Cell 1:991-1000. of either the PUT3 or the Ncd (DmNcd) proteins. These viruses regained virulence and lethality in mice. Moreover, a recombinant influenza virus expressing only the first 73 amino acids of the NS1 protein was able to replicate in mice lacking three IFN-regulated antiviral enzymes, PKR, RNaseL, and Mx, but not in wild-type (Mx-deficient) mice, suggesting that this attenuation was mainly due to an inability to GnRH Associated Peptide (GAP) (1-13), human inhibit the IFN system. Remarkably, a virus with an NS1 truncated at amino acid 73 but fused to the dimerization domain name of DmNcd replicated and was also highly pathogenic in wild-type mice. These results suggest that the main biological function of the carboxy-terminal region of the NS1 protein of influenza A virus is the enhancement of its IFN antagonist properties by stabilizing the NS1 dimeric structure. Folding of proteins into highly ordered structures is usually often essential for their functions. This is especially critical for proteins which perform multiple functions. In addition, multifunctional proteins usually show a modular organization, with different domains responsible for their different functions. RNA viruses typically encode a small number of proteins which need to take over the host cellular machinery in order to generate new infectious viruses. Therefore, most viral proteins perform multiple functions required for optimal viral replication. The NS1 protein of influenza A virus, a negative-strand RNA virus, appears to be one of those multifunctional proteins. GnRH Associated Peptide (GAP) (1-13), human At least three functional domains have been described in this 230-amino-acid protein: an RNA binding domain name, an eIF4GI binding domain name, and an effector domain name. In vitro studies showed that this core sequence of the RNA binding domain name of the NS1 protein is amino acids 19 to 38 (37). NS1 has been reported to bind to heterogeneous RNAs, including poly(A) RNA (39), viral genomic RNA (18, 29), the 5 untranslated region of viral mRNAs (36), U6 (40) and U6atac snRNA (51), and double-stranded RNA (dsRNA) (16, 26). The eIF4GI binding domain name requires amino acids 81 to 113. Binding of eIF4GI by the NS1 protein has recently been shown to facilitate the preferential translation of viral mRNAs (2). Previous reports suggested that this NS1 effector domain name, with a core sequence of amino acids 134 to 161 (37), blocks host mRNA splicing (24), polyadenylation (32, 42), and nuclear export (7, 10, 37). For these inhibitory functions on host mRNA processing, the RNA binding activity of NS1 seems not to be required MAP2 (24, 32). However, the biological significance of these NS1 functions in the context of an infectious virus has not been fully elucidated. It has been established that this induction of interferon alpha/beta (IFN-/) synthesis and secretion represents one of the first antiviral (innate) responses of the GnRH Associated Peptide (GAP) (1-13), human host (44). IFN-/ induces the transcriptional activation of many genes, some of which play essential roles in the host antiviral defense. The importance of IFNs can be gleaned from the fact that most viruses encode one or more factors to combat the IFN system of the host in order to launch productive infections (1, GnRH Associated Peptide (GAP) (1-13), human 14, 23). The influenza A virus NS1 protein is usually one of these virally encoded IFN antagonists. A mutant influenza virus which has a deleted NS1 gene, delNS1, was generated and found to replicate efficiently in IFN-/-deficient systems (12). Previous studies also exhibited that contamination of different cell types with the delNS1 virus, but not with the wild-type PR8 virus, induces high levels of IFN-/ (45, 53). Furthermore, expression of the NS1 protein blocks dsRNA-, delNS1-, and Sendai virus-mediated activation of the IFN- promoter (53). It was also exhibited that expression of the NS1 protein prevents the virus- and dsRNA-mediated activation of NF-B (53) and IRF-3 (45), both of which are key transcription.