Regulation of the influenza A virus polymerase activity by post-translational ubiquitin-modification

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Influenza A viruses (IAV) are the causative agent of severe respiratory disease with high morbidity and mortality in humans. Today, only two classes of antiviral drugs are available for the treatment of acute influenza infections. Rapid generation of drug resistant viruses against these drugs is worrisome and new targets for antiviral approaches are urgently needed for the development of future antiviral drugs. During IAV infection replication of the viral genome as well as the synthesis of viral mRNA are accomplished by the viral RNA-dependent RNA polymerase in a complex and highly regulated processes. Therefore, the viral polymerase provides a vulnerable target for novel approaches to develop highly effective antiviral drugs. Recent evidence suggests that the cellular ubiquitin-proteasome system plays an important role in the regulation of viral replication and transcription processes by post-translational linkage of ubiquitin to lysine residues in the proteins of the viral polymerase. Using mass spectrometry we were able to identify 54 ubiquitylated lysines in the IAV polymerase in infected cells. Of these, 9 lysines are not only highly conserved in the polymerase of IAV but are also conserved in influenza B and C viruses, indicating, that these residues, and potentially their ubiquitin-modification, are essential for the enzymatic activity of the viral polymerase. Structure modeling on the basis of the published crystal structure of the IBV polymerase revealed that many of the modified lysines are located in functionally relevant regions of the polymerase proteins. This includes known regions for protein-interactions, RNA-promoter binding, cap-binding, nuclear localization signals and the active center of the PA-endonuclease. However, the functional relevance of these modifications for the regulation of the polymerase activity are yet unknown. The aim of this project is to determine which ubiquitylation sites are involved in the regulation of the activity of the viral polymerase and to unravel the underlying functional mechanisms. Using siRNA- as well as inhibitor studies in cell culture, we aim to identify the cellular E3 ligases responsible for the modifications with regulatory functions. Finally, we will perform inhibitor studies in a newly established human lung tissue system in our laboratory and lay the basis for novel antiviral approaches based on the target-specific inhibition of cellular E3 ligases in the human lung.