Characterization of regulatory sites for the assembly of viral ribonucleoprotein complexes during the replication of the influenza A virus RNA genome

• Applicant: Dr. Franziska Günl
• Subject Area: Virology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 523956052

Influenza A viruses (IAV) are respiratory pathogens that cause annually recurring epidemics and occasional pandemics associated with high morbidity and mortality. Despite major progress in vaccine and antiviral research, the emergence of resistant strains poses a serious threat to human and animal health, demonstrating a constant urgency to identify novel drug targets. Deciphering the functionality of viral proteins and their interplay with the host cell is therefore pivotal. Given its central role during the expression of viral proteins and the replication of the viral genome, the RNA-dependent RNA polymerase (RdRP) of influenza viruses represents a prominent target for the development of effective antivirals. As part of the Orthomyxoviridae genus, IAV have a segmented single-stranded RNA genome (vRNA) of negative polarity. Each of the eight vRNA segments is encapsidated with multiple copies of NP proteins and bound to one copy of the heterotrimeric RdRP, together forming viral ribonucleoprotein complexes (vRNPs) - the replicational machinery of IAV. vRNA replication proceeds through the synthesis of a full-length complementary intermediate, the cRNA, which is packaged into cRNPs and serves as a template for vRNA synthesis. During this process, the viral RdRPs form replication platforms, in which they assemble to asymmetric dimers that mediate the binding of nascent RNA strands and the formation of new RNPs. The host protein ANP32 bridges these dimers and regulates NP recruitment. In a recent study, we identified a ubiquitin-targeted lysine located in the thumb region of PB1 that charge-dependently modulates the balanced replication of the eight vRNA segments. We thereby highlighted a functional region with the potential to regulate the encapsidation of new RNA strands, the assembly of RNPs, and the initiation of RNA synthesis. In this project, we aim to characterize the region of c/vRNP assembly in detail. To identify sites of regulatory importance we will carry out a mutational screen in the proposed RNP assembly region. A variety of RNA and protein biochemistry methods are going to be applied to characterize regulatory sites for their impact on c/vRNA synthesis in the single and multi-segment context, the binding of RNA promoters, RdRP oligomerization as well as the binding of NP and ANP32 to the polymerase. The results of these functional assays are going to be complemented by biophysical and structural studies using X-ray crystallography and single-particle cryo-EM. In combination, this will provide in-depth insights into RNP assembly that is essential to understand the progress of vRNA replication by the IAV polymerase.

• DFG Programme: WBP Fellowship
• International Connection: United Kingdom
• Host: Professor Dr. Ervin Fodor
• Participating Person: Professor Dr. Jonathan M. Grimes