Influenza virus is a serious public health threat causing significant morbidity and mortality. Seasonal infections are punctuated by pandemic outbreaks with the potential for widespread infection and disease. This is exemplified by the emergence of the 2009 H1N1 pandemic virus that rapidly became the dominant circulating strain. In spite of this risk, there are only two main classes of approved antivirals, and only the neuraminidase inhibitors are efficacious against currently circulating strains. There is a clear need for the discovery of new antiviral therapies and the identification of novel antiviral targets. The influenza virus polymerase is an attractive target for antiviral development. The polymerase assembles with genomic RNA and the viral nucleoprotein (NP) to form large ribonucleoprotein (RNP) complexes. The RNPs mediate both transcription of viral mRNA and replication of the viral genome. RNP assembly and polymerase activity are essential for viral replication, yet how these events are regulated has long remained unclear. We have generated data suggesting that post-translational modifications of NP and the viral polymerase dynamically regulate the formation of RNPs, and the resultant synthesis of viral RNAs. The overall goal of this proposal is to understand how post-translational modifications regulate influenza virus RNP assembly and polymerase activity. Our first objective (Aim 1) is to establish the mechanisms by which phosphorylation regulates NP oligomerization. These experiments identify host kinases and phosphatases that regulate NP phosphorylation and demonstrate how this controls the transition of NP from a monomer to the higher-order oligomer present in the RNP. We will also define the temporal patterns of NP phosphorylation and how these impact the transition from transcription of viral mRNAs to replication of the viral genome. Our second objective (Aim 2) is to determine the functional consequences of novel post-translational modifications we recently identified on the viral polymerase and NP. Experiments will determine the host factors mediating this modification and the biological impact during infections. Completion of this aim will define a completely new regulatory mechanism controlling RNP activity. The results from this proposal will establish a mechanistic understanding of the viral and host factors regulating assembly and function of the RNP and identify exciting new targets that can be exploited for the rational development of anti-influenza virus therapies.