Influenza A and B viruses cause a highly contagious respiratory disease in humans. Influenza B viruses infect only humans, whereas influenza A viruses infect many avian and mammalian species. Influenza A viruses are responsible for the periodic wide-spread pandemics that result in high mortality rates. The 1918 pandemic was the most devastating. Highly pathogenic H5N1 influenza A viruses are prime candidates for causing the next pandemic. The NS1 proteins of influenza A virus (NS1A protein) and of influenza B virus (NS1B protein) are multifunctional proteins that suppress cellular antiviral responses and are virulence factors. Many of the functions of the NS1A protein differ from those of the NS1B protein. The overall aim is to elucidate how multiple functions of the NS1A and NS1B proteins play important roles during infection. The NS1A protein has a binding site for double-stranded RNA (dsRNA) and binding sites for several cellular proteins, including CPSF30, a cellular factor required for the processing of cellular pre-mRNAs. CPSF30 binding, which results in the inhibition of the production of all cellular mRNAs, including interferon-2 (IFN-2) mRNA, is the primary, if not the only, mechanism by which the NS1A protein of a human influenza A virus (isolated in 1972) inhibits IFN-2 mRNA production. One aim is to determine whether this mechanism is shared by all other human influenza A viruses, including H5N1 and 1918 viruses. This research will entail the generation of multiple recombinant viruses expressing NS1A proteins containing specific amino acid changes. Recent structural results on the dsRNA- and CPSF30-binding sites of the NS1A protein described in this grant application may lead to the development of new antivirals directed at these binding sites and also to the development of better live attenuated virus vaccines against H5N1 viruses. For the latter purpose, one aim is to generate viruses whose attenuation is fine-tuned by site-specific amino acid changes in these two NS1A binding sites. Motivated by genetic experiments that showed an unexpected functional interaction between the NS1A protein and the viral polymerase, another aim is to use biochemical approaches to elucidate the molecular mechanisms underlying this interaction. The IFN-induced ISG15 protein, which is conjugated to multiple cellular proteins, has antiviral activity against both influenza A and B viruses. One aim is to identify the viral and/or cellular proteins whose conjugation to ISG15 accounts for the antiviral action of ISG15 conjugation against influenza A virus. Because the NS1B protein, unlike the NS1A protein, binds ISG15 and prevents its conjugation to target proteins, research on the antiviral function of ISG15 conjugation against influenza B virus will analyze the defects in replication of a recombinant influenza B virus that expresses a NS1B protein lacking an ISG15 binding site. As an integral part of this research, the role of the dsRNA-binding activity and other functions of the NS1B protein will also be determined. Elucidation of the molecular mechanisms by which ISG15 conjugation inhibits influenza A and B viruses may lead to therapies that exploit these mechanisms.