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, 14 (1), e1006877
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A New Mechanism of Interferon's Antiviral Action: Induction of Autophagy, Essential for Paramyxovirus Replication, Is Inhibited by the Interferon Stimulated Gene, TDRD7

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A New Mechanism of Interferon's Antiviral Action: Induction of Autophagy, Essential for Paramyxovirus Replication, Is Inhibited by the Interferon Stimulated Gene, TDRD7

Gayatri Subramanian et al. PLoS Pathog.

Abstract

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Setting up of high throughput genetic screen of human ISG shRNA library to identify anti-SeV ISGs.
(A) HeLa cells stably expressing shRNA against IRF9 or a non-targeting (NT) control were pre-treated with human IFN-β for 16 h, when the cells were infected with SeV (moi:10). SeV C protein expression was analyzed by immunoblot at 16 hpi. (B) HeLa cells stably expressing shRNA against IRF9 or NT, were pre-treated with IFN-β for 16 h, when the cells were infected with SeV; 16 h later flow cytometric analyses were performed after immunostaining the cells with anti-SeV antibody. (C) A strategy to quantify percent SeV infectivity using flow cytometric procedure, as described in (B). The numbers indicate each quadrant and their respective cell population. (D) Percent SeV infectivity in NT or IRF9 shRNA-expressing HeLa, infected with SeV in the absence or the presence of IFN-β pre-treatment. (E) Our strategy to screen the human ISG shRNA library to isolate anti-SeV ISGs.
Fig 2
Fig 2. Secondary validation of the primary screen results to identify the most effective anti-SeV ISGs.
(A) The z-scores of all the ISG shRNAs based on their anti-SeV activities; all z-scores were normalized to that of shIRF9. (B) The z-scores of the top 25 primary hits that were used for secondary validation. (C) HeLa cells, stably expressing the shRNAs against specific ISGs (indicated above by the numbers, 1–25) were treated with IFN-β for 16 h, when the cells were infected with SeV and analyzed for SeV C protein by immunoblot at 16 hpi. (D) The secondary validated anti-SeV ISGs, their z-scores and known functions.
Fig 3
Fig 3. TDRD7 inhibits SeV replication in human and mouse cells.
(A) QRT-PCR analyses of Tdrd7 induction by various treatments or SeV infection in mouse macrophages (RAW264.7). Treatments: IFN-β: 1000 U/ml, SeV (moi:10), polyI:C+LF: poly(I:C) transfected with Lipofectamine 2000 (LF) or naked poly(I:C). (B) QRT-PCR analyses of Tdrd7 mRNA levels in the lungs of mock-infected (NT, PBS-treated) or SeV (52 strain) infected mice after 2 days of infection. (C) LA4 cells stably expressing two different Tdrd7 shRNAs (#1 and #2), were infected with SeV for the indicated time, when the SeV C protein levels were analyzed by immunoblot. (D) ARPE19 cells stably expressing TDRD7-specific shRNA, were analyzed for SeV C protein expression after SeV infection. Lower panel shows the TDRD7 expression by immunoblot. (E) HT1080 (Wt) or TDRD7 knockout (TDRD7-/-) cells were infected with SeV and analyzed for SeV C protein expression by immunoblot (upper panel). TDRD7 protein expression is shown in the lower panel by immunoblot. (F) HEK293T cells, stably expressing V5.TDRD7 (lower panel), were analyzed for SeV C expression (upper panel) by immunoblot after SeV infection. TDRD7 protein expression is shown in the lower panel by immunoblot. (G) L929 cells stably expressing Tdrd7, were analyzed for infectious SeV production at the indicated time post SeV infection. NT, non-targeting, EV, empty vector, * indicates p<0.05. The results presented here are representatives of at least three biological repeats.
Fig 4
Fig 4. SeV triggers various stages of autophagy to support virus replication.
(A) Various stages and molecular markers of cellular autophagy. (B-D) L929 cells were infected with SeV Cantell (moi: 10) for the indicated time, when the cell lysates were analyzed for p62, SeV C (B), LC3 (C), phospho ULK1 (Ser757 or Ser317) (D) by immunoblot. (E) L929 cells were pre-treated with 3-MA (1 mM) or rapamycin (2 μM) and infected with SeV Cantell (moi:10); SeV P mRNA levels were analyzed by qRT-PCR at the indicated time post infection. (F) HT1080 cells, stably expressing ATG5-specific shRNA, were infected with SeV for the indicated time, when SeV C (upper panel) or p62 (middle panel) were analyzed by immunoblot. Lower panel shows the ATG5 levels in these cells by immunoblot. NT, non-targeting, * indicates p<0.05. The results presented here are representatives of at least three biological repeats.
Fig 5
Fig 5. TDRD7 inhibits autophagy induced by viral and non-viral stimuli, to control SeV replication.
(A, B) L929 cells, stably expressing V5.Tdrd7, were infected with SeV Cantell (moi:10), and analyzed for p62 (A) and LC3 (B) by immunoblot. LC3-II/Actin levels were quantified by Image J. (C) TDRD7-/- human cells expressing ATG5-specific shRNA were left untreated or treated with hIFN-β for 16 h, when the cells were infected with SeV and SeV C protein expression was analyzed by immunoblot. ATG5 protein expression is shown in lower panel by immunoblot. (D) HEK293T cells stably expressing V5.TDRD7 were serum-starved (SS) and LC3 levels were analyzed after 16 h by immunoblot. LC3-II/Actin ratio are indicated below the Actin panel. (E) L929 cells stably expressing V5.Tdrd7 were serum-starved (SS) for the indicated time, when p62 levels were analyzed by immunoblot. (F) L929 cells stably expressing V5.Tdrd7 were transfected with GFP-LC3 and serum-starved (SS) for 8 h, when the cells were fixed and analyzed by confocal microscopy. The cytoplasmic puncta structures are shown by arrows. (G) L929 cells stably expressing V5.Tdrd7 were treated with Rapamycin (Rapa) for the indicated time, when LC3-II levels were analyzed by immunoblot. NT, non-targeting, EV, empty vector, * indicates p<0.05. The results presented here are representatives of at least three biological repeats.
