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. 2012 Aug;86(16):8859-71.
doi: 10.1128/JVI.00597-12. Epub 2012 Jun 13.

Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor prevents AU-rich-element-mediated mRNA decay

Jennifer A Corcoran  1 , Denys A Khaperskyy, Benjamin P Johnston, Christine A King, David P Cyr, Alisha V Olsthoorn, Craig McCormick
Affiliations

Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor prevents AU-rich-element-mediated mRNA decay

Jennifer A Corcoran et al. J Virol. 2012 Aug.

Abstract

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3' untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells.

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Figures

Fig 1
Fig 1
vGPCR stabilizes reporter ARE-containing mRNA. (A) HeLa Tet-Off cells were cotransfected with pTRE2-Rluc (no ARE), pTRE2-Fluc-ARE, and an expression plasmid for kaposin B, vGPCR, a signaling inactive mutant of vGPCR (vGPCRR143A), or an empty vector control. At 24 h posttransfection, Dox was added to halt reporter gene transcription. Twenty-four hours after Dox addition, cell lysates were harvested and analyzed for firefly, Renilla, or normalized (firefly/Renilla) luciferase activity as described in Materials and Methods. Results are displayed in relative light units (RLUs) and are the averages from three independent experiments ± the standard error. (B) Lysates from transfected HeLa Tet-Off cells were harvested and immunoblotted with anti-vGPCR (1:1,000 dilution) or anti-β-actin (1:5,000 dilution) antibody. (C) HeLa Tet-Off cells were cotransfected with pTRE2-BBB and pTRE2-BBB-ARE, with or without an expression plasmid for vGPCR. At 18 h posttransfection, Dox was added to halt reporter gene transcription, and total RNA was harvested after 0, 0.5, 1, and 2 h. The amounts of reporter RNAs were detected simultaneously with a probe to the β-globin ORF. (D) Log2 values of the band intensities from panel B were plotted versus time and fitted with the linear regression curves. From the linear regression analyses, the slope value m was obtained. The inverted negative values of each m value are the calculated half-life values shown on the plot (t1/2 = −1/m).
Fig 2
Fig 2
vGPCR increases the half-life of endogenous ARE-containing mRNAs. (A to E) HUVE cells were transduced with recombinant retroviruses expressing vGPCR, a signaling inactive mutant of vGPCR (vGPCRR143A), or the empty vector control virus. After 2 days of selection with puromycin and 1 day of recovery, cell monolayers were examined by light microscopy (scale bar in panel A = 200 μm). Transduced cell monolayers were also analyzed as follows: cells were seeded on coverslips overnight and then fixed and immunostained using anti-vGPCR antibody (scale bar = 10 μm, thick arrows indicate high-expressing cells while thin arrows indicate low-expressing cells) (B); cells were lysed in protein sample buffer, and 25 μg of protein was analyzed by SDS-PAGE and immunoblotting with anti-Cox-2 antibody at 1:500, anti-vGPCR antibody at 1:2,000, or anti-β-actin antibody at 1:5,000 (C); ELISA analysis of cell-free supernatants was done for the presence of human IL-8 and IL-6, which are encoded by ARE-containing RNAs (D); cells were harvested for total RNA isolation, and the steady-state levels of the Cox-2 and IL-6 transcripts were determined by RT-qPCR (E). Error bars represent standard deviations (n = 3 experiments). (F) HUVE cells transduced as described in the legends to panels A to E were either treated with actinomycin D, which stops de novo transcription, for 1, 2, or 3 h, or not treated and harvested for total RNA. Each sample was analyzed by RT-qPCR to determine the decay rates of the Cox-2 transcript. Values are presented as fractions of the transcript levels at the time of actinomycin D addition, and the error bars represent standard deviations (n = 3 experiments). (G) Log2 values of the data from panel E were plotted versus time independently for all three replicates and fitted with the linear regression curves. From the linear regression analyses, the slope value m was obtained. The inverted negative values of each m value are the calculated half-life values shown on the plot (t1/2 = −1/m).
Fig 3
Fig 3
