Deng Hongyu's research group revealed that liquid-liquid phase separation mediates the formation of herpesvirus assembly intervals

Liquid-liquid phase separation (LLPS) plays an important role
in human health and disease as an effective mechanism for membrane-free organelle formation.
As an intracellular parasite, in order to promote its own reproduction and transmission, the virus will also establish a special membrane or non-membrane structure in its infected cells to improve its own replication efficiency
.
Viral assembly is an essential but complex process in the viral life cycle, especially for viruses with large genomes that encode numerous structural proteins, such as herpesviruses
.

Herpesviruses are ancient DNA viruses that can establish persistent infections
in humans and animals.
A mature herpesvirus particle is divided from the inside to the outside: double-stranded DNA genome, capsid, interstitium and capsule.

In the vast cytoplasmic space, the orderly and efficient assembly of dozens of structural proteins into mature virions is a difficult task, and the mechanism is still poorly
understood.

The team of Deng Hongyu of the Institute of Biophysics of the Chinese Academy of Sciences has been systematically studying the molecular mechanism of herpesvirus assembly, and has identified a variety of viral proteins such as interstitial proteins
that play a key role in viral morphogenesis.
His latest research found that γ herpes virus forms a special region, the "virus assembly interval", through the liquid-liquid phase separation mechanism in infected cells, and the viral interstitial protein ORF52 with phase separation properties is crucial
for the formation of virus assembly interval 。 The study, which is the first to reveal the important role of phase separation in the assembly of γ herpesviruses, is titled "Liquid-liquid Phase Separation Mediates the Formation of Herpesvirus Assembly Compartments," published online in the Journal of Cell Biology on October 17, 2022 on
.

To track viral morphogenesis in real time, the researchers first constructed an MHV-68 recombinant virus that expresses a fused fluorescent protein as a marker
.
Live-cell imaging and photoelectric correlation experiments showed that in the late stage of infection, viral assembly intervals begin to form in the cytoplasm, recruiting interstitial proteins and capsules containing viral glycoproteins to improve the efficiency of viral assembly; Over time, the virus assembly interval gradually increased
.
A series of experiments such as fluorescence bleaching recovery and chemical reagent treatment have shown that the virus assembly interval is a special structure
with dynamic and liquid phase properties.

By screening a variety of interstitial proteins encoded by viruses, the researchers found that only ORF52 has the ability of liquid-liquid phase separation, and the deletion of ORF52 leads to the complete disappearance of the viral assembly interval in infected cells, indicating that ORF52 is crucial
for the formation of viral assembly interval.
In addition, it was found that while both DNA and RNA can drive ORF52 phase separation, RNA is more likely than DNA to be involved in the formation
of viral assembly intervals in the later stages of viral replication.

The researchers further constructed a truncated body of ORF52 protein expression on the viral genome or mutated the conserved basic amino acid clusters in the C-terminal disordered region of ORF52 protein, and performed ultrastructure and functional analysis on the cells infected by the mutant virus strain, and the results showed that the liquid-liquid phase separation of ORF52 played an important role
in the formation of virus assembly intervals and the maturation of virus particles.

In summary, this study is the first to find that γ herpes virus forms a virus assembly interval through a liquid-liquid phase separation mechanism in the late stage of infection, in which the viral interstitial protein ORF52 plays a crucial role
.
These findings expand the scope of phase separation in cell biology and provide a new direction
for the study of the mechanism of herpesvirus morphogenesis.

Pattern diagram.
ORF52 mediates the formation of viral assembly intervals through liquid-liquid phase separation mechanism and promotes herpesvirus assembly
.

Zhou Sheng, special research assistant (postdoctoral fellow) at the Institute of Biophysics, Chinese Academy of Sciences, is the first author of the paper, and researcher Deng Hongyu is the corresponding author
of the paper.
The research work was supported
by the National Natural Science Foundation of China, the Ministry of Science and Technology, and the Chinese Academy of Sciences.

Article link: https://doi.
org/10.
1083/jcb.
202201088

(Contributed by: Deng Hongyu Research Group)