In a twist on the question, “Which came first, the chicken or the egg?”, scientists have long faced a similar question about how human adenovirus replicates: “Which comes first, assembly of the viral particle, or packaging of the viral genome?” Now, in a new study published in Nature, researchers at Children’s Hospital of Philadelphia (CHOP) have answered that question, showing that viral proteins use a process called phase separation to coordinate production of viral progeny.
Emerging evidence suggests that membraneless compartments form inside virus-infected cells by phase separation. These membraneless compartments, known as biomolecular condensates (BMCs), can regulate biological processes by concentrating or sequestering biomolecules in an enriched dense phase, while limiting their concentration in the light phase. Although BMCs have been linked to several viral processes, there was insufficient evidence that phase separation contributes functionally to the assembly of infectious viral offspring in infected cells.
To investigate the potential role of BMCs in this process, the researchers studied adenovirus, a nuclear-replicating DNA virus. Because the adenovirus proteins involved in genome replication are distinct from those involved in particle assembly and genome packaging, the researchers reasoned focusing on this virus would allow them to dissect and more easily identify the role of phase separation in specific viral processes.
Through a variety of techniques, including homopropargylglycine (HPG) labeling and fluorophore click chemistry, the researchers demonstrated that the adenovirus 52 kDa protein – a dedicated assembly/packaging protein – makes its own membraneless structures through phase separation and plays a critical role in the coordinated assembly of new infectious particles. They showed that not only does the 52 kDa protein organize viral capsid proteins into nuclear BMCs, but also that this organization is essential for the assembly of complete, packaged particles containing viral genomes.
Additionally, the researchers performed experiments with a mutant adenovirus lacking the 52 kDa protein and showed that incomplete capsids formed in the absence of viral BMCs. Thus, the researchers were able to show that by altering the formation of these membraneless structures within the cell, the “assembly line” producing viral offspring no longer functioned properly.