A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation

Abstract

RNA degradation is tightly regulated to selectively target aberrant RNAs, including viral RNA, but this regulation is incompletely understood. Through RNAi screening in Drosophila cells, we identified the 3'-to-5' RNA exosome and two components of the exosome cofactor TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex, dMtr4 and dZcchc7, as antiviral against a panel of RNA viruses. We extended our studies to human orthologs and found that the exosome as well as TRAMP components hMTR4 and hZCCHC7 are antiviral. While hMTR4 and hZCCHC7 are normally nuclear, infection by cytoplasmic RNA viruses induces their export, forming a cytoplasmic complex that specifically recognizes and induces degradation of viral mRNAs. Furthermore, the 3' untranslated region (UTR) of bunyaviral mRNA is sufficient to confer virus-induced exosomal degradation. Altogether, our results reveal that signals from viral infection repurpose TRAMP components to a cytoplasmic surveillance role where they selectively engage viral RNAs for degradation to restrict a broad range of viruses.

Keywords: RNA degradation; TRAMP; antiviral; arbovirus; exosome; intrinsic immunity.

Figure 1.

The RNA exosome is broadly antiviral in Drosophila cells. A panel of 177 genes with roles in RNA biology was depleted by RNAi in DL1 cells for 3 d and infected with VSV-GFP (A) (multiplicity of infection [MOI] = 0.1, 24 h) or SINV-GFP (B) (MOI = 2.5, 36 h) and screened by immunofluorescence measuring the percentage of infected cells. Robust Z-scores are shown for two replicates. These screens identified the positive control dArs2 (red) and the exosome component dRrp6 (green). (C) DL1 cells were treated with dsRNAs targeting the indicated genes or negative control dsRNA targeting β-galactosidase; infected with VSV-GFP (MOI = 0.1, 24 h), SINV-GFP (MOI = 2.5, 36 h), or RVFV (MOI = 0.1, 30 h); and subsequently processed for automated immunofluorescence microscopy for GFP or RVFV nucleocapsid (N). Representative images are shown with quantification of the percentage of infected cells. (D) Mean ± SEM of at least three experiments as shown in B, normalized to control. Mean percent infection in control cells was 5.40% (VSV), 3.80% (SINV), and 5.62% (RVFV). (*) P < 0.05 compared with control by Student's t-test. (E) Cells were infected as above and processed for RT-qPCR for VSV N, SINV Nsp1, or RVFV N compared with the housekeeping gene Rp49. Mean ± SEM normalized to control is shown. n ≥ 3. (*) P < 0.05, compared with control by Student's t-test.

Figure 2.

TRAMP orthologs dMtr4 and dZcchc7 are antiviral in Drosophila. (A) DL1 cells were treated with dsRNAs targeting the indicated genes or negative control dsRNA targeting β-galactosidase; infected with VSV-GFP (MOI = 0.1, 24 h), SINV-GFP (MOI = 2.5, 36 h), or RVFV (MOI = 0.1, 30 h); and subsequently processed for automated immunofluorescence microscopy for GFP or RVFV N. Representative images are shown with quantification of the percentage of infected cells. (B) Mean ± SEM of at least three experiments as shown in A, normalized to control. (*) P < 0.05, compared with control by Student's t-test. (C) Cells were infected as above and processed for RT-qPCR for VSV N, SINV Nsp1, or RVFV N compared with the housekeeping gene Rp49. Mean ± SEM normalized to control is shown. n ≥ 3. (*) P < 0.05, compared with control by Student's t-test. (D) Adult flies depleted of exosome or TRAMP genes in the fat body (YP1-Gal4>IR) or controls (YP1-Gal4>+) were challenged with RVFV for 6 d and then processed for Northern blot. A probe that identified the S segment genome/anti-genome and the N mRNA was used. The housekeeping gene RpS6 was used as a loading control. (E) Quantification of RVFV N mRNA from three or more experiments as shown in D. Mean ± SEM normalized to control. (*) P < 0.05, compared with control by Student's t-test.

Figure 3.

The RNA exosome and TRAMP orthologs are antiviral in human cells. (A) U2OS cells were transfected with the indicated siRNAs; infected with VSV-GFP (MOI = 0.05, 14 h), SINV-GFP (MOI = 1, 16 h), or RVFV (MOI = 0.03, 18 h); and subsequently processed for RT-qPCR for VSV N, SINV Nsp1, or RVFV N compared with the housekeeping gene GAPDH. Mean ± SEM is shown normalized to control. n ≥ 3. (*) P < 0.05, compared with control by Student's t-test. (B) Cells were transfected with the indicated siRNAs, infected with VSV-GFP (MOI = 0.05, 14 h), and then processed for GFP immunoblot. A representative blot is shown. n ≥ 3. (C) Cells were transfected with the indicated siRNAs, infected with SINV-GFP (MOI = 1, 8 h), and then processed for GFP immunoblot. A representative blot is shown. n ≥ 3. (D) Cells were transfected with the indicated siRNAs, infected with RVFV (MOI = 0.3, 18 h), and then processed for RVFV Gn glycoprotein immunoblot. A representative blot is shown. n ≥ 3.

