key: cord-0966146-2hik788b
authors: Zhang, Yuan-peng; Zhang, Rong-wu; Chang, Wei-shan; Wang, Yan-yan
title: Cxcl16 interact with SARS-CoV N protein in and out cell
date: 2010-10-08
journal: Virol Sin
DOI: 10.1007/s12250-010-3129-x
sha: 5f7a653beae752fae3a898cbfce92cb2854a874a
doc_id: 966146
cord_uid: 2hik788b

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.

proliferation and delays the cell growth by disrupting mitosis [12] . 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 [4] . The SARS-CoV N protein binding proteins identified in cells include: hnRNPA1 [7, 9, 11] , 14-3-3 protein [11] , cyclin-CDK complex [12] , human Ubiquitin binding enzyme 9 (hUbc9) [3] and cyclophilin A [6] . 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 [15] . 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 al investigated 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 [1] . 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 coprecipitation (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 [2] . In this paper we continue studying the interaction of SARS-CoV N and CXCL16 both in and out of the cell.

HEK293FT cells were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum and penicillin-streptomycin. SARS-CoV-N eukaryotic expression plasmid pDC516N was produced by Dr.

Jack Gauldie [11] , the University of Ontario, using plasmid pcDNA3.1V5His. Eukaryotic expression vector pcDNA3.1HisA/CXCL16 was produced by our laboratory [2] . 

CXCL16, pDC516N, pcDNA3.1HisA/SARS-N-GFP were used as a template, and PCR amplification showed fragments of about 821bp, 1,280bp, 2,016bp, respectively (Fig.1) . After double enzyme digestion, ligation, transformation, colony PCR and restriction endonuclease digestion (Fig. 2) and sequence analysis the positive clones were confirmed. pEGFP-N1/SARS-N, pdsRed2-N1/CXCL16, pcDNA3.1HisA/ SARS-N-GFP had the correct sequences. The vector was therefore successfully constructed. (Fig. 3) . showing green fluorescence was mainly located in the cytoplasm Co-location of the two kinds of protein in the cytoplasm showed an orange fluorescence (Fig. 4) .

pdsRed2-N1/CXCL16 was used to transfect HEK- This changed to orange fluorescence when the two areas overlapped (Fig. 5) .

CXCL16 is a newly discovered cell chemokine, composed of 254 amino acids, with a relative molecular mass of about 30,000 Da [14] . CXCL16 with chemotactic function exists in the membrane-bound form or secreted form. CXCL16 produces a strong chemotaxis for activated CD8 + T cells, and can also promote the interaction between antigen-presenting cells (APC) and T cells, induce the immune response of specific cell subsets, and regulate the migration of activated T cells in the spleen red pulp [10] . In addition, with the application of CXCL16 monoclonal antibodies, it was discovered that tonsil and CD19+B cells could express the chemokine, indicating that CXCL16 were involved in the cell-cell interactions in chronic inflammation. The only CXCL16 receptor was named CXCR6, and CXCR6-positive T cells play a very important role in some particular diseases [13] . SARS- invasion, and high CXCL16 expression in the mucous membrane areas may affect the ability of HIV infective particle to use the CXCL16 receptor as an assistant receptor [8] . SARS-CoV N protein is also likely to play the role of a viral chemokine-binding protein. 

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