Biomolecular Fluorescence Complementation Profiling and Artificial Intelligence Structure Prediction of the Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Interaction

• Published in: International journal of molecular sciences Vol. 23; no. 17; p. 9647
• Main Authors: Maeda, Yoshiko, Watanabe, Tadashi, Izumi, Taisuke, Kuriyama, Kazushi, Ohno, Shinji, Fujimuro, Masahiro
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 25.08.2022; MDPI
• Subjects: Acids; AlphaFold2; Artificial Intelligence; artificial intelligence (AI) structure prediction; bimolecular fluorescence complementation (BiFC); Complementation; Effusion; Epstein-Barr virus; Flow cytometry; Fluorescence; Gene expression; Gene Expression Regulation, Viral; Genes; Herpes viruses; Herpesvirus 8, Human - genetics; Humans; Initiation complex; Kaposi's sarcoma; Kaposis sarcoma; Kaposi’s sarcoma-associated virus (KSHV); Plasmids; Primary effusion lymphoma; Protein interaction; Proteins; Residues; RNA polymerase; Sarcoma; Sarcoma, Kaposi; Structural analysis; Transcription factors; viral pre-initiation complex (vPIC); Virus Replication - genetics; artificial intelligence (AI) structure prediction; bimolecular fluorescence complementation (BiFC); Kaposi’s sarcoma-associated virus (KSHV); AlphaFold2; viral pre-initiation complex (vPIC)
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23179647

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. During KSHV lytic infection, lytic-related genes, categorized as immediate-early, early, and late genes, are expressed in a temporal manner. The transcription of late genes requires the virus-specific pre-initiation complex (vPIC), which consists of viral transcription factors. However, the protein-protein interactions of the vPIC factors have not been completely elucidated. KSHV ORF18 is one of the vPIC factors, and its interaction with other viral proteins has not been sufficiently revealed. In order to clarify these issues, we analyzed the interaction between ORF18 and another vPIC factor, ORF30, in living cells using the bimolecular fluorescence complementation (BiFC) assay. We identified four amino-acid residues (Leu29, Glu36, His41, and Trp170) of ORF18 that were responsible for its interaction with ORF30. Pull-down assays also showed that these four residues were required for the ORF18-ORF30 interaction. The artificial intelligence (AI) system AlphaFold2 predicted that the identified four residues are localized on the surface of ORF18 and are in proximity to each other. Thus, our AI-predicted model supports the importance of the four residues for binding ORF18 to ORF30. These results indicated that wet experiments in combination with AI may enhance the structural characterization of vPIC protein-protein interactions.