Figure 4.

Human MTR4 and ZCCHC7 are exported to the cytoplasm upon viral infection. (A) U2OS cells were infected with RVFV (MOI = 10, 12 h) or mock-infected and processed for immunofluorescence microscopy for hZCCHC7 (green), RVFV N (red), and nuclei (blue). (B) Quantification of the percentage of cells with cytoplasmic hZCCHC7 punctae in mock- or RVFV-infected cells in at least three experiments as in A. Mean ± SEM is shown. (*) P < 0.05, compared with mock by Student's t-test. (C) U2OS cells were infected with SINV-mKate (MOI = 10, 5 h) or mock-infected and processed for immunofluorescence microscopy for hZCCHC7 (green), mKate (red), and nuclei (blue). (D) Quantification of the percentage of cells with cytoplasmic hZCCHC7 punctae in mock- or SINV-infected cells in at least three experiments as in C. Mean ± SEM is shown. (*) P < 0.05, compared with mock by Student's t-test. (E) U2OS cells were infected with SINV-GFP or VSV-GFP (MOI = 10, 8 h), subjected to nuclear/cytoplasmic fractionation and immunoblot, and probed for the nuclear protein lamin and the cytoplasmic protein tubulin to verify extract purity along with hMTR4 and hZCCHC7. A representative blot is shown. n ≥ 3. (F) U2OS cells were infected with RVFV (MOI = 10, 12 h), SINV-GFP (MOI = 10, 8 h), or VSV-GFP (MOI = 10, 8 h); subjected to nuclear/cytoplasmic fractionation and immunoblot; and probed for the nuclear protein lamin and the cytoplasmic protein tubulin to verify extract purity along with hZCCHC7 and hZCCHC8. A representative blot is shown. n ≥ 3. (G) U2OS cells were infected with VSV-GFP (MOI = 10, 8 h), SINV-GFP (MOI = 10, 8 h), or RVFV (MOI = 10, 12 h), and whole-cell lysates were processed for immunoblot. A representative image is shown. n = 2. (H) U2OS cells were transfected with siRNA specific to CRM1 or control, infected with RVFV (MOI = 10, 12 h), subjected to nuclear/cytoplasmic fractionation and immunoblot, and probed for the nuclear protein Lamin and the cytoplasmic protein tubulin to verify extract purity along with hMTR4 and hZCCHC7. A representative image is shown. n ≥ 3.

Figure 5.

Human MTR4 and ZCCHC7 form a cytoplasmic complex with the exosome upon infection. U2OS cells were transfected with an hMTR4-Flag expression vector or empty vector and infected with RVFV (MOI = 10, 12 h) or mock-infected, and then either whole-cell lysates (A) or cytoplasmic fractions (B) were processed for coimmunoprecipitation. Four percent input was loaded for the hMTR4 immunoblot, and 8% input was loaded for other proteins. Actin was used as a loading control. Representative blots are shown. n ≥ 3.

Figure 6.

Viral mRNA is bound by hZCCHC7. (A,B) U2OS cells were transfected with an hZCCHC7-Flag expression vector or empty vector, infected with RVFV (A) (MOI = 10, 12 h) or SINV-GFP (B) (MOI = 10, 8 h), and then fractionated. Cytoplasmic extracts were collected (input), and a fraction was subjected to Flag immunoprecipitation and processed for RT-qPCR. RNA quantification was normalized to vector control input or Flag immunoprecipitation. Fold change in hZCCHC7-bound RNA normalized to vector-bound RNA is presented. hDCP2 was used as an endogenous mRNA control, as it is not known to be regulated during RVFV or SINV infection. Mean ± SEM shown. (*) P < 0.05 by Student's t-test.

Figure 7.

The RNA exosome and hZCCHC7 target viral mRNAs for decay. (A) U2OS cells were infected with RVFV (MOI = 0.3, 18 h), and 3′ RACE was performed using primers that detect the RVFV small segment RNAs as indicated. Sequenced reads were aligned to RVFV, classified as full length or slightly shortened (<5 nucleotides), and plotted. Pooled data from three biological replicates are shown. (B) Pie chart of N mRNA reads from A that are full length (blue), encode a full-length ORF but truncated 3′ UTR (red), or encode a truncated ORF are shown (green). (C) U2OS cells were transfected with the indicated siRNAs and infected with RVFV (MOI = 1, 12 h). Infected cells were treated with 50 µg/mL cycloheximide and processed for RT-qPCR at the indicated time points. Mean ± SEM is shown normalized to hDCP2. n ≥ 3. (*) P < 0.05, compared with control by Student's t-test. (D) U2OS cells stably expressing cGFP reporters with the indicated 3′ UTRs were transfected with siRNAs targeting hRRP6 and hDIS3 or control and either uninfected or infected with RVFV (MOI = 10, 18 h). Cells were subsequently processed for automated immunofluorescence microscopy. Mean ± SEM is shown normalized to mock-infected control. n ≥ 3. (*) P < 0.05, compared with control by Student's t-test.