Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus
ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes six highly conserved core replication proteins essential for the viral lytic DNA synthesis, including ORF6 (single-stranded DNA-binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (prim...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02254-23 |
| _version_ | 1797393639134986240 |
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| author | Lee-Wen Chen Shie-Shan Wang Li-Yu Chen Hsiao-Yun Huang Si-min He Chien-Hui Hung Chun-Liang Lin Pey-Jium Chang |
| author_facet | Lee-Wen Chen Shie-Shan Wang Li-Yu Chen Hsiao-Yun Huang Si-min He Chien-Hui Hung Chun-Liang Lin Pey-Jium Chang |
| author_sort | Lee-Wen Chen |
| collection | DOAJ |
| description | ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes six highly conserved core replication proteins essential for the viral lytic DNA synthesis, including ORF6 (single-stranded DNA-binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor). Since the protein-protein interactions among KSHV core replication proteins are largely unknown, this study aimed to decipher their interrelationships. Herein, we propose a protein-protein interaction network of these six core replication proteins according to the results obtained from confocal fluorescence microscopy, coimmunoprecipitation, and mammalian two-hybrid (GAL4/VP16) reporter assays. In this interaction network, ORF40/41 plays a central role in the connection of different replication subcomplexes. In addition to the well-conserved helicase-primase subcomplex (consisting of ORF44, ORF56, and ORF40/41) and the replisome subcomplex (consisting of ORF9 and ORF59), our data also suggest that several discrete, stable subcomplexes exist in the cell nucleus. Among these replication subcomplexes in the nucleus, the tetrameric subcomplex composed of ORF44, ORF56, ORF40/41, and ORF6 exhibited the ability to trigger a DNA damage response. By using an established GAL4/VP16-based reporter system and confocal fluorescence microscopy, we additionally found that the heat shock protein 90 (Hsp90) inhibitor, radicicol, significantly inhibited the formation of both the helicase-primase subcomplex and the replisome subcomplex in a dose-dependent manner. Collectively, these data not only provide further insights into the interaction and assembly of KSHV-encoded core replication proteins but also suggest a critical role of Hsp90 in assisting the construction of the viral core replication machinery. IMPORTANCE Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell’s DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi’s sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation. |
| first_indexed | 2024-03-09T00:06:44Z |
| format | Article |
| id | doaj.art-e9240cb42d7844e2bc7e39fa7f50f217 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-09T00:06:44Z |
| publishDate | 2023-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-e9240cb42d7844e2bc7e39fa7f50f2172023-12-12T13:17:19ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-12-0111610.1128/spectrum.02254-23Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirusLee-Wen Chen0Shie-Shan Wang1Li-Yu Chen2Hsiao-Yun Huang3Si-min He4Chien-Hui Hung5Chun-Liang Lin6Pey-Jium Chang7Department of Respiratory Care, Chang Gung University of Science and Technology , Chiayi, TaiwanDepartment of Pediatric Surgery, Chang Gung Memorial Hospital , Chiayi, TaiwanGraduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University , Taoyuan, TaiwanGraduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University , Taoyuan, TaiwanDepartment of Pediatric Surgery, Chang Gung Memorial Hospital , Chiayi, TaiwanGraduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University , Taoyuan, TaiwanDepartment of Nephrology, Chang Gung Memorial Hospital , Chiayi, TaiwanGraduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University , Taoyuan, TaiwanABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes six highly conserved core replication proteins essential for the viral lytic DNA synthesis, including ORF6 (single-stranded DNA-binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor). Since the protein-protein interactions among KSHV core replication proteins are largely unknown, this study aimed to decipher their interrelationships. Herein, we propose a protein-protein interaction network of these six core replication proteins according to the results obtained from confocal fluorescence microscopy, coimmunoprecipitation, and mammalian two-hybrid (GAL4/VP16) reporter assays. In this interaction network, ORF40/41 plays a central role in the connection of different replication subcomplexes. In addition to the well-conserved helicase-primase subcomplex (consisting of ORF44, ORF56, and ORF40/41) and the replisome subcomplex (consisting of ORF9 and ORF59), our data also suggest that several discrete, stable subcomplexes exist in the cell nucleus. Among these replication subcomplexes in the nucleus, the tetrameric subcomplex composed of ORF44, ORF56, ORF40/41, and ORF6 exhibited the ability to trigger a DNA damage response. By using an established GAL4/VP16-based reporter system and confocal fluorescence microscopy, we additionally found that the heat shock protein 90 (Hsp90) inhibitor, radicicol, significantly inhibited the formation of both the helicase-primase subcomplex and the replisome subcomplex in a dose-dependent manner. Collectively, these data not only provide further insights into the interaction and assembly of KSHV-encoded core replication proteins but also suggest a critical role of Hsp90 in assisting the construction of the viral core replication machinery. IMPORTANCE Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell’s DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi’s sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.https://journals.asm.org/doi/10.1128/spectrum.02254-23KSHVDNA replication complexprimasehelicaseDNA polymeraseHsp90 |
| spellingShingle | Lee-Wen Chen Shie-Shan Wang Li-Yu Chen Hsiao-Yun Huang Si-min He Chien-Hui Hung Chun-Liang Lin Pey-Jium Chang Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus Microbiology Spectrum KSHV DNA replication complex primase helicase DNA polymerase Hsp90 |
| title | Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus |
| title_full | Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus |
| title_fullStr | Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus |
| title_full_unstemmed | Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus |
| title_short | Interaction and assembly of the DNA replication core proteins of Kaposi’s sarcoma-associated herpesvirus |
| title_sort | interaction and assembly of the dna replication core proteins of kaposi s sarcoma associated herpesvirus |
| topic | KSHV DNA replication complex primase helicase DNA polymerase Hsp90 |
| url | https://journals.asm.org/doi/10.1128/spectrum.02254-23 |
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