performed once. c, RT-qPCR to measure the mdg4 silencing efficiency for the ac-Gal4+tub-Gal80ts system at 29 °C based on the two-tailed t-test. Newly eclosed flies raised at 29˚C during pupal stage were used to extract RNA. Data are normalized to rp49 (RpL32) expression; the bars report mean ± standard deviation for two biological replicates. d, RT-qPCR to measure the mdg4 silencing efficiency for the ac-Gal4+tub-Gal80ts system at 25˚C based on the two-tailed t-test. Adult flies being shifted to 25˚C for 5 days were used to extract RNA. Data are normalized to rp49 (RpL32) expression; the bars report mean ± standard deviation for three biological replicates.

Source data

Extended Data Fig. 7 mdg4 activation renders hosts protection from virus infection.

a, By activating mdg4 RNAi from embryonic to adult stage, the survival rates were measured by raising flies on CrPV- or IIV-6-containing food for 20 days. sh-white flies served as controls. b, By activating mdg4 RNAi from embryonic to adult stage, the survival rates were measured after infecting adult files with different viruses with a single meal. c, RT-qPCR to quantify the fold change of DCV mRNA in sh-mdg4 flies at different time points after one-time infection, relative to sh-white controls based on the two-tailed t-test. d, RT-qPCR to quantify the fold change of DCV mRNA in sh-mdg4 flies on day 6 after one-time infection, relative to sh-white controls based on the two-tailed t-test. The bars in panel c and d report mean ± standard deviation for three biological replicates. Comparison of survival curves was completed using a Cox proportional-hazards model for panels a and b.

Source data

Extended Data Fig. 8 Relish activation renders hosts protection from virus infection.

a, By activating Relish RNAi from embryonic to adult stage, the survival rates were measured by raising flies on CrPV- or IIV-6-containing food for 20 days. sh-white flies served as controls. b, RT-qPCR to quantify the fold changes of DCV mRNA in sh-relish flies on day 6 after one-time infection, relative to sh-white controls based on the two-tailed t-test. c, RT-qPCR to quantify the fold changes of DCV mRNA in sh-relish flies on day 6 after one-time infection, relative to sh-white controls based on the two-tailed t-test. The bars in panels b and c report standard deviation for three biological replicates. d, By activating Relish RNAi only at adult stage, the survival rates were measured by raising flies on DCV- or FHV-containing food for 20 days. sh-white flies served as controls. e, RT-PCR experiments to monitor the amount of DCV in adult dcr-2 mutant (dcr-2L811fsX) flies after one-time infection. Two independent biological replicates were performed. f, RT-qPCR to quantify relish expression upon mdg4 depletion in fly pupae. Data are normalized to (RpL32) expression; the bars report mean ± standard deviation for 2 biological replicates. Expression was compared using a two-tailed t-test. Comparison of survival curves was completed using a Cox proportional-hazards model for panels a and d.

Source data

Extended Data Fig. 9 mdg4 products promote the translocation of Relish-N into nucleus.

a, By performing immuno-staining with the Relish-N antibody, which can detect both full-length and N-terminal fragment of Relish, very low–if any–signals were detected in midgut and anterior part of hindgut from sh-white, sh-mdg4, or sh-relish early pupae. b, RT-PCR to examine the levels of mdg4 full-length and Env transcripts in fat body cells. Two independent biological replicates were performed for a and b. c, Immuno-staining to detect the nuclear Relish-N signals in the fat body cells from sh-white and sh-mdg4 early pupae. While the animals for Fig. 7 were raised in germ-free condition, the pupae examined for this figure were non-germ-free. In both conditions, silencing mdg4 resulted in a significant decrease of the nuclear Relish-N in fat body cells. Box plots report the minimum, maximum, median, and interquartile ranges of the data. A two-tailed t-test was used to compare the relative intensities of each genotype. The data were collected from 3 individual animals per genotype.

Source data

Extended Data Fig. 10 A model to depict the activity of transposon during animal development and how its activation prepares the host for antiviral responses.

Our data indicate that the mdg4 retrotransposon selectively becomes active during metamorphosis to prepare the antiviral responses.

Supplementary information

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Wang, L., Tracy, L., Su, W. et al. Retrotransposon activation during Drosophila metamorphosis conditions adult antiviral responses. Nat Genet 54, 1933–1945 (2022). https://doi.org/10.1038/s41588-022-01214-9

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