Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila

Abstract

Innate immune responses are characterized by precise gene expression whereby gene subsets are temporally induced to limit infection, although the mechanisms involved are incompletely understood. We show that antiviral immunity in Drosophila requires the transcriptional pausing pathway, including negative elongation factor (NELF) that pauses RNA polymerase II (Pol II) and positive elongation factor b (P-TEFb), which releases paused Pol II to produce full-length transcripts. We identify a set of genes that is rapidly transcribed upon arbovirus infection, including components of antiviral pathways (RNA silencing, autophagy, JAK/STAT, Toll, and Imd) and various Toll receptors. Many of these genes require P-TEFb for expression and exhibit pausing-associated chromatin features. Furthermore, transcriptional pausing is critical for antiviral immunity in insects because NELF and P-TEFb are required to restrict viral replication in adult flies and vector mosquito cells. Thus, transcriptional pausing primes virally induced genes to facilitate rapid gene induction and robust antiviral responses.

Figure 1. NELF restricts viral infection in Drosophila cells

(A) Drosophila cells were treated with dsRNA against a control (Bgal), NELF-B, or NELF-D. Infected cells expressing a VSV-encoded GFP (MOI=0.2) or SINV- encoded GFP (MOI=5) are shown in green, nuclei in blue. (B) Quantification of images in (A) as normalized to controls. Mean ± S.D. of three independent experiments shown; *p < 0.05. Northern blot analysis of cells pretreated with the indicated dsRNAs and infected with (C) VSV or (D) SINV. (E) S2 and (F) Kc167 cells were treated as in (A). Quantification of images as percentage of infection, normalized to controls. Mean ± S.D. of three independent experiments shown; *p < 0.05. See also Figure S1.

Figure 2. Transcriptional pausing and release restricts viral infections in Drosophila cells

(A) Schematic of transcriptional pausing and release. (B) Drosophila cells treated with the indicated dsRNAs were challenged with VSV (MOI=0.2) or SINV (MOI=5) and monitored by fluorescence microscopy. (C) Quantification of images in (A). Mean ± S.D. of three independent experiments shown; *p < 0.05. Northern blot analysis of cells pretreated with the indicated dsRNAs and infected with (D) VSV or (E) SINV. See also Figure S2.

Figure 3. An antiviral transcriptional program is rapidly induced by viral infection

(A) Drosophila cells were infected with VSV (MOI=10) or SINV (MOI=25) for 4 hours. RT-qPCR was performed for Tep II and PGRP-SA, normalized to Rp49, and shown compared to uninfected controls. Mean ± S.D. of three independent experiments is shown; *p < 0.005. (B) Heat map of raw signal levels for genes differentially expressed at 4 hours post VSV-infection (MOI=10), performed in biological duplicates (q<0.005). Shown are 636 transcripts (540 upregulated, 96 downregulated) with at least 2.8-fold change in VSV-infected cells. Genes of interest are shown on the right. (C) Enriched GO terms for the 540 virally induced genes (p<0.05). See also Tables S1A-D.

Figure 4. Viral infection triggers a rapid and transcriptionally complex antiviral expression program

Drosophila cells were infected with (A) VSV (MOI=10) or (B) SINV (MOI=25). RT-qPCR was performed for the indicated genes at 4 h.p.i., normalized to Rp49, and shown relative to uninfected controls. Mean ± S.D. of three independent experiments is shown; **p < 0.005, *p<0.05. (C-D) Drosophila cells were untreated or pre-treated with 10 μg/ml Cycloheximide (CHX) and infected as in (A, B). RT-qPCR was performed for the indicated genes. Mean ± S.D. of three independent experiments is shown; *p<0.005.

Figure 5. A subset of virally induced genes is NELF and P-TEFb-dependent

(A-D) Drosophila cells were treated with the indicated dsRNAs and infected with VSV (MOI=10) or SINV (MOI=25). RT-qPCR of the indicated genes at 4 h.p.i., normalized to Rp49, and shown as relative to uninfected controls. Mean ± S.D. of three independent experiments is shown; **p< 0.005, *p<0.05. (E) Cells were treated with PGN for 6 hours. AttB expression was measured and normalized as stated above. Mean ± S.D. of three independent experiments is shown; *p<0.005. (F) Analysis of Cdk9-dependency of the 540 VSV-induced genes (279 genes, ≥1.5 fold down-regulation). Black and gray indicate the percentage of Cdk9-dependent and independent genes, respectively. See also Figure S3.

Figure 6. Virally induced, P-TEFb-dependent genes have chromatin features of transcriptionally paused loci

Mapping of Pol II (Rbp3), NELF (NELF-B), DSIF (Spt5), H3K4me3, and short RNA reads for (A) Tep II (B) Toll-8 (C) CG13325 and (D) Toll-5. ChIP of RNA Pol II for (E) Tep II (F) Toll-8 (G) CG13325 in cells treated with the indicated dsRNAs and either uninfected or infected with VSV (MOI=10). Primers span the promoter-proximal or downstream regions. The data is represented as a percentage of input. Mean ± SD for three independent experiments is shown; *p<0.05. (H) RT-qPCR of 5’ short reads for Tep II, Toll-8, and CG13325 with the indicated dsRNA treatment in Drosophila cells. Transcripts were normalized to Rp49 and shown as relative to controls. Mean ± S.D. of three independent experiments is shown; *p<0.05. (I) Comparison of VSV-induced, P-TEFb-dependent genes with pausing-associated chromatin features to the genome-wide distribution (*p<0.0001). See also Figure S4 and Table S2.

Figure 7. NELF and P-TEFb restrict viral infection in flies and mosquito cells

Adult flies of the indicated genotypes were challenged with (A and B) VSV or (C and D) SINV. Viral titers were measured with mean ± S.D. of three independent experiments shown; *p < 0.05. (E) Adult flies of the indicated genotypes were challenged with VSV. RT-qPCR of Tep II is shown, normalized to Rp49, and represented as relative to the uninfected mock-depleted controls. Mean ± S.D. of three independent experiments is shown; p<0.05. (F) Aedes aegypti Aag2 cells treated with the indicated dsRNAs were challenged with VSV (MOI=0.01) or SINV (MOI=0.5) and monitored by fluorescence microscopy (virus in green, nuclei in blue). (G) Quantification of images in (F) as normalized to controls. Mean ± S.D. of three independent experiments is shown; *p < 0.05. (H) Northern blot analysis of Aag2 cells pretreated with the indicated dsRNAs and infected with either VSV or SINV. See also Figure S5.