Abstract
Severe acute respiratory syndrome (SARS) is a newly emerged
infectious disease. The causative agent of SARS has been identified to be a new type of coronavirus, namely SARS coronavirus (SARS-CoV).SARS-CoV proteins including the ORF6 protein have been reported to inhibit interferon signaling response. The goal of this study is to identify human SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries and to investigate their interaction on the type I interferon antagonist function. At first, the recombinant ORF6 protein was generated in BL21(DE3) and purified by using IMAC (immobilized mental-affinity chromatography). After five rounds of biopanning, PI-3 kinase-related kinase SMG-1 protein was identified as SARS-CoV ORF6 interacting protein from phage displayed lung cDNA library. Confocal imaging revealed co-localization of SARS-CoV ORF6 protein with SMG-1 protein in HL-CZ cells. In vivo signaling pathway assay and real time RT-PCR showed that single gene expression of SARS-CoV ORF6-expressing cells blocked the INFα/β-induced responses, such as ISRE-responsive firefly luciferase activity and the expression of PKR. However, both gene expression of SARS-CoV
ORF6 and SMG-1 had a significantly higher relative activities of INFα/β-induced ISRE-responsive firefly luciferase than single gene expression of SARS-CoV ORF6 in HL-CZ cells. In addition, confocal imaging and Western blotting assays revealed that the translocation of STAT-1 into nucleus and the STAT-1 phophorylation were found in the transfected cells expressing both genes of ORF6 and SMG-1, but not in the ORF 6-expressing cells in response to INFα/β. Therefore, cell expression of PI-3 kinase-related kinase SMG-1 could restore the INFα /β responses in SARS-CoV ORF6 expressing cells. The interaction of SARS CoV ORF6 and SMG-1 may be responsible for the inhibition of type I IFN response by SARS-CoV.
