Dissecting the molecular mechanisms of coronavirus genome packaging

For all viruses, specifically packaging their genome into progeny virions is crucially important for efficient transmission between hosts. This process must be tightly regulated to prevent spurious incorporation of defective viral genomes, subgenomic RNAs or host nucleic acids, resulting in non-infectious particles. Many viruses encode specific packaging signals (PS) which interact with viral structural proteins during particle assembly. While PSs have been identified in model coronaviruses from mouse and swine, genome packaging in human coronaviruses is still poorly understood.
We focussed our initial analyses on Mouse Hepatitis Virus (MHV), for which there is a characterised PS and mutant, and its close relative, human coronavirus OC43. Purified virions were subjected to high throughput RNA structure mapping, to establish the first high quality full-genome MHV and
OC43 RNA structures. Next, we performed cross-linking and immunoprecipitation of the viral structural proteins Membrane and Nucleocapsid. This allowed us to examine structural protein binding sites around the PS, as well as to identify other regions of the genome which are specifically bound. Concurrently, we developed both reverse genetics and transfection-based virus-like particle
systems for OC43; these allowed us to mutationally characterise the interactions between the structural proteins and the PS, to unpick the mechanism of genome packaging.
Together, our work provides an initial framework to understand the essential features of two coronavirus PSs. In the future, this will allow us to analyse more divergent coronaviruses to establish the fundamental principles of genome packaging family-wide.