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. 2008 Feb 5;371(1):139-54.
doi: 10.1016/j.virol.2007.09.040. Epub 2007 Oct 26.

Reactivation of Kaposi's sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways

Jianping Xie  1 , Adetola Olalekan Ajibade, Fengchun Ye, Kurt Kuhne, Shou-Jiang Gao
Affiliations

Reactivation of Kaposi's sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways

Jianping Xie et al. Virology. .

Abstract

Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) promotes the progression of Kaposi's sarcoma (KS), a dominant malignancy in patients with AIDS. While 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced KSHV reactivation from latency is mediated by the protein kinase C delta and MEK/ERK mitogen-activated protein kinase (MAPK) pathways, we have recently shown that the MEK/ERK, JNK and p38 MAPK pathways modulate KSHV lytic replication during productive primary infection of human umbilical vein endothelial cells [Pan, H., Xie, J., Ye, F., Gao, S.J., 2006. Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection. J. Virol. 80 (11), 5371-5382]. Here, we report that, besides the MEK/ERK pathway, the JNK and p38 MAPK pathways also mediate TPA-induced KSHV reactivation from latency. The MEK/ERK, JNK and p38 MAPK pathways were constitutively activated in latent KSHV-infected BCBL-1 cells. TPA treatment enhanced the levels of activated ERK and p38 but not those of activated JNK. Inhibitors of all three MAPK pathways reduced TPA-induced production of KSHV infectious virions in BCBL-1 cells in a dose-dependent fashion. The inhibitors blocked KSHV lytic replication at the early stage(s) of reactivation, and reduced the expression of viral lytic genes including RTA, a key immediate-early transactivator of viral lytic replication. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. Furthermore, we showed that the activation of RTA promoter by MAPK pathways was mediated by their downstream target AP-1. Together, these findings suggest that MAPK pathways might have general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication.

