A single-molecule approach to RNA replication and virology, drug screening, and vaccine production and characterization.
Viruses synthesize copies of their genomes very rapidly, but in doing so also introduce many errors. A better understanding of the underlying mechanisms of this process could allow us to force viral polymerases towards higher or lower rates of misincorporation i.e. to make them introduce either more or fewer errors, thereby impeding virus survival. Overall, viral systems are excellent model systems for understanding the intricacies of DNA and RNA replication.
Our viral research focuses in particular on understanding the molecular processes that underline RNA replication of RNA-viruses, with the aim of gaining spatiotemporal insight into the molecular mechanisms of RNA-dependent-RNA polymerases (RdRp) responsible for carrying out RNA synthesis. In particular, we are interested in assessing how polymerases’ tendency towards misincorporation can be influenced by the presence of 1) RNA structures, 2) specific RNA sequences 3) and/or nucleotide analogs and, as such, provide ways of understanding viral adaptation and evolution, and ultimately contribute to the development of new antivirals.
We use high-throughput single-molecule techniques, in particular magnetic tweezers, to examine nucleotide synthesis by viral polymerases. The high-throughput character of these techniques allows us to achieve high statistics and, in consequence, test quantitative models for polymerase mechanisms. Our assays consist of tracking tethered RNA molecules as they are converted from double-stranded RNA to single-stranded RNA by a single RNA-dependent RNA polymerase. Due to the applied force on the bead, and the fact that single-stranded DNA is more extensible than the double-stranded helix, we see this as a length change over time. This can then be converted into a number of RNA nucleotides synthesized over time. Studying RdRp elongation dynamics permits the direct observation of the behavior of the polimerases that can include copy-back synthesis, back tracking, stability of the RdRp-nascent-RNA complex, and the dimensions of the RdRp nucleic-acid-binding channel.
Most recently, we have used this approach to study the RdRp of EV-A71 virus, and shown that this RdRp is particularly prone to copy-back synthesis, and, deriving from a similar mechanism, recombination. The figure below highlights in particular the recombination pathway. The occurrence of both phenomena was also increased when we added an antiviral, T-1106-TP. This observation highlights a previously unknown mechanistic impact of antivirals, and may provide further means of impacting viral proliferation.
As is clear from the ongoing SARS-Cov-2 pandemic, RNA viruses represent a threat to human health. For this reason, bio-medically relevant applications form an important motivation for our ongoing research in viral replication. These include the characterization of the methods of action of antiviral drugs as described above, the usage of force spectroscopy platforms for drug screening, and vaccinology.
Vaccines are currently the major means to prevent viral infection and global pandemics, yielding an imperative requirement for the development of safe, low cost, and scalable vaccine production process. A type of vaccine that has gained significant interest over the recent years, are virus-like particles (VLPs) vaccines representing one of the most promising alternatives to present vaccines. We are particularly interested in the biophysical characterisation of this particles, using techniques such as AFM, and (cryo)-EM will be required. This characterization is especially during the re-assembly of the individual proteins that constitute the capsid. This is a necessary step if one wants to completely avoid the risk of producing particles with RNA trapped inside.
Open student projects
- In vitro single-molecule studies of RNA-dependent RNA polymerase replication of negative strand RNA viruses (see more).
Researchers currently involved
- Louis Kuijpers
- Richard Janissen
- Theo van Laar
- Craig Cameron (Penn State University, USA)
- Shin-Ru Shih (Chang Gung University, Taiwan)
- Martin Depken (TU Delft)
- Arjen Jakobi (TU Delft)
- Wouter Roos (Groningen University)
- Leo van der Pol (Intravacc, Dutch Institute of Vaccinology)