Interaction and assembly of the DNA replication core proteins of Kaposi's sarcoma-associated herpesvirus

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.