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Figures

Fig. 1
Fig. 1
Constitutive activation of the ERK, JNK and p38 MAPK pathways in uninduced BCBL-1 cells, and transient enhanced activation of the ERK but not JNK and p38 pathways by TPA. (A) Western-blotting with specific antibodies to detect the phosphorylated forms of ERK (first set of panels), p38 (third set of panels) and JNK (sixth set of panels), and total ERK (second set of panels), p38 (fourth set of panels) and JNK (seventh set of panels) in cells induced with TPA for different lengths of time (0-144 h). An anti-β-tubulin antibody was used to normalize the sample loading (fifth and eighth panels). The ERK and p38 blots were run with one set of samples while the JNK blots were run with another set of samples. (B) Quantification of pERK levels in mock- and TPA-induced cells. (C) Quantification of pp38 levels in mock- and TPA-induced cells.
Fig. 2
Fig. 2
Inhibitors of MAPK pathways reduced the production of infectious virions in TPA-induced KSHV reactivation from latency in BCBL-1 cells. (A) Effects of inhibitors of MAPK pathways on TPA-induced virion production. Supernatants from cells treated with the inhibitors were titrated at 5 dpi for infectious virions by infecting 293 cells and recording GFP-positive cells at 2 days post-infection. Both JNK inhibitor II (JNK inhibitor) and SB203580 (p38 inhibitor) were used at 50 μM while U0126 (MEK inhibitor) was used at 10 μM. (B) Quantification of relative virus titers in supernatants from cells treated with inhibitors of MAPK pathways as described in (A). (C-D) The effects of inhibitors of MAPK pathways on the infectivity of KSHV inoculums. Virus inoculums were incubated with the inhibitors of MAPK pathways for 5 days at 37 °C before titration (C), or with freshly reconstituted inhibitors (D) immediate before titration. The same concentrations of the inhibitors described in (A) were used for the experiments. (E-G) Inhibitors of MEK (E), JNK (F) and p38 (G) pathways reduced the production of TPA-induced KSHV infectious virions in a dose-dependent manner. The experiments were independently carried out four times except those in (C and D) which were carried out twice, each with three repeats. Results presented were the averages with standard deviations from one experiment.
Fig. 3
Fig. 3
Inhibitors of MAPK pathways affected the early stages of TPA-induced KSHV reactivation from latency in BCBL-1 cells. (A) TPA-induced cells were treated with inhibitors of MEK, JNK, and p38 MAPK pathways at the onset of TPA induction or at 1, 2, 3, and 4 dpi, and supernatants were titrated for infectious virions at 5 dpi. (B) TPA-induced cells were treated with inhibitors of MAPK pathways at the onset of TPA induction. Both TPA and the inhibitors were washed away at 2 dpi and replaced with fresh medium without any inducer and inhibitors. The supernatants were collected and titrated for infectious virions at 5 dpi. (C) TPA-induced cells were treated with inhibitors of MEK, JNK, and p38 MAPK pathways at the onset of TPA induction or at 2, 4, 8, 16 and 24 hpi, and the supernatants were titrated for infectious virions at 5 dpi. All the experiments were independently carried out three times, each with three repeats. Results presented were the averages with standard deviations from one representative experiment.
Fig. 4
Fig. 4
Inhibitors of MAPK pathways suppressed the expression of viral lytic genes during TPA-induced KSHV reactivation from latency in BCBL-1 cells. (A) Inhibitors of MAPK pathways suppressed the expression of KSHV lytic transcripts but not latent transcripts during KSHV reactivation. Cells were induced with TPA in the presence of inhibitors of MAPK pathways, and analyzed for the expression of vFLIP, RTA, ORF59, vIL-6 and K8.1 transcripts at 48 hpi by RT-qPCR. (B) Inhibitors of MAPK pathways suppressed the protein expression of a KSHV lytic gene ORF59 during KSHV reactivation. Cells were treated as described in (A) and analyzed for the expression of ORF59 protein at 48 hpi by immunofluorescence antibody staining using a specific monoclonal antibody. (C) Quantification of the effects of inhibitors of MAPK pathways on the expression of ORF59 protein during KSHV reactivation as described in (B). (D) Inhibitors of MAPK pathways had no effect on the expression of KSHV latent protein LANA during KSHV reactivation. Cells were treated as described in (A) and analyzed for the expression of LANA protein at 48 hpi by immunofluorescence antibody staining using a specific monoclonal antibody. All the experiments were independently carried out three times, each with three repeats. Results presented in (A, C and D) were the averages with standard deviations from one representative experiment.
Fig. 5
Fig. 5
TPA-induced activation of the RTA promoter was mediated by multiple MAPK pathways. (A) Kinetics of TPA activation of the RTA promoter reporter. Bjab cells were transfected with a full-length RTA promoter reporter construct, and assayed for luciferase activity at 48 h post-transfection. Prior to harvest, cells were treated with 20 ng/ml of TPA for 0, 1, 2, 4, 6, 8, 12, and 15 h. (B) Inhibitors of MAPK pathways suppressed TPA activation of the RTA promoter reporter in a dose-dependent fashion. Bjab cells transfected with a RTA promoter reporter construct for 42 h were treated with 20 ng/ml of TPA for 6 h with or without inhibitors of JNK, MEK and p38 pathways, lysed, and assayed for luciferase activity. The inhibitors were used at the following concentrations: JNK inhibitor II (JNK inhibitor) at 12.5, 25, 50 and 100 μM; U0126 (MEK inhibitor) at 2.5, 5, 10, and 20 μM; and SB203580 (p38 inhibitor) at 12.5, 25, 50 and 100 μM. (C) Dominant negative constructs (DNs) of MAPK pathways inhibited TPA activation of the RTA promoter reporter in a dose-dependent fashion. Bjab cells transfected with a RTA promoter reporter construct together with a vector control (V) or different doses of DNs of JNK, MEK and p38 MAPK pathways for 42 h were treated with TPA for 6 h, lysed, and assayed for luciferase activity. (D) Overexpression of active forms (CAs) of ERK, JNK or p38 was sufficient to activate the RTA promoter, which was abolished by their respective DNs. Bjab cells transfected with a RTA promoter reporter construct together with a vector control or a CA of ERK, JNK or p38 with or without the presence of their respective DNs for 48 h were lysed and assayed for luciferase activity. The experiments were carried out three times, each with three repeats. Results presented were the averages with standard deviations from one experiment.
Fig. 6
Fig. 6
Identification of the dominant cis-element in RTA promoter activated by TPA. (A) Deletion analysis of the RTA promoter to identify the region responsive to TPA induction. Bjab cells transfected with different RTA promoter deletion reporter constructs for 42 h were treated with 20 ng/ml of TPA for 6 h, lysed, and assayed for luciferase activity. (B) Illustration of a wild type reporter (R-259) in the RTA promoter region (-15 to -259) responsive to TPA induction. R-259mut was a corresponding mutant reporter with the AP-1 site ablated. (C) Dominant negative constructs (DNs) of MAPK pathways inhibited TPA activation of the R-259 reporter while the mutant R-259mut reporter was not responsive to TPA treatment. Bjab cells transfected with R-259 or R-259mut reporter constructs together with a vector control (V) or DNs of the JNK, MEK and p38 MAPK pathways for 42 h were treated with TPA for 6 h, lysed and assayed for luciferase activity. (D) Inhibitors of MAPK pathways suppressed TPA activation of the R-259 reporter. Bjab cells transfected with R-259 or R-259mut reporter constructs for 42 h were treated with 20 ng/ml of TPA for 6 h with or without inhibitors of JNK, MEK and p38 pathways, lysed and assayed for luciferase activity. Both JNK inhibitor II (JNK inhibitor) and SB203580 (p38 inhibitor) were used at 50 μM while U0126 (MEK inhibitor) was used at 10 μM. (E) Overexpression of an active form (CA) of ERK, JNK or p38 was sufficient to activate the R-259 reporter but not the mutant R-259mut reporter. Bjab cells transfected with R-259 or R-259mut reporter constructs together with a vector control or a CA of ERK, JNK or p38 with or without their respective DNs for 48 h were lysed and assayed for luciferase activity. (F) DN of c-Fos or c-Jun inhibited TPA activation of the R-259 reporter. Bjab cells transfected with R-259 or R-259mut reporter constructs together with a vector control (V) or DN of c-Fos, c-Jun, or both for 42 h were treated with TPA for 6 h, lysed and assayed for luciferase activity.
Fig. 7
Fig. 7
Increased binding of AP-1 complexes to a putative AP-1 cis-element in the RTA promoter during TPA-induced KSHV reactivation from latency in BCBL-1 cells. Nuclear extracts from uninduced cells (lanes 3-4) or cells induced with 20 ng/ml of TPA for 6 h (lanes 5-16) were subjected to electrophoretic mobility shift assay using a 32P-labeled probe of the putative AP-1 cis-element from the RTA promoter (W) or its mutant with the putative AP-1 site mutated (M). The labeled probes alone were shown in lanes 1 and 2, respectively. In the competition assay, 100X excess amounts of unlabeled probes were added to the reactions (lanes 7-10). Addition of 1 μg of an antibody to c-Fos (lanes 11-12), or c-Jun (lanes 13-14), respectively, supershifted the AP-1-probe complex, while addition of 1 μg of IgG of a control antibody did not (lanes 15-16).
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