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. 2003 Jan;77(1):57-67.
doi: 10.1128/jvi.77.1.57-67.2003.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor has broad signaling effects in primary effusion lymphoma cells

Mark Cannon  1 , Nicola J Philpott, Ethel Cesarman
Affiliations

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor has broad signaling effects in primary effusion lymphoma cells

Mark Cannon et al. J Virol. 2003 Jan.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV-8]) is a gamma-2-herpesvirus responsible for Kaposi's sarcoma as well as primary effusion lymphoma (PEL). KSHV is a lymphotropic virus that has pirated many mammalian genes involved in inflammation, cell cycle control, and angiogenesis. Among these is the early lytic viral G protein-coupled receptor (vGPCR), a homologue of the human interleukin-8 (IL-8) receptor. When expressed, vGPCR is constitutively active and can signal via mitogen- and stress-activated kinases. In certain models it activates the transcriptional potential of NF-kappaB and activator protein 1 (AP-1) and induces vascular endothelial growth factor (VEGF) production. Despite its importance to the pathogenesis of all KSHV-mediated disease, little is known about vGPCR activity in hematopoietic cells. To study the signaling potential and downstream effects of vGPCR in such cells, we have developed PEL cell lines that express vGPCR under the control of an inducible promoter. The sequences required for tetracycline-mediated induction were cloned into a plasmid containing adeno-associated virus type 2 elements to enhance integration efficiency. This novel plasmid permitted studies of vGPCR activity in naturally infected KSHV-positive lymphocytes. We show that vGPCR activates ERK-2 and p38 in PEL cells. In addition, it increases the transcription of reporter genes under the control of AP-1, NF-kappaB, CREB, and NFAT, a Ca(2+)-dependent transcription factor important to KSHV lytic gene expression. vGPCR also increases the transcription of KSHV open reading frames 50 and 57, thereby displaying broad potential to affect viral transcription patterns. Finally, vGPCR signaling results in increased PEL cell elaboration of KSHV vIL-6 and VEGF, two growth factors involved in KSHV-mediated disease pathogenesis.

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Figures

FIG. 1.
FIG. 1.
Map of pTruf-Tet, a single plasmid designed to allow tetracycline-mediated induction of transgenes cloned into the unique NsiI site. ITR, inverted terminal repeat from AAV-2; Pcmv, CMV-derived promoter region; GFP, green fluorescent protein; pA, polyadenylation site from SV40; Ptre, tetracycline-responsive promoter; tTS, transcriptional silencer; rtTA, reverse tetracycline-responsive transcriptional activator; Neor, conveys neomycin resistance.
FIG. 2.
FIG. 2.
Establishment of doxycycline-inducible vGPCR-expressing PEL lymphoma cell lines. (A) Binding assay shows increased vGPCR protein expression with increasing doses of doxycycline. Cells were incubated with125I-GROα, and pellets were counted in a γ counter. The first two lanes verify that doxycycline has no independent effect on control BC-3 cells. (B) vGPCR expression slows the growth of PEL cells in culture. Cells were plated at 5 × 104 cells/ml with the doxycycline doses shown (in micrograms per milliliter) and counted daily by trypan blue exclusion. (Top) BC3.14 with increasing doxycycline doses and thereby increasing vGPCR expression. The percentage of dead cells does not vary between vGPCR-expressing and -nonexpressing cells. (Bottom) Control BC-3 cells incubated with 2 μg/of doxycycline per ml shows that doxycycline has no independent effect on cell growth. Shown is average of three independent experiments; error bars are omitted for clarity.
FIG. 3.
FIG. 3.
vGPCR signaling activates ERK-2 and p38. (A) Western blots for total and phosphorylated (active) ERK-1/2 show increasing enzyme activity with increasing doxycycline doses. BC-3.14 cells were grown at 37°C for 48 h in the presence of doxycycline doses as shown. Protein extracts (30 μg/well) were subjected to SDS-PAGE and blotted first with α-phospho-ERK1/2 and then stripped and reprobed with α-total ERK1/2. (B) Western blots for phosphorylated and total p38. Protein extracts (40 μg) from cells stimulated as above were first probed with α-phospho-p38 and then stripped and reprobed with α-total p38. Numbers above the bands represent the average fold induction in band intensity relative to baseline (three independent experiments).
FIG. 4.
FIG. 4.
vGPCR activates AP-1, CREB, and NF-κB in PEL cells. BC-3.14 cells were transfected with the appropriate luciferase reporter and plated with the doses of doxycycline shown with or without IP-10 (100 nM), an inverse agonist of vGPCR. At 48 h, lysates were assayed for luciferase activity. (A) Dose-response curve of AP-1 activation. The inset shows the response to doxycycline of the negative control plasmid pTA-luc for the AP-1 and NF-κB constructs. (B) CREB activation. The inset shows the negative control plasmid, pFC2-dbd. (C) NF-κB activation. The results shown represent at least three independent transfections. Values are normalized to the no-doxycycline point. (D) vGPCR causes the phosphorylation of IκBα and AKT. Cells were subjected to the doses of doxycycline shown for 48 h, after which lysates (40 μg) were transferred to PVDF, probed for phosphorylated enzyme, stripped, and probed for total enzyme. Numbers above the bands represent the average fold induction (two independent experiments) in band intensity relative to baseline. ∗, In panels A to C, P ≤ 0.05 relative to the baseline uninduced value.
FIG. 5.
FIG. 5.
vGPCR activates transcription by NFAT as well as the KSHV ORF 50 and ORF 57 promoters. BC-3.14 cells in exponential growth were transfected with the respective luciferase reporter construct and exposed to does of doxycycline and/or GROα as shown, to induce vGPCR expression and further increase signaling, respectively. After 48 h, lysates were assayed for luciferase activity as described in Materials and Methods. (A) NFAT activity. The negative control was as in the inset of Fig. 4A. (B) ORF 50 promoter. The inset shows the response to doxycycline of the negative control plasmid pGL2-control. (C) ORF 57 promoter. The inset shows the response to doxycycline of the negative control plasmid pG32-control. Results shown represent at least three independent transfections. ∗, P ≤ 0.05 relative to the baseline uninduced value.
FIG. 6.
FIG. 6.
vGPCR increases the expression of KSHV vIL-6 and VEGF. (A) BC-3.14 cells were transfected with the vIL-6-luciferase construct, incubated with or without doxycycline and with or without GROα (100 nM) for 48 h, and assayed for luciferase activity. Negative control is as in the inset in Fig. 5C. Results of three independent transfections are shown. (B) BC-3.14 cells were plated at 5 × 105 cells/ml in the doses of doxycycline shown. The cells were lysed in RIPA buffer after 48 h, and 40 μg of protein per lane was loaded onto the SDS-PAGE gel. Western blot analysis was performed using anti-vIL6 Ab, stripped, and then the blot was reprobed with anti-β-actin Ab to control for loading. Numbers above the bands represent average fold induction in band intensity relative to baseline (three independent experiments). (C) BC-3.14 cells growing exponentially were preincubated in full medium with or without doxycycline and with or without GROα (100 nM) for 36 h. They were then washed and replated in serum free medium with or without doxycycline and with or without GROα for another 12 h. Then 6 × 105 cells per point were plated in serum-free medium, and at 24 h, the supernatant was assayed for VEGF by ELISA. Results represent three independent experiments and are expressed as picograms per milliliter over baseline production. Uninduced cells expressed 535 pg of VEGF per ml. vGPCR-expressing cells stimulated with GROα expressed 1,054 pg/ml. ∗, P ≤ 0.001 relative to the baseline uninduced value.
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