Establishment and maintenance of Kaposi's sarcoma-associated herpesvirus latency in B cells

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma and is also associated with two B-cell lymphoproliferative diseases, primary effusion lymphoma and the plasmablastic form of multicentric Castleman's disease. KSHV is also found in the B-cell fraction of peripheral blood mononucleocytes of some KS patients. Despite in vivo infection of B cells and the ability of KSHV to infect many cell types in culture, to date B cells in culture have been resistant to KSHV infection. However, as shown here, the lack of infection is not due to the inability of B cells to support latent KSHV infection. When KSHV DNA is introduced into B cells, the virus is maintained as an episome and can establish and maintain latency over the course of months. As in all primary effusion lymphoma cell lines, there is a low level of spontaneous lytic replication in latently infected BJAB cells. Importantly, viral gene expression is similar to that of primary effusion lymphoma cell lines. Furthermore, the virus can be reactivated to higher levels with specific stimuli and transmitted to other cells, indicating that this is a productive infection. Thus B cells in culture are capable of establishing, maintaining, and reactivating from latency. These studies provide a controlled system to analyze how KSHV alters B cells during KSHV latency and reactivation.

FIG. 1.

Creation of KSHV-GFP1 recombinant virus. (A) Strategy for generation of KSHV-GFP1 recombinant virus. The structure of the first 30 kb of the KSHV genome is shown at the top. Viral genes are indicated as open boxes. The selective marker GFP-puro under the control of the CMV IE promoter was integrated between the ORF 11 and ORF K2 genes in the KSHV genome by homologous recombination in BCBL-1 cells with the recombination plasmid pKSGFP. Genomic structure of the resulting reconstituted virus KSHV-GFP1 is shown below. (B) Southern analysis of virion DNA isolated from induced BCBL-1 and KSHV-Vero supernatants. The probes used were the full-length vIL-6 gene (left panel), or the enhanced green fluorescence protein gene (right panel). (C) Photomicrograph of Vero cells infected with KSHV-GFP1 and selected with puromycin. (Left column) Phase. (Right column) Fluorescence for GFP.

FIG. 2.

Photomicrographs of BJAB cells and KSHV-BJAB cells 2 weeks and 2 months after puromycin selection, showing phase (left panel), eGFP fluorescence (middle-left panel), 4′,6′-diamidino-2-phenylindole (DAPI) (middle-right panel), and LANA red fluorescence (right panel).

FIG. 3.

KSHV is episomal in KSHV-BJAB cells. BCBL-1 and KSHV-BJAB cells were induced with Adeno-50 (Ad50) and 2 mM sodium butyrate (NaB). Forty-eight hours after induction, uninduced and induced cells were examined by Gardella gel electrophoresis to detect KSHV episomes. KSHV virion was used as a control. ³²P-labeled full-length vIL-6 was used as a probe.

FIG. 4.

Photomicrographs of KSHV-BJAB cells 48 h postinduction showing 4′,6′-diamidino-2-phenylindole (DAPI) (left panel), eGFP fluorescence (middle panel), and ORF59 red fluorescence (right panel). BJAB cells (upper panel) were included as a negative control. Quantification is described in the text. Ad50, Adeno-50; NaB, sodium butyrate.

FIG. 5.

Recombinant KSHV virus reconstituted from induced KSHV-BJAB cells can be transmitted to hTERT-immortalized microvascular endothelial (TIME) cells. Phase (upper panel) and eGFP fluorescence (lower panel) are shown. Three days postinduction, supernatant was briefly centrifuged to remove cell debris and then filtered through a 0.45-μm-pore filter. The resulting supernatant was used to infect TIME cells on a 12-well plate. Twenty-four hours postinfection, phase and fluorescence pictures were taken. Supernatant from BJAB cells was used to infect TIME cells (left panel) as a negative control. TIME cells infected with supernatants from uninduced (middle-left panel), TPA-induced (middle-right panel) and Adeno-50 (Ad50) plus 2 mM sodium butyrate (NaB)-induced (right panel) KSHV-BJAB cells are shown. Quantification is described in the text.

FIG. 6.

Northern blot analysis of uninduced and induced KSHV-BJAB cells. BCBL-1 cells and KSHV-BJAB cells were induced by Adeno-50 (Ad50) plus 2 mM sodium butyrate (NaB). Forty-eight hours postinduction, mRNA was extracted and analyzed. BJAB cells were included as a negative control. Uninduced and induced BCBL-1 cells were included as a positive control. (A) Northern blot probed with radiolabeled sequences containing viral cyclin (vCyclin), PAN RNA, and GAPDH consecutively. (B) Northern blot probed with vIL-6 and GAPDH consecutively.