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. 2007 Mar;3(3):e44.
doi: 10.1371/journal.ppat.0030044.

Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus

Fuqu Yu  1 , Josephine N Harada, Helen J Brown, Hongyu Deng, Moon Jung Song, Ting-Ting Wu, Juran Kato-Stankiewicz, Christian G Nelson, Jeffrey Vieira, Fuyuhiko Tamanoi, Sumit K Chanda, Ren Sun
Affiliations

Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus

Fuqu Yu et al. PLoS Pathog. 2007 Mar.

Abstract

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic Representation and Results of the Primary Screen System
(A) Overview of the screen approach. Individual cDNA clones were transfected into KSHV latently infected KS-1 cells in 384-well plates. Positive signals activate the RTA promoter and induce the expression of RTA protein from the endogenous viral genome. RTA can also activate its own promoter. In response to the endogenous RTA expression, the PAN promoter in the cotransfected PAN-69Luc reporter construct will be activated and the reporter activity can be analyzed. (B) Histogram of a representative screen result. The x-axis represents the relative luciferase intensity generated by transfected cDNAs normalized to the median luciferase intensity; the y-axis represents the frequency of signals falling in the binned range of luciferase intensity. The arrow indicates the group of signals that showed greater than a 5-fold increase in luciferase intensity. (C–E) Western blot showing the induction of KSHV lytic protein K8 expression; RT-Q-PCR showing the induction of lytic transcripts RTA/ORF50 (D) and PAN (E) upon transfection of the cDNA clones in the top hits list into KS-1 cells.
Figure 2
Figure 2. Effect of Ras on KSHV Lytic Transcript Levels Assessed by RT-Q-PCR
BC-3 cells were transfected with pCMV, RTA, or Ha-Ras (Q61L). At 24, 48, and 72 h post-transfection, levels of immediate-early lytic transcripts RTA/ORF50 (A), early lytic transcripts PAN (B), viral thymidine kinase (TK) (C), and late lytic transcript ORF65 (D) were measured by RT-Q-PCR. (The high level of ORF50 transcript level in the RTA transfected cells in [A] is largely due to the transfection of exogenous RTA cDNA expression clone.)
Figure 3
Figure 3. Effect of Chemical Inhibitors on KSHV Reactivation in PEL Cells
(A) Expression of KSHV lytic protein RTA or K8 24 h after transfection with pCMV, RTA, Ha-Ras (Q61L), Raf22W, and Raf22W/DDED. (B) The effects of the p38 pathway inhibitor SB203580 (20 uM), the JNK pathway inhibitor SP600125 (20 uM), and the PI3K/Akt pathway inhibitor LY294002 (20 uM) on Ras-induced K8 expression. (C) Dose-dependent inhibition of lytic protein K8 expression upon treatment with the Raf/MEK/ERK pathway–specific inhibitors PD98059 and U0126.
Figure 4
Figure 4. Effect of Ras on KSHV Lytic Promoter Activity Assessed by Reporter Assays
(A and B) The effect of increasing doses of U0126 (5 uM, 10 uM, 20uM) on Ras-induced RTA promoter activity in the absence (A) or presence (B) of endogenous RTA protein. (C) The effect of Ras on the promoter activity of four KSHV lytic genes PAN, Kpsn, ORF57, and v-IL-6 in the absence or presence of RTA protein. The fold induction represents the increase in luciferase intensity corrected over the background induction on pE4T basic construct. The data presented here are the average of three independent experiments. The promoter activity is the relative luciferase intensity compared to pcDNA3-transfected cells whose luciferase intensity was set as 1.
Figure 5
Figure 5. Effect of TPA on KSHV Reactivation in PEL Cells
(A) Fluorescence microscopy of rKSHV.219 latently infected JSC-1 cells after 48 h incubation with TPA (20 ng/ml) and increasing amount of U0126 pretreatment. (B) The expression of KSHV lytic protein K8 after 24 h incubation with TPA (20 ng/ml) and increasing amount of U0126 pretreatment. (C) BC-3 cells were cotransfected with MACS4.1 plasmid Ha-Ras (S17N) (Flag-tagged) 24 h before incubation with TPA (20 ng/ml). Sixteen hours later, the successfully transfected cells were then enriched by a MACSelection system as +ve portion, and untransfected cells were indicated as −ve portion. Ha-Ras (S17N) and K8 expression was assessed by Western blot analysis with anti-Flag and anti-K8 antibody, respectively.
Figure 6
Figure 6. Effect of U0126 on KSHV Spontaneous Reactivation in BC-3-G Cells by Flow Cytometry
Three independent experiments were conducted at 18 h (A) and 42 h (B) time points; a representative experiment result for the 18-h time point is shown here (C): (a) untreated parental cell line BC-3; (b) BC-3-G untreated; (c) BC-3-G + 0.1% dimethyl sulfoxide (DMSO); (d) BC-3-G + 5 uM U0126; (e) BC-3-G + 10 uM U0126; (f) BC-3-G + 20 uM U0126; (g). BC-3-G + 20 ng/ml TPA; (h) BC-3-G + 20 ng/ml TPA + 5 uM U0126; (i) BC-3-G + 20 ng/ml TPA + 10 uM U0126; (j) BC-3-G + 20 ng/ml TPA + 20 uM U0126.
Figure 7
Figure 7. Effect of Ets-1 on KSHV Reactivation by Western Blot, Reporter Assays, and RT-Q-PCR
(A) Expression of K8 protein in BC-3 cells 48 h after transfection of ets-1 or DN-ets-1. Expression of Ets-1 and DN-Ets-1 was detected using an anti-Flag antibody. (B) The activation of the RTA promoter upon expression of increasing amount of Ets-1 or DN-Ets-1 (10 ng, 50 ng, 100 ng, and 250 ng) in 293T cells by reporter assay. (C) The effect of increasing amount of DN-Ets-1 on RTA promoter activity induced by Ets-1 (left), Ha-Ras (Q61L), or v-Ki-Ras2 (right). The firefly luciferase readings were normalized to Renilla luciferase reading, and the relative luciferase induction compared to vector alone was presented here. (D) Levels of lytic transcripts RTA/ORF50, PAN, and viral thymidine kinase (TK) upon transfection of Ha-Ras (Q61), DN-Ets-1, or a combination of both. (E) EMSA assessing Ets-1 DNA binding ability using BC-3 cell nuclear extract after incubation with TPA (20 ng/ml) for indicated time. The binding of Oct-1 probe was used as loading control. NS, non-specific binding. (F) Competition experiments confirming the specificity of binding to Ets-1 probe. NS, non-specific binding.
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