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. 2011 Feb;42(1):37-45.
doi: 10.1007/s11262-010-0544-x. Epub 2010 Oct 26.

SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway, and its C-terminal region is critical for the antagonism

Xiaolu Lu  1 , Ji'an Pan, Jiali Tao, Deyin Guo
Affiliations

SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway, and its C-terminal region is critical for the antagonism

Xiaolu Lu et al. Virus Genes. 2011 Feb.

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein which can inhibit the synthesis of type I interferon (IFN), but the molecular mechanism of this antagonism remains to be identified. In this study, we demonstrated that the N protein of SARS-CoV could inhibit IFN-beta (IFN-β) induced by poly(I:C) or Sendai virus. However, we found that N protein could not inhibit IFN-β production induced by overexpression of downstream signaling molecules of two important IFN-β induction pathways, toll-like receptor 3 (TLR3)- and RIG-I-like receptors (RLR)-dependent pathways. These results indicate that SARS-CoV N protein targets the initial step, probably the cellular PRRs (pattern recognition receptors)-RNAs-recognition step in the innate immune pathways, to suppress IFN expression responses. In addition, co-immunoprecipitation assays revealed that N protein did not interact with RIG-I or MDA5. Further, an assay using truncated mutants revealed that the C-terminal domain of N protein was critical for its antagonism of IFN induction, and the N deletion mutant impaired for RNA-binding almost completely lost the IFN-β antagonist activity. These results contribute to our further understanding of the pathogenesis of SARS-CoV.

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Figures

Fig. 1
Fig. 1
SARS-CoV N protein inhibits IFN-β production induced by poly(I:C) and SenV. 293T cells (1 × 105) were transfected with a plasmid expressing Flag-N or the empty vector and pIFN-β-luc, pRL-TK. At 24 h post-transfection, a cells were transfected with poly(I:C) or b infected with SenV. 18 h later, reporter assays were performed. c, d Reporter assays were performed similarly as in (a) and (b) except that an increasing amount of Flag-N-expressing plasmids and MDA5- or RIG-I-expressing plasmid were transfected. The data represent the mean ± S.D. of three independent experiments. * P < 0.05
Fig. 2
Fig. 2
N cannot inhibit IFN-β promoter activation induced by overexpressing signaling molecules in innate immune pathways. 293T cells (1 × 105) were co-transfected with Flag-N or empty vector, pIFN-β-luc, pRL-TK and indicated plasmid expressing RIG-I-2CARD (a), MAVS, TRIF (b), TBK1, or IKKε (c). At 24 h post-transfection, reporter assays were performed. The data represent the mean ± S.D. of three independent experiments. * P < 0.05
Fig. 3
Fig. 3
N reduces IFN-β mRNA synthesis induced by Sendai virus and poly(I:C), but not by overexpressing signaling molecules in innate immune pathways. 293T cells (1 × 105) were similarly treated as in Figs. 1 and 2 without pIFN-β-luc and pRL-TK in transfections. The mRNAs for IFN-β in transfected cells were detected by real-time PCR. The data represent the mean ± S.D. of three independent experiments. * P < 0.05
Fig. 4
Fig. 4
N inhibits the nuclear translocation of IRF-3 promoted by poly(I:C), but not by MAVS. 293T cells (1 × 105) were transfected with indicated plasmids, together with a plasmid expressing the IRF-3 and GFP fusion (GFP-IRF-3) (left panels) to follow IRF-3’s localization. At 24 h post-transfection, cells were either analyzed by microscopy (b) or tranfected with poly(I:C) for another 12 h before microscopy analysis (a). N proteins were detected with monoclonal antibodies against Flag (right panels), and DAPI staining was used to show the localization of nucleus (middle panels). The experiments were repeated for three times with similar results
Fig. 5
Fig. 5
N inhibits the dimer formation of IRF-3 promoted by poly(I:C), but not by MAVS. a 293T cells (4 × 105) were transfected with plasmid expressing Flag-IRF-3 (1 μg) and indicated plasmids (2 μg). At 24 h post-transfection, cells were mock treated or transfected with poly(I:C) (4 μg). 12 h later, cell extracts were prepared and detected by Western blot analysis. IRF-3 dimers and monomers were separated by native PAGE and then detected with monoclonal antibodies against Flag. b 293T cells (4 × 105) were transfected with plasmids expressing Flag-IRF-3 (1 μg) and HA-MAVS (1 μg) and indicated plasmids (2 μg), and 18 h later, cell extracts were prepared and detected by Western blot analysis similarly as in (a). pcDNA3.0-NS3/4A and its empty vector pcDNA3.0 (V-3) were set as a control. V-M stands for the empty vector pCMV-Myc, which was used for expression of N. The experiments were repeated for three times with similar results
Fig. 6
Fig. 6
N does not interact with RIG-I or MDA5. a 293T cells (2 × 106) were co-transfected with indicated expression plasmids (8 μg each). Cell lysates were immunoprecipitated with control rabbit IgG or rabbit anti-Flag (αFlag) antibody as indicated. The immunoprecipitates were analyzed by Western blots with mouse anti-Myc antibody (top panels) and mouse anti-FLAG antibody (middle panels). The expression levels of transfected proteins in the lysates were analyzed by Western blots with indicated antibodies (bottom panel). The experiments were repeated for three times with similar results. b 293T cells (2 × 106) were co-transfected with indicated expression plasmids (8 μg each). At 24 h post-transfection, cells were transfected with poly (I:C) (left panels) or infected with Sendai virus (right panels) for 6 h. Then, co-immunoprecipitation assays were performed similarly as in (a)
Fig. 7
Fig. 7
Reporter assays using truncated N expression constructs. a Truncated constructs of N. The open reading frame of N was truncated in six constructs corresponding to the structural domains. b Western blot analysis was performed to determine the expression of SARS-CoV N protein and N truncation mutants. c The truncated N plasmids, pIFN-β-luc, pRL-TK were co-transfected into 293T cells (1 × 105) for 24 h before SenV infection. Reporter assays were performed 18 h after infection. The data represent the mean ± S.D. of three independent experiments. * P < 0.05. Meanwhile, cell extracts were prepared, and the expression of β-actin protein was determined by Western blot as the expression control
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