Visiting Group Leader & Senior Scientist
Our goal is to understand the molecular mechanisms whereby the genomic RNA of influenza-like viruses is, on the one hand, the template for transcription and replication of the viral genome by its RNA-dependent RNA polymerase and, on the other hand, an Achilles’ heel, whose recognition as non-self can trigger an innate immune response to counter the viral infection. Molecular warfare between the virus and the host-cell occurs at many levels. Influenza polymerase has a unique mechanism of transcription priming called ‘cap-snatching’, which involves pirating short-capped oligomers from nascent cellular Pol II transcripts; this contributes to shutdown of host-cell gene expression. The cell counters RNA viruses with innate immune pattern-recognition receptors, such as the RNA helicase RIG-I, which recognise particular viral RNA structural motifs (e.g. 5′ triphosphate-dsRNA) as non-self, thus activating a signalling pathway leading to interferon production and establishment of the anti-viral state. In response, viruses deploy proteins as counter-counter-measures to dampen the immune response, for instance, by supressing the RIG-I signalling pathway.
In 2014, we determined the first crystal structures of the complete heterotrimeric influenza polymerase (Pflug et al., Nature 2014) and proposed a mechanism of how cap-snatching is performed (Reich et al., Nature 2014). We have also elucidated how influenza polymerase interacts with the phosphorylated C-terminal domain (CTD) of Pol II during cap-snatching (Lukarska et al., Nature 2017, Krischuns et al., PLoS Pathog 2022). In 2011 we published the first structure-based mechanism of the activation of RIG-I, showing how RNA binding resulted in a major conformational change that liberated the N-terminal CARD domains for downstream signalling (Kowalinski et al., Cell 2011). Subsequently we determined structures of related innate immune receptors MDA5 and LGP2 (Uchikawa et al., Mol. Cell 2016). Previously, we worked on aminoacyl-tRNA synthetases, which play an essential role in protein synthesis by charging specifically their cognate tRNA(s) with the correct amino acid and editing mischarged amino acids if necessary (Palencia et al., NSMB 2012). Our work led to the understanding of the mechanism of action of a new anti-fungal compound targeting the editing activity of leucyl-tRNA synthetase (Rock et al., Science 2007), and to the design of new antibiotics that target multi-resistant gram negative bacteria, tuberculosis and apicomplexan parasites (Palencia et al., Antimicrob. Agents Chemother. 2016).
Our current goal is to derive models explaining the detailed mechanisms of transcription and replication of the viral genome (vRNA) by influenza-like viral polymerases. To achieve this we use X-ray crystallography and single-particle cryoEM to determine structures after trapping successive states along the active transcription or replication pathways. For transcription by influenza polymerase, we have recently determined a series of high-resolution structures corresponding to the transcription initiation, elongation and poly-adenylation/termination and recycling states (Kouba et al., NSMB 2019, Wandzik et al., Cell 2020). In parallel, we are doing the same for viral replication, which is unprimed and occurs in two-steps via an intermediate complementary RNA (cRNA). These studies are being extended to viral RNPs (the physiological RNA synthesis units) to understand the behaviour of the viral nucleoprotein during replication and transcription and to include host factors important for viral replication. We complement structural studies with in vitro polymerase enzymology and in-cell studies using mini-replicon systems, and, in collaborations, recombinant viruses and live-cell imaging.
There are several other aspects of these projects of particular interest.