SARS coronavirus (SARS-CoV) has been identified as an etiologic agent of Severe Acute Respiratory Syndrome. Its genome consists of a positive strand RNA with a length of 30, 000 nucleotides, which contains 10 overlapping Open Reading Frames (ORF) and a bilateral non-coding area. The structural proteins encoded by coronavirus include Nucleocapsid protein (N), Envelope protein (E), Membrane protein (M) and Spike protein (S). The multiple roles of N protein include participation in the formation of SARS-CoV nucleocapsid, modulation of genome RNA synthesis and transcription and translation of sub-genome RNA; it also inhibits host cell proliferation and delays the cell growth by disrupting mitosis. SARS-CoV N Protein interacts with a number of proteins within the virus or the host cells, which interferes with the cellular cycle and transforms the cellular vital activities by influencing multiple signal transduction pathways of the host cells. The SARS-CoV N protein binding proteins identified in cells include: hnRNPA1[7, 9, 11], 14-3-3 protein, cyclin-CDK complex, human Ubiquitin binding enzyme 9 (hUbc9)and cyclophilin A. Recently, N protein was reported to associate with Smad3 and to promote Smad3-p300 complex formation by interfering with the complex formation between Smad3 and Smad4. However, considering what is known about the host genome, the proteomics and the cellular signal transduction pathways represent only a portion of the interactors. Brunn et alinvestigated the interaction between SARS-CoV and host cells and discovered that the cross-reactions of the different components of the virus and the interaction between virus and host cells make up a complicated network. Therefore, it is necessary to select the SARS-CoV interaction protein in the host cells with proper modalities, which in return greatly contributes to the elucidation of the pathogenesis of SARS-CoV and the search for new drug targets. Chang et al employed the SARS-CoV N protein as bait in the Y2H system, and selected 15 interactors of SARS-CoV nucleocapsid from the human embryo-lung cDNA library of Two-Hybridization (LLH). Finally, Immunological co-precipitation (CO-IP) was used to verify the interaction between CXCL16 and SARS-CoV N protein and that this interaction could be blocked by SARS-CoV N antibody. In this paper we continue studying the interaction of SARS-CoV N and CXCL16 both in and out of the cell.
Cxcl16 Interact With SARS-CoV N Protein In and Out Cell
- Received Date: 22 January 2010
- Accepted Date: 30 June 2010
Abstract: Our study investigated the host cell protein which can interact with SARS-CoV N protein, and explored the functional connections. The eukaryotic expression vectors pEGFP-N1/SARS-CoVN and pdsRed2-N1/ CXCL16 were constructed and used to co-transfect HEK293FT cells by the calcium phosphate method. The HIS-tagged fusion protein SARS-CoVN-GFP was then built and purified for the binding assay in vitro. The co-localization of SARS-CoVN and CXCL16 in the cytoplasm of HEK293FT cells was also shown using confocal laser scanning microscopy. It is suggested that their interaction might be through direct combination. Under a fluorescence microscope, it was observed that the purified fusion protein SARS-CoVN-GFP was attached to the cell membrane of CXCL16-transfected cells, indicating that SARS-CoVN and CXCL16 can be mutually combined.