The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination

Abstract

Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein.IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response.

Keywords: RIG-I; SARS coronavirus; TRIM25; interferon; nucleocapsid.

FIG 1

SARS-CoV N protein interacts with TRIM25. (A) Cell extracts prepared from 293T cells transfected with Flag-SARS N protein (Flag-N) or Flag vector were subjected to anti-Flag immunoprecipitation. The immunoprecipitates were resolved using SDS-PAGE electrophoresis and Coomassie blue staining. (B) The stained bands marked by arrows in Fig. 1A were digested with trypsin and analyzed by LC-MS/MS. Two peptides (SDLGAVAKGLSGELGTR and EPEELGK) matching the TRIM25 protein sequence were identified. (C and D) 293T cells transfected with the indicated plasmids were infected with or without SeV (C), and the cell lysates were subjected to anti-Flag immunoprecipitation (IP). The immunoprecipitates were analyzed by immunoblotting (IB) with anti-TRIM25 (C) or anti-Myc (D). (E) HeLa cells transfected with GFP-N were fixed with 4% paraformaldehyde and incubated with anti-TRIM25 and anti-GFP antibodies. An in situ PLA assay was conducted as described and imaged with a confocal microscope at ×100 magnification. Red dots indicate the interaction.

FIG 2

The interaction domain of the SARS-CoV N protein and TRIM25. (A) Anti-Flag immunoprecipitates prepared from lysates of 293T cells expressing Flag-TRIM25 and GFP-tagged full-length or truncated SARS CoV N protein were analyzed by immunoblotting with anti-Flag and anti-GFP. (B) Anti-Flag immunoprecipitates prepared from lysates of 293T cells expressing Myc-N protein and the Flag-B box/CCD or Flag-SPRY domain were analyzed by immunoblotting with anti-Flag and anti-Myc. (C) Anti-Flag immunoprecipitates prepared from lysates of 293T cells expressing the GFP-N or Flag-tagged TRIM25 domain were analyzed by immunoblotting with anti-Flag and anti-GFP.

FIG 3

The interaction between TRIM25 and RIG-I is inhibited by the SARS-CoV N protein. (A) 293T cells were transfected with Flag–RIG-I and Myc-TRIM25 with or without Myc-N. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Flag or anti-Myc. (B) 293T cells were transfected with Flag-TRIM25, Myc-RIG-I, and the indicated amount of GFP-N. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Flag, anti-Myc, or anti-GFP. (C) HeLa cells expressing the GFP-N or GFP plasmid were fixed with 4% paraformaldehyde and incubated with anti-TRIM25 and anti-RIG-I antibodies. The in situ PLA assay was conducted as described in the text, and the results were imaged with a confocal microscope at magnification of ×100. Red dots indicate the interaction. (D) Red dots indicating positive PLA signals were counted in 30 randomly selected cells. The data are expressed as the means ± SD (*, P < 0.05).

FIG 4

TRIM25-mediated RIG-I ubiquitination is suppressed by the SARS-CoV N protein. (A, B, and D) 293T cells transfected with the indicated plasmids for 36 h were infected with or without SeV at an MOI of 2 for 12 h. The anti-Flag immunoprecipitates prepared from the cell extracts were analyzed by immunoblotting with the indicated antibodies. (C) 293T cells were transfected with the vector, Flag-2CARD, or Myc-N. At 24 h posttransfection, whole-cell lysates were subjected to immunoblotting with anti-IRF3 and anti-p-IRF3(S396) antibodies.

