H5N1 Avian Influenza Virus NS1 Protein Down Regulates IL-6 And IL-8 Expression Via Interference With NF-κB Activity

Objective: H5N1 strains of avian influenza A virus have already caused the deaths of more than 250 people since the outbreak of infection in Southeast Asia in 1997 corresponding to a death rate of about 60% for known infections. These viruses, which have now spread from Asia to Europe and Africa, are strong candidates for causing the next flu pandemic if they acquire the ability for efficient human-to-human transmission. A major research goal has been to identify the molecular basis of the virulence of H5N1 viruses in humans.The NS1 protein of H5N1 virus is synthesized in infected cells but not incorporated into virus particles. Studies have suggested that NS1 protein contributes to the virulence of influenza virus during infection by multiple mechanisms. These mechanisms include the inhibiton of the PKR and the blocking of IFN-βproduction. As the NS1 prtoein is considered to be an IFN antagonist, we hypothesized that the NS1 protein might also be involved in the control of the production of other pro-inflammatory cytokines. So DNA microarry was used to examine the efftecs of NS1 protein on the host cell cytokine expression.Methods: Real-time PCR and ELISA were used to detect the expression of IL-6 and IL-8 in the cells transfected with pcDNA3.0 or pcDNA3.0-NS1. IL-6 promoter, and site-specific mutants for NF-κB binding site of IL-6 promoter; IL-8 promoter, and site-specific mutants for NF-κB binding sites of IL-8 promoter were subcloned into to the luciferase expression vector respectively. The luciferase assay was performed to study whether NS1 protein affects the activity of the IL-6 and IL-8 promoters through NF-κB bingding sites. To investigate the effect of NS1 protein on NF-κB activity, the pNF-κB-Luc plasmid was contransfected with pcDNA3.0 or pcDNA3.0-NS1 to A549 cells. At 36 h posttransfection, cells were harvested for luciferase assay. To study the molecular mechamism of NF-κB suppression in NS1-expression cells, the total quantity of NF-κB in 293T cells were examined by wentern blot analysis using anti-p50 and anti-p65 antibodies. To deternmine the localization of NF-κB, cytoplasmic and nuclear fractions were prepared and detected by western blot analysis using the anti-p50 and anti-p65 antibodies.Results:1. NS1 protein inhibits the expression of IL-6 and IL-8.DNA microarray was used to identify the cellular genes regulated by NS1 protein by other group in our lab. The genes of IL-6, IL-8, COX-2, CCL-20, were down-regulated by NS1 protein detected by the gene chip. The suppression of IL-6 and IL-8 by NS1 were further confirmed by Real-time PCR and ELISA.2. NS1 protein inhibits the expression of IL-6 and IL-8 through the NF-κB transcript factor.IL-6 and IL-8 are all NF-κB-mediated down-stream genes and NF-κB can bind directly to their promoters as a transcript factor. Here we use luciferase assay to detect whether the suppression of IL-6 and IL-8 by NS1 proteins was related with NF-κB. The results indicates that NS1 protein does affect the activity of the IL-6 and IL-8 promoters through NF-κB sites.3. NS1 protein suppresses NF-κB activityThe effect of NS1 protein on NF-κB activity was investigated by a luciferase assay. The results showed that NS1 protein significantly suppressed luciferase activity in A549, HepG2 and 293T cell lines.4. NS1 protein dose not alter the expression of NF-κB but affects its nuclear translocation.To investigate the molecular mechanism of NF-κB suppression in NS1-expressing cells, the total quantity of NF-κB in 293T cells transfected with pcDNA3.0 or pcDNA3.0-NS1 was examined by western blot. Also the cytoplasmic and nuclear fractions of the cells were prepared and detected by western blot analysis using the anti-p50 and anti-p65 antibody. The result indicates that NS1 dose not alter the expression of NF-κB, but inhibits the translocation of p50 and p65 to the nucleus.Conclusions: H5N1 avian influenza virus NS1 protein suppresses IL-6 and IL-8 expression via interference with NF-κB activity. NS1 dose not alter the expression of NF-κB, but affects its nuclear translocation.