Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: Application for genetic analysis

Fu Chun Zhou, Yan Jin Zhang, Jian Hong Deng, Xin Ping Wang, Hong Yi Pan, Evelyn Hettler, Shou Jiang Gao

Research output: Contribution to journalArticle

203 Citations (Scopus)

Abstract

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 ≈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 ≈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.

Original languageEnglish (US)
Pages (from-to)6185-6196
Number of pages12
JournalJournal of Virology
Volume76
Issue number12
DOIs
StatePublished - 2002

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Human herpesvirus 8
Bacterial Artificial Chromosomes
Human Herpesvirus 8
bacterial artificial chromosomes
genetic techniques and protocols
Infection
infection
virion
Virion
cells
Herpesviridae Infections
Kaposi's Sarcoma
sarcoma
Green Fluorescent Proteins
green fluorescent protein
genome
Acetates
Artificial Chromosomes
pathogenesis
acetates

ASJC Scopus subject areas

  • Immunology

Cite this

Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome : Application for genetic analysis. / Zhou, Fu Chun; Zhang, Yan Jin; Deng, Jian Hong; Wang, Xin Ping; Pan, Hong Yi; Hettler, Evelyn; Gao, Shou Jiang.

In: Journal of Virology, Vol. 76, No. 12, 2002, p. 6185-6196.

Research output: Contribution to journalArticle

Zhou, Fu Chun ; Zhang, Yan Jin ; Deng, Jian Hong ; Wang, Xin Ping ; Pan, Hong Yi ; Hettler, Evelyn ; Gao, Shou Jiang. / Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome : Application for genetic analysis. In: Journal of Virology. 2002 ; Vol. 76, No. 12. pp. 6185-6196.
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abstract = "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 ≈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 ≈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.",
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