Fig 6
Fig 6. TDRD7 inhibits autophagy-inducing kinase AMPK, whose activity is required for SeV replication.
(A) L929 cells, transfected with Flag.VPS34 and V5.Tdrd7, were infected with SeV for the indicated time, when VPS34 was immunoprecipitated from the cell lysates and PI3K III activity was analyzed as described in Materials and Methods. (B) L929 cells stably expressing V5.Tdrd7 were transfected with GFP.p40.phox and then serum-starved (SS) for 8 h, when the cells were fixed and analyzed by confocal microscopy. The GFP.p40.phox puncta were counted from at least 100 cells and the results are presented on the right panel. (C) L929 cells stably expressing V5.Tdrd7 were serum-starved for 8 h, when pULK1 (Ser317) was analyzed by immunoblot. (D) L929 cells stably expressing V5.Tdrd7 were serum-starved for 8h, when pULK1 (Ser757) was analyzed by immunoblot. (E) HeLa cells expressing AMPK shRNA were infected with SeV and viral protein (SeV C) expression was analyzed by immunoblot at 16 hpi. Lower panel indicates the AMPK levels in these cells. (F) L929 cells ectopically expressing HA-AMPK (lower panel) were infected with SeV and viral protein expression was analyzed at 16 hpi by immunoblot. (G) L929 cells were pre-treated with various concentrations of Compound C for 1h, and then infected with SeV. Viral protein (SeV C) expression and pAMPK (on Thr172) and AMPK levels were analyzed at 16 hpi by immunoblot. (H) HeLa cells were pre-treated with various concentrations of Compound C for 1h, and then infected with SeV. Viral protein (SeV C) expression and pAMPK (on Thr172) and AMPK levels were analyzed at 16 hpi by immunoblot. (I) L929 cells stably expressing V5.Tdrd7 were serum-starved (SS) for the indicated time, when pAMPK (Thr172) levels were analyzed by immunoblot. (J) L929 cells stably expressing V5.Tdrd7 were infected with SeV for the indicated time, when pAMPK (Thr172) levels were analyzed by immunoblot. EV, empty vector, NT, no treatment, * indicates p<0.05. The results presented here are representatives of at least three biological repeats.
Fig 7
Fig 7. TDRD7 inhibits HPIV3 and RSV replication by anti-AMPK activity.
(A) HeLa cells were infected with rgHPIV3 (moi:1) and analyzed for LC3 and p62 by immunoblot. (B, C) HT1080 cells stably expressing shRNA against ATG5 were infected with rgHPIV3 (moi:1); GFP at 24 hpi (B) and viral HN protein (C) expression were analyzed at the indicated time. (D, E) HeLa cells expressing V5.TDRD7 were infected with rgHPIV3 (moi:1), GFP (D) and viral HN protein (E) expression were analyzed at 24 hpi. IFN-β pre-treatment was used as a positive control. (F) HeLa cells expressing V5.TDRD7 were infected with RSV (moi:1) and viral protein expression was analyzed at 48 hpi. Arrows indicate the polyclonal serum detecting various viral proteins. (G) HeLa cells expressing AMPK shRNA were infected with rgHPIV3 (moi:1) and viral protein (HN) expression was analyzed by immunoblot at 24 hpi. Lower panel indicates the AMPK levels in these cells. (H) HeLa cells expressing AMPK shRNA were infected with rgHPIV3 (moi:1) and GFP expression was analyzed by fluorescence microscopy at 24 hpi. (I) HeLa cells expressing AMPK shRNA were infected with rrRSV and virus-encoded red fluorescent protein expression was analyzed by fluorescence microscopy at 24 hpi. (J) HeLa cells were pre-treated with various concentrations of Compound C for 1h, and then infected with rgHPIV3. Viral protein (HN) and pAMPK (Thr172) levels were analyzed at 24 hpi by immunoblot. (K, L) HeLa cells were pre-treated with Compound C (CC, 10 μM) for 1h, and then infected with rgHPIV3 (K) or rrRSV (L). Infectious virus particle release in the culture supernatants was analyzed by fluorescence focus assay (expressed in ffu/ml). NT, non-targeting, EV, empty vector, * indicates p<0.05. The results presented here are representatives of at least three biological repeats.
Fig 8
Fig 8. A newly identified antiviral ISG, TDRD7 inhibits paramyxovirus-induced autophagy to control virus replication.
The model shows a new mode of action of an ISG, IFN-induced TDRD7 to control paramyxovirus replication by inhibiting cellular autophagy pathway. Paramyxoviruses trigger cellular autophagy by activating the autophagy-inducing kinase, AMPK, by phosphorylation on Thr172. AMPK directly phosphorylates ULK1 on Ser317 to activate autophagy pathway. Activated AMPK also inhibits mTOR, which phosphorylates ULK1 on Ser757 to inhibit autophagy. The newly identified antiviral ISG, TDRD7 inhibits virus-induced autophagy by inhibiting the activation of AMPK, to suppress paramyxovirus replication. Autophagy, induced by nutrient starvation or rapamycin, is also inhibited by TDRD7. Therefore, autophagy inhibition is a new mechanism of the IFN system to control virus replication.

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