Research overview
A number of pathogenic species are viruses or bacteria. The activities of both viruses and bacteria require synthesizing copies of their genomes. A better understanding of the underlying mechanisms of DNA or RNA replication processes of viruses and bacteria could allow us to develop new treatments against pathogenic viruses and bacteria by impeding the correct and efficient replication of their genomes. Overall, viral and bacterial systems are excellent model systems for understanding the intricacies of DNA and RNA replication.
Our research on viral replication 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.
Our research on bacterial replication focuses now on studying the spatial-temporal impact that DNA-damaging drugs and UV radiation have on the different components of the replisome, and on accessory helicases, in live bacterial cells. The fundamental knowledge from comprehending these mechanisms is essential to addressing the biology of cancer-associated processes, where there is extensive genome instability resulting from replication anomalies. The resulting insights into replication dynamics may also provide more specific drug targets for antibiotics discovery.
Approach
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.
We employ an array of different techniques depending on the biological questions that we wish to answer. In the past, we have used in vitro approaches to get direct insight into how individual components of the replication machinery contribute to the efficiency and reliability of DNA replication. High-throughput single-molecule techniques and purified components were used to study for example the termination of bacterial replication (see past projects). Currently, we focus on in vivo approaches to examine the dynamics of replication as it occurs within the complexity of the cell.
The in vivo aspect of these studies is key, because our studies will hence probe the natural cellular context, which includes physiological concentrations and representative interaction dynamics within multi-protein complexes that will complement the data available from in vitro experiments. The fundamental knowledge from comprehending these mechanisms is essential to addressing the biology of cancer-associated processes, where there is extensive genome instability resulting from replication anomalies. The resulting insights into replication dynamics may also provide more specific drug targets for antibiotics discovery.
The bacterium E. coli is our experimental workhorse, involving genetic engineering and molecular biology. We use live cell, single-molecule fluorescence, confocal and super-resolution microscopy approaches to address our research questions. To make this possible, we make use of different fluorescence proteins that can be tagged to the replisome as well as other proteins involved in replication. When necessary, microfluidic devices are used for performing long time-lapse fluorescence microscopy. In these devices, E. coli cells are immobilized in growth channels perpendicular to a main trench through which growth medium is actively pumped.
Recent works
We have used magnetic tweezers 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.
Monitoring synthesis by Viral Polymerase (RdRp)
Illustration of viral recombination. Our work on EV-A71 RdRp illustrates that a combination of backtracking and polymerase flexibility leads to an enhanced incidence of both copy-back synthesis and recombination in EV-A71 virus (image credit: University of North Carolina/TU Delft).
As is clear from the 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 characterization 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.
Researchers currently involved
- Louis Kuijpers
- Belén Solano
- Theo van Laar
Current collaborators
- Craig Cameron (Penn State University, USA)
- Shin-Ru Shih (Chang Gung University, Taiwan)
- Marco Vignuzzi (Institut Pasteur, France)
- Arjen Jakobi (TU Delft)
- Wouter Roos (Groningen University)
- Leo van der Pol (Intravacc, Dutch Institute of Vaccinology)