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. 2004 Sep;78(18):10187-92.
doi: 10.1128/JVI.78.18.10187-10192.2004.

Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

Einat Deutsch  1 , Adina Cohen, Gila Kazimirsky, Sara Dovrat, Hadara Rubinfeld, Chaya Brodie, Ronit Sarid
Affiliations

Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

Einat Deutsch et al. J Virol. 2004 Sep.

Abstract

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.

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Figures

FIG. 1.
FIG. 1.
Effect of TPA and inhibitor of PKC on KSHV reactivation. Northern blot hybridizations with T1.1 and KSHV/ORF45 probes of total RNA extracted from BCP-1 (A) and BCBL-1 (B) cells 24 h after treatment. Cells were subcultured at 2 × 105 cells per milliliter, incubated overnight, and exposed to 20 ng of TPA (Sigma Chemical Co., St Louis, Mo.)/ml or 5 μM GF 109203X (Calbiochem, San Diego, Calif.) for 24 h or exposed to 5 μM GF 109203X for 30 min before the addition of TPA for 24 h. Untreated cells were used as controls. The GAPDH transcript was analyzed as a control for equal RNA loading. Protein extracts were prepared from BCP-1 cells, and equal amounts of protein (30 μg) were loaded per lane. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer of proteins to nitrocellulose, blots were probed for vIL-6 by Western blot analysis. Actin antibody was used to control for equal loading (C). The results shown are representative of those from three similar experiments.
FIG. 2.
FIG. 2.
Effect of the PKCδ inhibitor rottlerin on TPA-dependent virus lytic induction. Cells were pretreated with 5 μM rottlerin (Calbiochem) for 30 min followed by 24 h of treatment with 20 ng of TPA/ml. RNA extracts from BCP-1 (A) and BCBL-1 (B) cells were then analyzed for the T1.1 early transcript by Northern blot hybridization, and protein extracts from BCP-1 cells were assayed for the expression of KSHV/Rta and vIL-6 by Western blot analysis (C). As shown, TPA induced virus reactivation, whereas pretreatment with rottlerin inhibited the TPA-induced virus reactivation. The results shown are representative of those from three similar experiments.
FIG. 3.
FIG. 3.
Expression and translocation of PKCδ in BCP-1 and BCBL-1 cells that were treated with TPA. The expression of the PKCδ isoform was examined by using anti-human PKCδ (nPKCδ C-20; Santa Cruz) rabbit polyclonal immunoglobulin G in protein extracts from BCP-1 (A) and BCBL-1 (B) cells growing under standard growth conditions and from cells that were treated with TPA for 30 min, 60 min, and 24 h. The membrane was then probed with antiactin antibody. Fixed BCP-1 (C) and BCBL-1 (D) cells were incubated with rabbit anti-PKCδ antibody followed by an anti-rabbit antibody conjugated to fluorescein isothiocyanate. Propidium iodide (PI) staining was used to mark nuclei. Cells were visualized by confocal microscopy (Bio-Rad MRC 1024 confocal scan head mounted on a Nikon microscope). The results are from one of three similar experiments.
FIG. 3.
FIG. 3.
Expression and translocation of PKCδ in BCP-1 and BCBL-1 cells that were treated with TPA. The expression of the PKCδ isoform was examined by using anti-human PKCδ (nPKCδ C-20; Santa Cruz) rabbit polyclonal immunoglobulin G in protein extracts from BCP-1 (A) and BCBL-1 (B) cells growing under standard growth conditions and from cells that were treated with TPA for 30 min, 60 min, and 24 h. The membrane was then probed with antiactin antibody. Fixed BCP-1 (C) and BCBL-1 (D) cells were incubated with rabbit anti-PKCδ antibody followed by an anti-rabbit antibody conjugated to fluorescein isothiocyanate. Propidium iodide (PI) staining was used to mark nuclei. Cells were visualized by confocal microscopy (Bio-Rad MRC 1024 confocal scan head mounted on a Nikon microscope). The results are from one of three similar experiments.
FIG. 4.
FIG. 4.
Dominant-negative PKCδ expressed by an adenovirus vector inhibits TPA-mediated KSHV reactivation. Cells were infected with a recombinant adenovirus vector that expresses dominant-negative PKCδ (Adeno-DN-PKCδ). Twenty-four hours after the adenoviral transduction, cells were either treated with TPA or left untouched. RNA extracts from BCP-1 (A) and BCBL-1 (B) cells were then analyzed for the T1.1 early transcript. Expression of the ectopically expressed mouse dominant-negative PKCδ and vIL-6 was monitored in BCP-1 cells by Western blot analysis 24 h after the addition of TPA (C). Infection with empty adenovirus vector (Adeno-CV) was used as a control. Actin antibody was used to control for equal loading. The results shown are representative of those from three similar experiments.
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