Dual Role of the pestiviral non-structural protein NS4A in RNA Replication and virion morphogenesis

• Applicant: Professor Dr. Norbert Tautz
• Subject Area: Virology
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 280799249

For all viruses of the Flaviviridae family the non-structural proteins (NSPs) are in addition to the structural proteins essential for the formation of infectious virions. Accordingly, the NSPs have a dual function in genome replication as well as in virion morphogenesis. Aim of this proposal is the elucidation of the molecular basis of this multifunctionality. For pestiviruses the NS2-3 cleavage decides on its function: while uncleaved NS2-3 is required for the production of infectious particles, free NS3 is essential for RNA replication. Based on a virus mutant which is capable of virion production despite of complete NS2-3 cleavage, we could demonstrate that NS3/NS4A surface interactions are decisive for the decision whether this protein complex functions in virion morphogenesis or RNA replication. Moreover, in a detailed functional study we studied in the C-terminal region of BVDV NS4A which amino acids are essential for its function as (I) NS3 protease cofactor, (II) replicase modul, or (III) component in virion morphogenesis. These analyses are to be finalized. The central aspect of the application is the identification of the composition of the replicase- and packaging-complexes, with special focus on the identification of the viral interaction partners of the two monovalent NS3/NS4A complexes (active either in the replicase or in virion morphogenesis complexes) and the protein surfaces involved in this interaction. Techniques to be used are protein-„crosslinking“ via „orthogonal protein labeling“ und „biotin-ligase protein fusions“. Current studies have shown that gain-of-function mutations for virion morphogenesis in the absence of uncleaved NS2-3, identified in NS2 and NS3, enhance the NS2/NS3 interaction. Furthermore, it was observed that a C-terminal truncation of NS4A induced a massive increase in NS2/NS3 interaction. Together, these findings indicate that NS2 and the C-terminal part of NS4A compete for binding to the same or an over-lapping surface area of NS3. Accordingly, the protein partner binding to this NS3 surface area decides on the functionalization of the complexes for either RNA replication or virion morphogenesis. The NS2/NS3 interaction shall be further characterized by mutagenesis of the relevant NS3 and NS4A surfaces. Critical amino acids will be tested for their functionality in protein binding using orthogonal protein labeling/crosslinking. Understanding the molecular basis for the multifunctionality of viral proteins is of high scientific relevance since the underlying principle of the functionalization of protein complexes by selective recruitment of interaction partners to multifunctional interaction surfaces will most likely be also applied in the polyproteins of other positive-strand RNA viruses and retroviruses.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Olaf Isken