Human Respiratory Syncytial Virus NS 1 Targets TRIM25 to Suppress RIG-I Ubiquitination and Subsequent RIG-I-Mediated Antiviral Signaling

Abstract

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling.

Keywords: Interferon; Nonstructural Protein 1; RIG-I; Respiratory Syncytial Virus; TRIM25.

Figure 1

Suppression of RIG-I dependent interferon signaling by RSV NS1. (A) HEK293T cells were transfected with RIG-I-2Cards (RIG-IN; 10 ng/well) together with increasing amount of RSV NS1 (63, 125, 250 ng/well) as indicated together with Interferon (IFN)-β promoter firefly luciferase and Thymidine kinase (TK) renilla luciferase reporter plasmids. Promoter activities were determined by dual-luciferase assays. Data were presented as the mean ± SEM. * p < 0.05. (B–D): HEK293T (B) A549 (C) and HEp-2 (D) cells were transfected with RIG-IN together with increasing amounts of NS1 (63 and 250 ng/well). Total RNAs were prepared 24 h after transfection and subjected to RT-qPCR to determine mRNA levels of IFN-β and ISG15. Data were presented as the mean ± SEM. * p < 0.05. All experiments were repeated at least three times. The results show the most representative data from a single experiment conducted in triplicate.

Figure 2

Inhibition of RIG-I ubiquitination by RSV NS1. (A) HEK293T cells were transfected with vector (pEBG-GST; 7 μg/dish) or GST-RIG-I-2Cards (GST-RIG-IN; 10 μg/dish) and incubated for 24 h. Cells were infected with RSV (m.o.i. = 4) as indicated and further incubated for 24 h. Cell lysates were subjected to GST-pulldown and immunoblotting using indicated antibodies to analyze the ubiquitination of GST-RIG-IN. Ubiquitinated forms were detected using anti-ubiquitin antibody (P4D1). (B) RSV NS1 and NS2 expression plasmids were transfected to HEK293T cells together with RIG-IN and TRIM25 as indicated. Cell lysates were subjected to GST-pulldown assays. Ubiquitination of RIG-IN was analyzed by immunoblotting of GST pulldown samples and whole cell lysates (WCL). (C) RIG-IN and TRIM25 were transfected with increasing amounts of RSV NS1. RIG-IN ubiquitination was analyzed as in (B). Ubiquitination of RIG-I was quantitatively analyzed by densitometry and normalized to pulled-down GST-RIG-IN (unubiquitinated form) of each sample. All experiments were conducted at least three times with similar results.

Figure 3

Interaction between RSV NS1 and TRIM25. (A) To test whether RSV NS proteins interact with TRIM25, V5-TRIM25 was expressed with RSV NS1-FLAG or NS2-HA in HEK293T. Whole cell lysates (WCL) were subjected to co-immunoprecipitation (co-IP) and immunoblotting using indicated antibodies. (B) FLAG-tagged RSV NS1 was overexpressed in HEK293T. Cell lysates were immunoprecipitated with anti-FLAG antibody and analyzed by immunoblotting. (C) HEK293T cells were transfected with FLAG-tagged NS1 and V5-tagged TRIM25 expression plasmids. Localization of NS1 and TRIM25 were visualized by primary antibodies and FITC- and PE-labeled secondary antibodies as described in Material and Methods. The lower panel shows the intensity of NS1 (green) and TRIM25 (red) along the white line of the upper panel images. (D) TRIM25 truncated mutants including V5-tagged RING, B-box/CCD and SPRY domain constructs were expressed in HEK293T cells together with RSV NS1. Co-IP was performed followed by immunoblotting. The arrowheads indicate the bands with correct size of each construct. (E) Wild type TRIM25 or SPRY deletion (ΔSPRY) mutant plasmid was transfected with RSV NS1-FLAG followed into HEK293T cells. Interaction was analyzed by co-IP and immunoblotting as indicated. All experiments were conducted at least three times with similar results.

Figure 4

Inhibition of RIG-IN interaction with MAVS-CARD-PRD by RSV NS1. (A) GST-RIG-IN or GST vector was transfected to HEK293T cells together with MAVS-CARD-PRD-FLAG plasmid as indicated. After 24h, cells were infected with RSV (moi=4) as indicated and further incubated for 24 h. Cell lysates were subjected to GST-pulldown and immunoblotting using indicated antibodies to analyze the ubiquitination of GST-RIG-IN. (B) MAVS-CARD-PRD-FLAG, RSV NS1-V5 and GST-RIG-IN or GST vector plasmids were transfected into HEK293T cells as indicated. The lysates (WCL) were analyzed by co-IP and immunoblotting. Ubiquitinated forms were detected using anti-ubiquitin antibody (P4D1). (C) Densitometric analysis of the precipitated MAVS-CARD-PRD-FLAG. The levels of co-precipitated MAVS-CARD-PRD-FLAG were analyzed by densitometry using Vision-Capt software and normalized to the levels of pulled-down GST-RIG-IN. (D) HA- and V5-tagged TRIM25 (10 μg/Φ10 cm dish) with or without FLAG-tagged NS1 (0, 4 or 8 μg/Φ10 cm dish) were transfected into HEK293T cells as indicated. Interaction between HA-TRIM25 and V5-TRIM25 was analyzed by co-IP and immunoblotting using indicated antibodies. All experiments were conducted at least three times with similar results.

Figure 5

Ablation of interferon-β promoter suppressive effect of NS1 by ectopic expression of TRIM25. (A,B) RSV NS1 was expressed with increasing amount of TRIM25 in HEK293T cells. To induce interferon-β promotor activity, low molecular weight polyI:C (A) or RIG-IN (B) were transfected at the same time as indicated. Interferon (IFN)-β promoter firefly luciferase and Thymidine kinase (TK) renilla luciferase reporter plasmids were co-transfected. Interferon-promoter activities were analyzed by luciferase assays. Data were presented as the mean ± SEM. * p < 0.05. The experiments were repeated at least three times. The results show the most representative data from a single experiment conducted in triplicate.