In vitro single-molecule studies of RNA-dependent RNA polymerase replication of negative strand RNA viruses
RNA viruses represent an existing and emerging global threat to human and animal health. Unfortunately, we lack sufficient knowledge of the molecular mechanism of RNA virus replication to specifically target intervention stratgies against these viruses.
What is the goal of our this project?
The overarching goal is to create fundamental knowledge on the dynamic molecular mechanisms of negative-strand RNA virus proliferation. Our model system is influenza A virus, a member of the Orthomyxoviridae family of viruses. Influenza A viruses outbreaks are on the rise and are driven by the continuing emergence of novel strains. Recent advances in the biochemistry of influenza A virus RNA-dependent RNA polymerase (RdRp) have opened up new avenues for the study of its mechanics and kinetics. With this project, we focus on two key goals: (i) quantitative characterization of the kinetics of influenza virus RdRp during RNA synthesis and (ii) assessment of how local RNA sequence or structure influence these kinetics and potentially lead to nucleotide misincorporation.
What tools do we use?
The large size and complexity of the multi-subunit RdRps often encountered in negative-strand RNA viruses have long hampered their systematic study relative to that of positive-strand RNA viruses. In recent years, however, advances in the biochemistry of these RdRps have permitted the expression and purification of recombinant active forms and subsequent structural determination via crystallization or electron microscopy. Advances have been particularly great for influenza virus RdRps, which were the first of any negative-strand RNA virus to be crystallized. This now makes it possible to, for the first time, gain insight into the molecular mechanisms that underlie the functioning of these enzyme complexes by performing systematic in vitro studies. We employ state-of-the-art RdRp purification and labeling, fluorescence microscopy, in-vitro RdRp replication assays using synthetic transcripts in bulk and in single-molecule assays.
What is the significance to society of what we do?
Influenza viruses constitute a constant threat to global health. Despite the impact of influenza viruses, basic understanding of viral replication is still lacking. This proposal constitutes fundamental research aimed at enhancing our understanding of RNA synthesis by the influenza virus RdRp. More generally, the knowledge generated will also pertain to the mechanisms employed by multi-subunits RdRps of other negative-strand RNA viruses. By providing insights into viral adap tation and evolution mediated through the influenza virus RdRp, this project will also have implications for public health, animal health, and biomedical research.
What will your specific project look like?
We are looking for student colleague(s) (preferably MEP level) interested in quantitatively exploring influenza virus RdRp replication. You will be optimizing single-molecule assays with recombinant influenza virus polymerase and synthetic constructs to study the replication dynamics. This project will be carried out in close collaboration with Dr. Mathilde Richard, virologist at the ErasmusMC.
Prof. Nynke Dekker, biophysicist
Dr. Mathilde Richard, virologist at the ErasmusMC, department of Viroscience