Deng Hongyu's research group revealed the molecular mechanism of herpesvirus interstitial protein involved in virus assembly

Herpesviruses are a double-stranded DNA virus with a capsule that can infect almost all vertebrates, including humans, and some invertebrates
.
Herpesviruses can be divided into three subfamilies
: α, β, and γ.
Among them γ herpesvirus, also known as tumor-associated herpesvirus, is closely
related to the occurrence of some tumors.
EBV and KSHV are γ herpes viruses capable of infecting humans and are closely related
to the occurrence of diseases such as nasopharyngeal carcinoma and Kaposi's sarcoma, respectively.
Murine herpesvirus 68 (MHV-68) is also a γ herpesvirus and is a good model
for studying γ herpesviruses.

The structure of the herpes virus is, from the inside out, the genomic DNA, the capsid, the interstitial protein layer, and the capsule.

The assembly and release of the herpes virus begins within the nucleus of the
host cell.
First, the genomic DNA is wrapped by a protein shell to form a nucleocapsid, and then enters the cytoplasm through capsulation-decapsulation with the assistance of a virus-encoded nuclear release complex; Viral particles undergo interstitialization in the cytoplasm to obtain a series of interstitial proteins, and germinate on the intracellular membrane system to obtain capsules; Finally, mature viral particles are released outside
the cell by exocytosis.
The interstitialization of herpesviruses is a very critical step in the maturation process of the virus, requiring complex interactions
between interstitial proteins and nucleocapsids and between interstitial proteins and interstitial proteins.
However, the mechanism of herpesvirus assembly remains unclear
.

The research group of Deng Hongyu of the Institute of Biophysics of the Chinese Academy of Sciences has been committed to the systematic study of the molecular mechanism of herpesvirus assembly and release, and has identified the nuclear release complex and a number of interstitial proteins necessary for herpesvirus assembly, such as ORF33, ORF45 and ORF52, and found that ORF52 mediates the formation
of "virus assembly interval" in the cytoplasm through the liquid-liquid phase separation mechanism.
On October 27, 2022, his research group published a paper entitled "The interaction between tegument proteins ORF33 and ORF45 plays an essential role in cytoplasmic virion maturation" in the Journal of Virology of a gammaherpesvirus", further revealing that MHV-68's two interstitial proteins, ORF33 and ORF45, interact, which are necessary
for the maturation of the virus in the cytoplasm.

Previous studies of the MHV-68 protein interaction network found that the interstitial protein ORF33 interacted
with ORF45.
The researchers demonstrated that ORF33 and ORF45 interact
under both plasmid co-transfection conditions and viral infection conditions through co-immunoprecipitation experiments.
When expressed alone, ORF45 is mainly localized within the nucleus, and ORF33 is localized to the nucleus and cytoplasm
.
When ORF33 and ORF45 are co-expressed, most of the ORF33 in the cytoplasm is transferred into the nucleus and colocalized
with ORF45.
In virus-infected cells, ORF33 and ORF45 are colocalized
in both the nucleus and cytoplasm.

To further explore the function of the ORF33-ORF45 interaction during MHV-68 maturation, the researchers identified 23 amino acids at the C-terminal of ORF45 that are critical
for its interaction with ORF33.
Next, the researchers constructed a mutant virus (ORF45DC23) with 23 amino acids missing at the C-terminal of ORF45 at the level of the viral genome by bacterial artificial chromosome (BAC) technology, and found that the 23 amino acids at the C-terminal of ORF45 are necessary
for the transfer of ORF33 into the nucleus and the production of infectious virions.
Observation of viral morphogenesis by electron microscopy showed that 23 amino acids at the C-terminal of ORF45 were necessary
for the maturation of the virus in the cytoplasm.
In addition, the researchers also identified 17 amino acids at the N-terminal of ORF33 that are essential for its interaction with ORF45, and that the 17 amino acids at the N-terminal of ORF33 are necessary
for the localization of ORF33 and the maturation of the virus in the cytoplasm.

Fig.
The interstitial proteins ORF33 and ORF45 interactions are necessary for the maturation of viral particles in the cytoplasm

In summary, the study showed that MHV-68 mesenchymal protein ORF33 interacted with ORF45, and 23 amino acids at the C-terminal of ORF45 and 17 amino acids at the N-terminal of ORF33 were essential
for the interaction.
The interaction of ORF33 with ORF45 is necessary
for the localization of ORF33 and the maturation of viral particles in the cytoplasm.
These findings reveal the molecular mechanisms by
which herpesvirus interstitial protein interactions are involved in the maturation of viral particles.

Researcher Deng Hongyu of the Institute of Biophysics, Chinese Academy of Sciences is the corresponding author of this paper, and Jia Xing and Sun Long, graduated doctoral students of the Institute of Biophysics, Chinese Academy of Sciences, are co-first authors
.
The research was supported
by funding from the Ministry of Science and Technology, the National Natural Science Foundation of China and the Chinese Academy of Sciences.

Article link: https://doi.
org/10.
1128/jvi.
01073-22

(Contributed by: Deng Hongyu Research Group)