Phase Separation: The Surprising Process Behind DNA Virus Replication Revealed by New Study

Researchers from the Children’s Hospital of Philadelphia (CHOP) have found that viral proteins use a process known as phase separation to coordinate the complex process of replicating viral genomes and packaging them into viral particles.

Their findings, published in Nature, shed light on how viruses hijack host cellular processes to produce infectious offspring and may have implications for gene therapy delivery and potential therapeutic interventions.

The findings answer “a fundamentally important question: how a viral nucleic acid gets inside a particle so that viral offspring can be delivered to cells,” says Matthew Charman, PhD, a research associate in the Weitzman Lab at Children’s Hospital of Philadelphia. “These findings have broad implications, from potential therapeutic interventions to improved gene therapy delivery, in addition to expanding our understanding of basic cell biology.”

Viruses exploit cellular processes to reproduce and create contagious offspring crucial for viral propagation. In order to achieve this, they must replicate their genetic material and encapsulate it into viral particles for the infection cycle to progress. Nevertheless, it is not well understood how genome replication, particle assembly, and genome packaging are synchronized in a crowded nuclear environment.

“If we think of viral replication as an old-fashioned milk assembly line, we know how the milk bottles are formed and that they come out filled, but prior to this study, the process of filling them was somewhat of a black box,” adds senior author Matthew D. Weitzman.

This study suggests “that the viral particle forms around the viral genome. Extending the analogy, many have assumed that the bottle must be made before being filled, but it turns out the bottle is actually formed around the milk. Led by Dr. Charman, we have shown that a biophysical process known as phase separation allows this process to occur in an orderly, coordinated fashion.”

Recent studies suggest that phase separation generates membraneless compartments in virus-infected cells. These compartments, also known as biomolecular condensates (BMCs), can concentrate biomolecules in an enriched dense phase, which is crucial for regulating biological processes. Despite the linkage of BMCs to several viral processes, there has been insufficient evidence that phase separation contributes functionally to the assembly of infectious viral offspring in infected cells. To explore the potential role of BMCs in this process, researchers investigated adenovirus, a nuclear-replicating DNA virus. The distinct involvement of adenovirus proteins in genome replication and particle assembly and genome packaging allowed researchers to dissect and identify the role of phase separation in specific viral processes.

Using advanced techniques such as HPG labeling and fluorophore click chemistry, the researchers established that the adenovirus 52 kDa protein, which is responsible for assembly and packaging, creates its own membraneless structures through phase separation. This protein plays a crucial role in the coordinated assembly of new infectious viral particles.

The study revealed that the 52 kDa protein not only organizes viral capsid proteins into nuclear BMCs but also plays a pivotal role in the formation of complete, packaged particles containing viral genomes.

Furthermore, the researchers conducted experiments with a mutant adenovirus that lacked the 52 kDa protein and observed that incomplete capsids formed when viral BMCs were absent.

These findings demonstrate that altering the formation of membraneless structures within the cell can hinder the production of viral offspring by disrupting the orderly assembly line.

“Now knowing these steps, the question becomes: could we reengineer viruses based on this biological process to, for example, become better delivery vehicles for innovations like gene therapy?” Dr. Charman adds. “Understanding how viruses are made opens up a world where we could not only potentially target those viruses more effectively in the future but also create gene therapy tools that lack the limitations of current delivery approaches.”

Source:10.1038/s41586-023-05887-y

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