Comprehensive Analyses of Intraviral Epstein-Barr Virus Protein-Protein Interactions Hint Central Role of BLRF2 in the Tegument Network

Yuya Hara; Takahiro Watanabe; Masahiro Yoshida; H M Abdullah Al Masud; Hiromichi Kato; Tomohiro Kondo; Reiji Suzuki; Shutaro Kurose; Md Kamal Uddin; Masataka Arata; Shouhei Miyagi; Yusuke Yanagi; Yoshitaka Sato; Hiroshi Kimura; Takayuki Murata

doi:10.1128/jvi.00518-22

Comprehensive Analyses of Intraviral Epstein-Barr Virus Protein-Protein Interactions Hint Central Role of BLRF2 in the Tegument Network

J Virol. 2022 Jul 27;96(14):e0051822. doi: 10.1128/jvi.00518-22. Epub 2022 Jul 11.

Authors

Yuya Hara^#¹, Takahiro Watanabe^#¹, Masahiro Yoshida¹, H M Abdullah Al Masud^{1

2}, Hiromichi Kato¹, Tomohiro Kondo¹, Reiji Suzuki³, Shutaro Kurose¹, Md Kamal Uddin¹, Masataka Arata¹, Shouhei Miyagi¹, Yusuke Yanagi¹, Yoshitaka Sato^{1

4}, Hiroshi Kimura¹, Takayuki Murata^{1

5}

Affiliations

¹ Department of Virology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
² Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chattogram, Bangladesh.
³ Department of Complex Systems Science, Graduate School of Informatics, Nagoya University, Nagoya, Aichi, Japan.
⁴ PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
⁵ Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

^# Contributed equally.

Abstract

Protein-protein interactions (PPIs) are crucial for various biological processes. Epstein-Barr virus (EBV) proteins typically form complexes, regulating the replication and persistence of the viral genome in human cells. However, the role of EBV protein complexes under physiological conditions remains unclear. In this study, we performed comprehensive analyses of EBV PPIs in living cells using the NanoBiT system. We identified 195 PPIs, many of which have not previously been reported. Computational analyses of these PPIs revealed that BLRF2, which is only found in gammaherpesviruses, is a central protein in the structural network of EBV tegument proteins. To characterize the role of BLRF2, we generated two BLRF2 knockout EBV clones using CRISPR/Cas9. BLRF2 knockout significantly decreased the production of infectious virus particles, which was partially restored by exogenous BLRF2 expression. In addition, self-association of BLRF2 protein was found, and mutation of the residues crucial for the self-association affected stability of the protein. Our data imply that BLRF2 is a tegument network hub that plays important roles in progeny virion maturation. IMPORTANCE EBV remains a significant public health challenge, causing infectious mononucleosis and several cancer types. Therefore, the better understanding of the molecular mechanisms underlying EBV replication is of high clinical importance. As protein-protein interactions (PPIs) are major regulators of virus-associated pathogenesis, comprehensive analyses of PPIs are essential. Previous studies on PPIs in EBV or other herpesviruses have predominantly employed the yeast two-hybrid (Y2H) system, immunoprecipitation, and pulldown assays. Herein, using a novel luminescence-based method, we identified 195 PPIs, most of which have not previously been reported. Computational and functional analyses using knockout viruses revealed that BLRF2 plays a central role in the EBV life cycle, which makes it a valuable target for drug development.

Keywords: BLRF2; EBV; PPI; lytic cycle; tegument.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Epstein-Barr Virus Infections* / virology
Herpesvirus 4, Human* / genetics
Herpesvirus 4, Human* / physiology
Humans
Protein Interaction Maps*
Viral Proteins* / genetics
Virus Replication

Substances

Viral Proteins