FIG 5

The SARS-CoV N protein inhibits TRIM25-mediated RIG-I activation and interferon production. (A, B, C, and F) 293T cells were cotransfected with the IFN-β-Luc firefly luciferase reporter plasmid, the Renilla luciferase control reporter plasmid pRL and the indicated plasmids for 36 h. The transfected cells were infected with or without SeV at an MOI of 2 for 12 h (C and F). The luciferase activity of the cell lysates was analyzed with the dual luciferase reporter assay system (Promega) and was measured on a Monolight 2010 luminometer. (D and E) A549 cells transfected with Myc-N and Flag-TRIM25 were infected with or without SeV at an MOI of 2 for 36 h. The mRNA was extracted, and the IFN-β mRNA expression level was determined by RT-PCR (D). The IFN-β concentrations in the cell culture supernatants were measured using a VeriKine human IFN-β enzyme-linked immunosorbent assay (ELISA) kit (E). (G) 293T cells cotransfected with the IRSE-Luc reporter plasmid, pRL, and the other indicated plasmids were infected with or without SeV at an MOI of 2 for 24 h. The luciferase activity of the cell lysates was analyzed with the dual luciferase reporter assay system and was measured using a Monolight 2010 luminometer. (H to J) Total RNA was extracted from A549 cells expressing the indicated plasmids. The ISG15, ISG56, and IP10 mRNA expression levels were determined by RT-PCR. All results in panels A to I are expressed as the means ± SD from three independent experiments (ns, nonsignificant; *, P < 0.05; **, P < 0.01, ***, P < 0.001). (K to M) A549 cells were transfected with 0.5 μg of Myc-vector (left and middle) or Myc-N (right) for 24 h. The cells were infected with SeV at an MOI of 2 for 16 h (middle and right) or not infected (left). Cell culture supernatants were harvested and incubated with Vero E6 cells for 2 h prior to infection of the Vero E6 cells with NDV-GFP at an MOI of 2. After 24 h, the GFP fluorescence of the Vero E6 cells was detected by flow cytometry. The cell culture supernatants were harvested and incubated with Vero E6 cells for 2 h prior to infection of the Vero E6 cells with NDV-GFP at an MOI of 2. The rate of NDV infection of the Vero E6 cells was measured by flow cytometry after 24 h of infection.

FIG 6

N protein promotes virus replication by inhibiting TRIM25-mediated IFN-β production. (A) A549 cells were transfected with TRIM25 siRNA or scramble siRNA. At 24 h posttransfection, the cells were infected with recombinant SARS-CoV at an MOI of 0.05 PFU per cell for 24 h. The virus particles released into the cell culture supernatants were determined by viral genomic RNA level as assayed by qPCR. The TRIM25 mRNA expression level was determined by RT-PCR (right). (B) A549 cells transfected with Flag-TRIM25 or Flag vector were infected with recombinant SARS-CoV as described for panel A. The virus particles released into the cell culture supernatants were quantified by viral genomic RNA level as assayed by qPCR. (C) A549 cells transfected with GFP vector, GFP-N, or GFP-N(1–361) were infected with SARS-CoV as described for panel A. The virus particles released into the cell culture supernatants were quantified by viral genomic RNA level as assayed by qPCR. (D) Calu-3 cells were transfected with full-length N protein or N(1–361), and 24 h after transfection, the cells were infected with mouse-adapted SARS-CoV at an MOI of 0.05 PFU per cell in the presence or absence of IFN-β-specific neutralizing antibody. Viral RNA from viral particles in the supernatant was quantified by qPCR. The results are expressed as the means ± SD from three independent experiments (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). The significance of results for cells not treated with the antibody from panel D was analyzed using the t test.

FIG 7

The MERS-CoV N protein interacts with TRIM25 and inhibits RIG-I interferon signaling. (A and B) 293T cells were transfected with 2 μg of the indicated plasmids for 36 h. Whole-cell lysates were subjected to anti-Flag immunoprecipitation and immunoblotting with anti-Flag or anti-GFP antibodies. (C and D) 293T cells were cotransfected with the IFN-β-Luc firefly luciferase reporter plasmid, the Renilla luciferase control reporter plasmid pRL, Flag-2CARD, and increasing amounts of the GFP-MERS-N plasmid (C) or GFP-MERS-N (aa 1 to 170) or GFP-MERS-N (aa 171 to 413) (D) for 36 h. The luciferase activity of the cell lysates was analyzed with the dual luciferase reporter assay system (Promega) and was measured with a Monolight 2010 luminometer. The results are expressed as the means ± SD from three independent experiments.

FIG 8

Schematic of N protein inhibiting type I interferon production by interfering with TRIM25-mediated RIG-I ubiquitination.