FIG. 1.

Molecular cloning of infectious KSHV and establishment of a genetic manipulation system. (A) Schematic illustration of molecular cloning of KSHV genome. Replacement plasmid (RP) pMHGP36, containing the BAC vector, Hyg, GFP cassette, and the L36 locus as the flanking sequences, was integrated into the PmeI site between orf18 and orf19 in the long unique region (LUR) of the viral genome by homologous recombination in KSHV-infected BCBL-1 cells. (B) Strategy for the isolation of infectious KSHV. pMHGP36 was transfected into KSHV-infected BCBL-1 cells to generate recombinant virus. Cell clones were selected for hygromycin resistance, GFP expression, and production of infectious virions after TPA induction. Episomal DNA from selected cell clones was then prepared and transformed into E. coli to obtain recombinant KSHV BAC36. The rescued episomes were transfected into 293 cells and induced to produce infectious virions, which were used to infect normal 293 cells. BAC36-infected 293 cell lines were then established with hygromycin selection. The selected cells were induced to produce infectious recombinant virions. TR, terminal repeat.