Infectious virion formation is an amazing natural process that underlies one of the major lifestyle stages of a very important group of pathogens. We have discovered a conserved mechanism that is used by single-stranded RNA viruses, such as HIV and coronavirus, to regulate their assembly using multiple sites/motifs across their genomes to make cognate interactions with their coat protein subunits. Such sites are evolutionarily conserved across viral strain variants making them legitimate drug targets. The precise details of how this mechanism promotes assembly in different viral families is distinct. We are using interdisciplinary combinations of biophysics (single molecule spectroscopy; cryo-EM), biochemistry (X-ray footprinting; in vitro reassembly), genetics (SELEX; strain conservation analysis) and physical theory (mathematical modelling) to determine the roles of the RNA-coat protein contacts in a number of important human pathogens, as well as model systems. These RNA sites, termed Packaging Signals (PSs), function co-operatively and it is the ensemble of PSs that produce the desired results of efficient and selective assembly.
Knowledge of the various roles of PSs in assembly is being exploited to recode therapeutic cargoes so that they can be produced cheaply and delivered efficiently in gene therapy applications.
Current major projects
- Examining the roles of RNA PSs in virion/nucleocapsid assembly/disassembly
- Visualising PS-CP contacts directly via asymmetric cryo-electron microscopy
- Correlating X-ray RNA footprinting of genomes at differing viral lifecycle stages with cryo-EM/CLEM
- Recoding cargoes to be efficient assembly substrates
Detailed research programme
In collaboration with our mathematical modelling and bioinformatics colleagues at the University of York, led by Prof Reidun Twarock, we are pursuing the following research goals, within a wider vision of resetting the paradigm of virion assembly and thus opening up novel therapeutic and translational opportunities.
Examining the roles of RNA PSs in virion/nucleocapsid assembly/disassembly
RNA Packaging Signal-mediated virion assembly relies on the collective action of PS motifs, which can be very sparse, dispersed across a viral RNA genome to induce virion formation. This can be in the context of other cellular machinery or in its absence. We are using genome variants and biophysical techniques, such as single molecule fluorescence spectroscopy, to determine the molecular mechanisms underlying the PS-mediated assembly used by various families of viruses. Disrupting the functions of PSs using RNA-binding ligands as antiviral drugs is being used to harness their therapeutic potential in viruses such as Hepatitis B Virus.
Visualising PS-CP contacts directly via asymmetric cryo-electron microscopy
All virions are intrinsically asymmetric since even those with highly symmetrical coat protein shells encapsidate genomes that have little or no inherent symmetry. The vast majority of virion structures determined to date use the symmetry-averaging technique applied by my post-doctoral mentor, Steve Harrison at Harvard, to improve the signal to noise in virus electron density. One consequence of this, that was self-evident from the beginning, is that virtually all asymmetric features are lost from the electron density maps, including those of the genome and its interaction(s) with CPs. These seem essential for assembly and must get broken during RNA uncoating. We are using various tricks, such as forming uncoating complexes with cellular receptors, to further break symmetry, allowing modern cryo-EM to reveal for the first time the details of the molecular machinery viruses use to take over their hosts.
Correlating X-ray RNA footprinting of genomes at differing viral lifecycle stages with cryo-EM/CLEM
Viral RNA genomes undergo multiple large scale conformational rearrangements during the viral lifecycle, from compact structures suited to encapsidation in infectious virions to the extended polynucleotides required for replication/translation substrates. These events are naturally linked to key steps in the lifecycle such as virion assembly, or uncoating after initial binding to cellular receptors. We are developing the tool of X-ray induced RNA footprinting to follow these events in real time. Such footprints do not require addition of exogenous modification reagents, can be carried out on frozen samples that can be compared directly to cryo-EM reconstructions, and are complete within 100msec, i.e. are completed so quickly that modification cannot lead to conformational changes. We are working across different timescales and technologies to correlate the XRF patterns with events happening as viruses contact and enter/exit host cells.
Recoding cargoes to be efficient assembly substrates
Viruses are capable of selectively packaging large nucleic acid molecules, i.e. their genomes, against a backdrop of cellular competitor RNAs. Understanding the “syntax” of PS-mediated assembly in various viral families allows us to recode non-viral nucleic acid cargoes so that they become good assembly substrates. These can then be delivered to cells with the same tropism as the natural virus, opening up an efficient way to deliver therapeutic genes, or gene editing tools. We are developing such systems to contribute to the growing field of gene therapy in the clinic.