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Journal of Virology, June 2002, p. 6185-6196, Vol. 76, No. 12
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.12.6185-6196.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Efficient Infection by a Recombinant Kaposi's Sarcoma-Associated Herpesvirus Cloned in a Bacterial Artificial Chromosome: Application for Genetic Analysis

Fu-Chun Zhou,1 Yan-Jin Zhang,1 Jian-Hong Deng,1 Xin-Ping Wang,1 Hong-Yi Pan,1 Evelyn Hettler,1 and Shou-Jiang Gao1,2,3,4*

Departments of Pediatrics,1 Microbiology,2 Children's Cancer Research Center, The University of Texas Health Science Center at San Antonio,3 San Antonio Cancer Institute, San Antonio, Texas 782294

Received 7 January 2002/ Accepted 11 March 2002

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 {approx}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 {approx}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.

* Corresponding author. Mailing address: Department of Pediatrics, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229. Phone: (210) 567-5248. Fax: (210) 567-6305. E-mail: gaos{at}uthscsa.edu.

Journal of Virology, June 2002, p. 6185-6196, Vol. 76, No. 12
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.12.6185-6196.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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