Figure: Model of the activated state of RIG-I with bound dsRNA (centre) and ATP (top-right). The helicase domains (green and cyan), the insertion domain (yellow) and the C-terminal domain (gold) all contribute to RNA binding, which displaces the CARD domains thus allowing downstream signalling and interferon expression.
The Cusack group uses X-ray crystallography to study the structural biology of protein-RNA complexes involved in RNA virus replication, innate immunity and cellular RNA metabolism.
Previous and current research
We study the molecular mechanisms whereby the RNA of viruses such as influenza 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 has a unique mechanism of transcription priming called ‘cap-snatching’, which involves pirating short-capped oligomers from nascent cellular Pol II transcripts; this leads to shut-down 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 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 interfering with the RIG-I signalling pathway.
In 2011 we published the first structure-based mechanism of activation of RIG-I, showing how RNA binding resulted in a major conformational change that liberated the N-terminal CARD domains for downstream signalling. In 2014 we published the first crystal structures of the complete heterotrimeric influenza polymerase and proposed a mechanism of how cap-snatching is performed. 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. Our work led to the understanding of the mechanism of action of a new anti-fungal compound targeting leucyl-tRNA synthetase, and to the design of new antibiotics that target multi-resistant gram negative bacteria, tuberculosis and apicomplexan parasites.
Future projects and goals
Our current goal is to obtain structural snapshots of influenza virus polymerase performing transcription of the viral genome (vRNA) by freezing states corresponding to cap-snatching, transcription initiation, elongation and poly-adenylation. In parallel we will do the same for viral replication, which is unprimed and occurs via an intermediate complementary RNA (cRNA). This will involve understanding how the behaviour of the polymerase bound to vRNA differs from that when bound to cRNA. We complement structural studies with in vitro polymerase enzymology and in-cell studies using mini-replicon systems, recombinant viruses, and live-cell imaging.
There are several other aspects of this project of particular interest. Firstly, study of how the influenza polymerase interacts with host proteins (e.g. transport factors, helicases, and splicing factors), which help it function efficiently in the cellular context, can give insight into the polymerase mutations required for an avian virus to adapt to be able to infect humans. Secondly, our structural work on influenza polymerase has opened up the area of structure-based drug design of novel anti-virals targeting multiple sites on the polymerase. To exploit this we co-founded a Vienna-based company called SAVIRA and the project is now being pursued by Roche in Basel. Thirdly, we have extended this work to polymerases of related segmented negative-strand RNA viruses such as the large family of bunyaviruses, which includes several emerging human pathogens. Finally, we are studying the nuclear cap-binding complex (CBC), which binds to the 5’ cap of nascent Pol II transcripts and mediates interaction with nuclear RNA processing and transport machineries. One aim of this is to understand how influenza polymerase can compete with CBC for access to capped RNAs emerging from Pol II.