BIOCHEMICAL AND GENETIC-EVIDENCE FOR COMPLEX-FORMATION BETWEEN THE INFLUENZA-A VIRUS NS1 PROTEIN AND THE INTERFERON-INDUCED PKR PROTEIN-KINASE

The interferon (IFN)-induced protein kinase (PKR) functions as a gatek eeper of mRNA translation initiation and is, therefore, a key mediator of the host IFN-induced antiviral defense system. Many viruses have i nvested countermeasures against PKR, Some apparently use more than one mechanism. The influenza virus can repress PKR activity through the u se of at least two factors, the cellular p58(IPK) protein and the vira l NS1 protein. The exact mode of action of the latter has not been est ablished. Here, using a coprecipitation assay, we found that PKR could form a complex with NS1 in crude cell extracts prepared from influenz a virus-infected HeLa cells. The NS1-PKR interaction was verified by u sing the yeast two-hybrid system and an in vitro binding assay. Deleti on analysis mapped the NS1 binding site to the N-terminal 98 residues of PKR regulatory region. Furthermore, an NS1 mutant, which lacks PKR inhibitory activity, did not bind PKR, Finally, the functional role of NS1 in PKR inhibition was substantiated using an in vivo assay for PK R activity. These results support the role of NS1 in PKR modulation du ring viral infection that is mediated through a complex formation betw een the two proteins.