As intracellular parasites, viruses must modulate the environment of infected cells to facilitate processes which are crucial for their own propagation, including virion assembly. This is an essential yet complex step in the life cycle of the virus, and it is particularly so for viruses with large genome sizes (i.e., large coding capacities for many structural proteins) and complicated virion structures such as herpesviruses.

Herpesviridae is a large family of DNA viruses that infect humans and other animals. A mature herpesvirion contains, from the inside to the outside, double-stranded DNA genome, capsid, tegument and envelope. Assembly of the dozens of structural proteins into mature virions in the vast cytosolic space is a daunting task, and the underlying mechanisms remained poorly understood.

In a study published in Journal of Cell Biology, a research group led by Prof. DENG Hongyu from the Institute of Biophysics of the Chinese Academy of Sciences demonstrated the formation of cytoplasmic virion assembly compartments (cVACs) which recruit viral tegument proteins and host vesicles containing viral glycoproteins for virus maturation during γ-herpesvirus infection, and showed that cVACs are membraneless compartments generated through a process of liquid-liquid phase separation (LLPS), mediated by a tegument protein, revealing the critical role of phase separation in γ-herpesvirus assembly.

Prof. DENG's group has been investigating the mechanisms involved in virion assembly and has identified a number of tegument proteins which play essential roles in virion morphogenesis. To investigate cytoplasmic virion assembly of γ-herpesviruses, the researchers constructed recombinant murine gammaherpesvirus 68 (MHV-68) expressing a fluorescently-tagged tegument protein. Using live-cell imaging and correlative light and electron microscopy, they discovered the formation of cVACs which start to accumulate in the cytoplasm as viral infection proceeds. They showed that cVACs are membrane-less structures and are formed by LLPS.

By examining a series of viral tegument proteins, the researchers identified ORF52, an abundant tegument protein in virion, that possesses LLPS properties. Analysis of an ORF52-null viral mutant indicated that ORF52 is required for cVAC formation as well as complete tegumentation and secondary envelopment of viral particles.

Furthermore, the researchers found that both DNA and RNA can drive LLPS of ORF52 in vitro. However, during the late stage of viral replication, they found that RNA colocalizes with ORF52 puncta and is likely to promote LLPS of ORF52.

Finally, by deleting domains or mutating a conserved cluster of basic amino acids in the intrinsically disordered region of ORF52 followed by ultrastructural and functional analyses, the researchers demonstrated that the LLPS properties of ORF52 are crucial for cVAC formation and virion production.

The study adds to the cellular processes that are facilitated by phase separation, and it provides novel insights into cVACs biogenesis and lays a foundation for further investigation of the complete details of herpesvirion morphogenesis.