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

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 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.

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.

FIG. 2.

Genetic analysis of BAC36. (A) Field inversion gel electrophoresis of PmeI-digested BAC DNA. BAC DNA was isolated from chloramphenicol-resistant E. coli colonies transformed with cl1, cl2, and cl3 episomal DNA recovered from 293 cell lines. pMHGP36 was included as a control. (B, F, and G) KpnI-digested BAC DNA from bacterial colonies recovered from 293 cell lines was resolved on a 0.8% agarose gel. Episomal DNA prepared from BCBL-1 cells was used as the control. (C, D, E, and H) Southern hybridization with the following probes: pMHGP (C); L36 (D); and inserts from six cosmid clones covering the entire BC-1 KSHV genome (E and H). Restriction bands identified on gels or by Southern blot hybridization are indicated with short lines. The band marked with a square was only present in BCBL-1 cells, which was shifted to the band marked with a triangle in BAC DNA because of the insertion of the replacement plasmid. Panels C and D show the same membrane from panel B; panel E shows the membrane from panel F; panel H shows the membrane from panel G. Lanes M, size markers.

FIG. 2.

Genetic analysis of BAC36. (A) Field inversion gel electrophoresis of PmeI-digested BAC DNA. BAC DNA was isolated from chloramphenicol-resistant E. coli colonies transformed with cl1, cl2, and cl3 episomal DNA recovered from 293 cell lines. pMHGP36 was included as a control. (B, F, and G) KpnI-digested BAC DNA from bacterial colonies recovered from 293 cell lines was resolved on a 0.8% agarose gel. Episomal DNA prepared from BCBL-1 cells was used as the control. (C, D, E, and H) Southern hybridization with the following probes: pMHGP (C); L36 (D); and inserts from six cosmid clones covering the entire BC-1 KSHV genome (E and H). Restriction bands identified on gels or by Southern blot hybridization are indicated with short lines. The band marked with a square was only present in BCBL-1 cells, which was shifted to the band marked with a triangle in BAC DNA because of the insertion of the replacement plasmid. Panels C and D show the same membrane from panel B; panel E shows the membrane from panel F; panel H shows the membrane from panel G. Lanes M, size markers.

FIG. 3.

Determination of KSHV genomic copy number in BAC36-infected cells by Southern blot hybridization. BamHI-digested DNAs from 293 cells, BAC36-infected 293 cells, and KSHV-infected BC-1 and BCBL-1 cells were resolved in an agarose gel, transferred to a nylon membrane, and hybridized with KS330, KS631, and vIRF probes. Then 10 μg of DNA from approximately 5 × 10⁵ BC-1 cells containing 2 × 10⁷ to 4 × 10⁷ KSHV genomes was used as the copy number control. Purified BAC36 DNA (15 ng) was also used as a control. β-Actin was used to calibrate any loading differences between cell lines. BAC36-infected 293 cell lines were determined to harbor 1 to 2 copies of BAC36 per cell.

FIG. 4.

Detection of KSHV gene expression in BAC36-infected cells by Northern blot hybridization. Total RNA was extracted from uninduced and TPA-induced BAC36-infected 293 cells, 293 cells, and BCBL-1 cells, resolved on agarose gels, transferred to nylon membranes, and hybridized with riboprobes of different KSHV genes. The β-actin probe was used to monitor RNA loading.

FIG. 5.

Detection of KSHV gene expression by IFA. Uninduced (A, E, G, and H) and TPA-induced (B, C, D, and F) BAC36-infected 293 cells (A, B, E, and F), 293 cells (D and H), and BCBL-1 cells (C and G) were stained for LNA (E to H) or mCP (A to D) protein expression. Cells shown in panels A′, B′, E′, and F′, which were from the same fields as those shown in panels A, B, E, and F, respectively, expressed GFP, indicating the presence of BAC36.

FIG. 6.

Infection of 293 cells, HeLa cells, and E6E7-immortalized human dermal microvascular endothelial cells (EC) with culture supernatant from TPA-induced, BAC36-infected 293 cells. Cells were observed 2 days postinfection with a fluorescence microscope.

FIG. 7.

Generation of a KSHV mutant with a deletion of the vIRF gene. (A) A Kan cassette PCR product flanking sequences from outside the vIRF gene was electroporated into E. coli previously transformed with BAC36 and plasmid pGETrec. Episomal DNA isolated from bacterial colonies containing the KSHV mutant in which the vIRF gene was replaced with Kan after a second round of transformation into E. coli to eliminate the pGETrec plasmid was analyzed by PCR assay, restriction enzyme digestion, and Southern blot hybridization. The mutant virus was then reconstituted in 293 cells and used for phenotype analysis. (B) PCR assay to confirm the deletion of the vIRF gene in BAC36. BAC36 produced a band of 1.3 kb, while the mutant virus produced a band of 1.7 kb because of the size differences between the vIRF gene and Kan. (C) KpnI digestion of BAC36 and mutant virus and Southern blot hybridization with Kan as the probe.

FIG. 7.

Generation of a KSHV mutant with a deletion of the vIRF gene. (A) A Kan cassette PCR product flanking sequences from outside the vIRF gene was electroporated into E. coli previously transformed with BAC36 and plasmid pGETrec. Episomal DNA isolated from bacterial colonies containing the KSHV mutant in which the vIRF gene was replaced with Kan after a second round of transformation into E. coli to eliminate the pGETrec plasmid was analyzed by PCR assay, restriction enzyme digestion, and Southern blot hybridization. The mutant virus was then reconstituted in 293 cells and used for phenotype analysis. (B) PCR assay to confirm the deletion of the vIRF gene in BAC36. BAC36 produced a band of 1.3 kb, while the mutant virus produced a band of 1.7 kb because of the size differences between the vIRF gene and Kan. (C) KpnI digestion of BAC36 and mutant virus and Southern blot hybridization with Kan as the probe.