Development and Characterization of an Inducible Bacterial Artificial Chromosome System for Studying Lytic Replication and Pathogenesis of Kaposi's Sarcoma-Associated Herpesvirus.

Bacterial artificial chromosome (BAC) is widely used to manipulate herpesvirus genome and generate recombinant virus. Here, we developed a new KSHV BACmid, namely as iBAC, by replacing the EGFP with TET3G transactivator under EF1α promoter and inserted Tet response elements in the promoter of RTA in the original KSHV BAC16 clone and characterized KSHV lytic replication in SLK-iBAC cells. SLK-iBAC cells developed more efficient lytic replication and generated more progeny virus than iSLK-BAC16 cells upon the same conditions of doxycycline treatment. Since SLK-iBAC cells only occupied hygromycin selection marker, it is convenient to generate cellular gene knockout via lentivirus-mediated CRISPR-Cas9 or stably express viral or cellular gene via lentivirus followed by antibiotic selection, making iBAC system a better tool to identify cellular targets of viral proteins in the context of virus infection or study the role of viral or cellular genes for KSHV lytic replication and pathogenesis. In addition, iBAC is color-free and can be utilized to track subcellular localization of viral proteins or colocalization between different viral proteins by introducing fusing fluorescent proteins into the BAC backbone. Therefore, the new KSHV iBAC is a powerful inducible tool to study KSHV lytic replication and pathogenesis in cell model.