Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.
Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma and several other malignancies. The lack of an efficient infection system has impeded the understanding of KSHV-related pathogenesis. A genetic approach was used to isolate infectious KSHV. Recombinant bacteria artificial chromosome (BAC) KSHV containing hygromycin resistance and green fluorescent protein (GFP) markers was generated by homologous recombination in KSHV-infected BCBL-1 cells. Recombinant KSHV genomes from cell clones that were resistant to hygromycin, expressed GFP, and produced infectious virions after induction with tetradecanoyl phorbol acetate (TPA) were rescued in Escherichia coli and reconstituted in 293 cells. Several 293 cell lines resulting from infection with recombinant virions induced from a full-length recombinant KSHV genome, named BAC36, were obtained. BAC36 virions established stable latent infection in 293 cells, harboring 1 to 2 copies of viral genome per cell and expressing viral latent proteins, with approximately 0.5% of cells undergoing spontaneous lytic replication, which is reminiscent of KSHV infection in Kaposi's sarcoma tumors. TPA treatment induced BAC36-infected 293 cell lines into productive lytic replication, expressing lytic proteins and producing virions that efficiently infected normal 293 cells with a approximately 50% primary infection rate. BAC36 virions were also infectious to HeLa and E6E7-immortalized human endothelial cells. Since BAC36 can be efficiently shuttled between bacteria and mammalian cells, it is useful for KSHV genetic analysis. The feasibility of the system was illustrated through the generation of a KSHV mutant with the vIRF gene deleted. This cellular model is useful for the investigation of KSHV infection and pathogenesis.
Zhou
Fu-Chun
FC
Department of Pediatrics, The University of Texas Health Science Center at San Antonio, 78229, USA.
Zhang
Yan-Jin
YJ
Deng
Jian-Hong
JH
Wang
Xin-Ping
XP
Pan
Hong-Yi
HY
Hettler
Evelyn
E
Gao
Shou-Jiang
SJ
D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016679:Genome, Viral; D006367:HeLa Cells; D006566:Herpesviridae Infections / Q000821:virology; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D009154:Mutation; D011995:Recombination, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion; D017735:Virus Latency
D014764:Viral Proteins