Phase transitions underlying viral replication: roles of biomolecular condensates in virus assembly

There is an urgent need for new drugs to combat viruses that threaten human, animal and plant health. Most existing antivirals inhibit virus attachment/entry or target key enzymes, and new antiviral targets are needed to develop novel treatments and counter antiviral resistance. One key process in the life cycle of many viruses is the formation of dynamic organelles called viral factories. There is increasing evidence that some viral factories form via liquid-liquid phase separation (LLPS), including SARS-CoV-2, influenza and measles virus. These compartments concentrate viral replication enzymes and sequester replication intermediates from the immune sensors. Targeting phase separation is an emerging paradigm that may underlie discovery of broad-spectrum antivirals.

We have recently discovered that rotaviruses, a large evolving class of RNA viruses, employ a similar mechanism of formation of replication factories via LLPS1. Rotaviruses (RVs) remain a major cause of acute gastroenteritis in children, and despite vaccines being available are responsible for >200,000 child deaths annually, predominantly in low-income countries. Rotavirus replication factories represent complex functional colloids formed by spontaneous interactions of at least two viral proteins, during the process modulated by posttranslational modifications and nucleic acids binding. This research project will focus on dissecting the physicochemical properties of these viral condensates to understand how their dynamic conformations and posttranslational modifications that affect charge mediate assembly of viral factories, and in doing so, identify targets for future therapeutic intervention.

To quantitatively describe the formation of these condensates, we will examine the observed phase transitions of binary and tertiary mixtures of recombinantly produced viral proteins, as well as viral RNAs in vitro using the recently developed high throughput microfluidics platform PhaseScan1,2. These findings will lead us to define a new model of viral replicative condensate formation that addresses protein-specific attributes (posttranslational modifications, conformation), and their highly selective RNA composition (partitioning of cognate viral transcripts and exclusion of non-viral RNAs). We will apply mass spectrometry (MS)-based proteomics tools (Orthogonal Organic Phase Separation3 and proximity-based biotinylation approaches) to define the molecular composition of viral replicative condensates in vivo. To further validate our findings, a super-resolution DNA-PAINT microscopy approach4 will be used to define the complete molecular picture of viral replication factories in situ. Identified protein residents of viral biomolecular condensates during early and late infection points will be examined by the recently developed state-of-the-art machine learning approach5 to reveal the amino acid features that drive the LLPS. The roles of these residues in the formation of viral replicative condensates will be further tested via mutagenesis. In addition, we will identify and examine the roles of posttranslational modifications on the phase behaviour of the viral biomolecular condensates. The insights gained from these approaches will underlie the search for compounds that could serve as drug templates for prospective therapies for RNA viruses, and improve our fundamental understanding of the synergistic interactions of viral proteins that spontaneously form complex biocondensates that underlie viral replication. Partitioning of small molecule compounds into these biomolecular condensates will be screened in collaboration with the mass-spectrometry facility at the University of Leeds, to determine the degree of partitioning of such molecules into the condensates. Successful hits will be then tested in vitro (PhaseScan) and in vivo (virus replication assays) to determine their potency in disrupting phase separation and viral replication.