Activation of AP-1 signal transduction pathway by SARS coronavirus nucleocapsid protein

Abstract

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia and subsequently proven to be the causative agent of the disease now referred to as severe acute respiratory syndrome (SARS). The complete genome of the SARS coronavirus (SARS-CoV) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) shares little homology with other members of the coronavirus family. To determine if the N protein is involved in the regulation of cellular signal transduction, an ELISA-based assay on transcription factors was used. We found that the amount of transcription factors binding to promoter sequences of c-Fos, ATF2, CREB-1, and FosB was increased by the expression of SARS-CoV N. Since these factors are related to AP-1 signal transduction pathway, we investigated whether the AP-1 pathway was activated by SARS-CoV N protein using the PathDetect system. The results demonstrated that the expression of N protein, not the membrane protein (M), activated AP-1 pathway. We also found that SARS-CoV N protein does not activate NF-kappaB pathway, demonstrating that activation of important cellular pathways by SAS-CoV N protein is selective. Thus our data for the first time indicate that SARS-CoV has encoded a strategy to regulate cellular signaling process.

Fig. 1

(A) Diagram of TransFactor inflammation profiling kit method. Cell extracts are added to 96-well plates coated with the wildtype DNA elements of individual transcription factors. Bound protein samples are then detected and quantified by primary and secondary antibody incubation. (B) Diagram of the PathDetect method for in vivo signal transduction assessment. The luciferase reporter gene is driven by a basic TATA promoter and an inducible enhancer bound proteins which recognize the AP-1 specific enhancer element are detected by an increase in reporter gene transcription.

Fig. 2

Detection of nucleocapsid protein by Western blotting analysis. Expressed proteins were analyzed by SDS–polyacrylamide gel (4–12% Bis–Tris) electrophoresis and visualized by autoradiography after nitrocellulose membrane transfer. Lane 1, mock transfection of Huh7 cells; lane 2, pcDNA_3.1(−) transfection vector alone; lane 3, pcDNA_3.1(−) transfection vector carrying the SARS-CoV N protein.

Fig. 3

Effect of SARS-CoV N protein on the in vitro activation of pro-inflammatory transcription factors c-Fos, FosB, ATF2, and CREB-1. Absorbance measurements of samples were made at 655 nm after use of the TransFactor Inflammation Profiling Kit (Clontech Laboratories). Lanes 1, mock transfection; lanes 2, pcDNA_3.1(−) vector; lanes 3, pcDNA_3.1(−) + N.

Fig. 4

PathDetect assay on AP-1 activation. Vero and Huh7 cells transfected by dilutions of pcDNA_3.1(−), pcDNA-3.1, and pcDNA_3.1(−) + N. The reporter plasmid pAP-1Luc was co-transfected with the above plasmids. Forty-eight hours after transfection, luciferase activities were measured from the cell lysates of transfected samples. Values shown are means of five experiments subtracted from the blank control.

Fig. 5

PathDetect assay on AP-1 activation. Vero and Huh7 cells transfected pcDNA_3.1(−), pcDNA-3.1, and pcDNA_3.1(−) + N. The reporter plasmid pNFκB-Luc was co-transfected with the above plasmids. The plasmid expression MEK kinase (pFC-MEKK) supplied by the manufacture was used as positive control for NFκB activation. Forty-eight hours after transfection, luciferase activities were measured from the cell lysates of transfected samples. Values shown are means of five experiments subtracted from the blank control.