Objective To study the interactions between the highly pathogenic avian influenza H5N1 nucleoprotein (H5N1 NP) and NF-κB-inducing kinase (NIK),and to reveal the effect of H5N1 NP on NIK-induced NF-κB transcriptional activity.Methods The gene encoding NIK protein was amplified by RT-PCR from total RNA of HeLa cell line.Eukaryotic expression plasmid pCMV-Myc-NIK and prokaryotic expression plasmid pGEX-4T-1-NP (GST-NP) were constructed by cloning from HeLa cell cDNA and pcDNA3-Flag-NP vector,respectively.Co-immunoprecipitation (co-IP) and GST pull-down were used to test the interactions between H5N1 NP and NIK.Dual-luciferase reporter gene analysis system was used to test the effect of H5N1 NP on NIK-induced NF-κB transcriptional activity.Results Co-IP and GST pull-down showed that pCMV-Myc-NIK and pGEX-4T-1-NP (GST-NP) could express Myc tagged NIK protein and GST tagged NP protein in HEK293T cells and E.coli,respectively,and that H5N1 NP was associated with NIK in vivo and in vitro.Dual-luciferase reporter gene analysis suggested that H5N1 could inhibit NIK-induced NF-κB transcriptional activity.Conclusion H5N1 NP interacts with NIK and inhibits NIK-induced NF-κB transcriptional activity.This finding can facilitate further study of H5N1.