MK2 inhibition disrupts vGPCR-mediated ARE-mRNA stabilization. (A) HUVE cells were transduced with recombinant retroviruses expressing vGPCR or the empty vector control virus and selected as described for Fig. 2. Transduced cells were treated with chemical inhibitors of the kinases p38 (SB203580; 1 or 10 μM), MK2 (compound III; 1 or 10 μM), or dimethyl sulfoxide (DMSO) as a vehicle control for 1 h before lysis in 1× protein sample buffer. Additional wells of cells were treated with sorbitol (0.4 M), to activate the stress-inducible p38 MAPK pathway. A total of 10 μg of total protein was subjected to 12% SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of heat shock protein 27 (Hsp27) (ser82; 1:1,000) and β-actin (1:5,000). One representative experiment of three is shown. (B) HeLa Tet-Off cells were cotransfected with pTRE2-Rluc (no ARE), pTRE2-Fluc-ARE, and an expression plasmid for vGPCR or an empty vector control. At 23 h posttransfection, cells were treated with chemical inhibitors of the kinases p38 (SB203580; 10 μM), MK2 (compound III; 10 μM), or DMSO for 1 h. At 24 h posttransfection, Dox was added to halt reporter gene transcription. Twenty-four hours after Dox addition, cells were lysed and analyzed for firefly, Renilla, and normalized (firefly/Renilla) activity as described in Materials and Methods. Results are displayed in relative light units (RLUs) and are the averages from three independent experiments ± the standard errors.
Fig 4
Fig 4
MK2/3 knockout cells do not support vGPCR-mediated ARE-RNA stability. Wild-type (wt) or MK2/3 knockout (MK2/3−/−) mouse embryonic fibroblasts (MEFs) were transduced with recombinant retroviruses expressing vGPCR or the empty vector control virus and selected as described in Fig. 2. Postselection, cells were treated with actinomycin D to stop de novo transcription and harvested for total RNA isolation after 0, 1, 2, and 3 h. Each sample was analyzed by RT-qPCR to determine the decay rates of the Cox-2 transcript during the chase. Values are presented as fractions of the transcript levels at the time of actinomycin D addition.
Fig 5
Fig 5
vGPCR disrupts the formation of cytoplasmic PBs in a Rho-dependent fashion. (A) HUVE cells were treated with arsenite for 30 min to induce stress and the formation of PBs. After fixation and permeabilization, PBs were immunostained with two antibodies directed against resident proteins: anti-hedls antibody (green), which also binds to nuclear S6K, and anti-DDX6 antibody (red). (B and C) HUVE cells, transduced with empty vector and selected as described in Fig. 2, were treated with the RhoA activators lysophosphatidic acid (LPA) (B, C), nocodazole (C), or Rho activator II (C) as described in Materials and Methods. After fixation, cells were stained with anti-hedls antibody (green) to visualize PBs, and Alexa 555-conjugated phalloidin (red) to visualize actin. (C and D) To quantify the effect of Rho activation on PB accretion, the number of cells with normal (approximately 1 μm in diameter)-sized PBs per each field of view was counted. After transduction with vGPCR, the number of vGPCR-positive cells with normal PBs was counted. For each treatment, 100 to 200 cells were counted from three independent experiments. The fold reduction in the number of cells with PBs was calculated as described in Materials and Methods. (D to F) After transduction with vGPCR (wild type or signaling-inactive mutant R143A) or control retroviruses, selected cells were either mock treated in serum-free medium or treated with 1 μg/ml of C3 transferase, an irreversible inhibitor of RhoA subfamily GTPases, for 6 h, followed by 1 h in normal medium. Additional wells were treated with an inhibitor of the RhoA-associated kinase, ROCK, for 1 h, or not treated. These cells were fixed and triple stained with antibodies to the PB resident hedls protein (green) and vGPCR (falsely colored blue) and Alexa 647-conjugated phalloidin (red) to visualize actin stress fibers. Images are representative of two (R143A mutant) or five (vector and wild-type vGPCR) independent experiments (scale bar = 10 μm). Insets clearly indicate the presence or absence of PBs in vGPCR-expressing cells under different conditions.
Fig 5
Fig 5
vGPCR disrupts the formation of cytoplasmic PBs in a Rho-dependent fashion. (A) HUVE cells were treated with arsenite for 30 min to induce stress and the formation of PBs. After fixation and permeabilization, PBs were immunostained with two antibodies directed against resident proteins: anti-hedls antibody (green), which also binds to nuclear S6K, and anti-DDX6 antibody (red). (B and C) HUVE cells, transduced with empty vector and selected as described in Fig. 2, were treated with the RhoA activators lysophosphatidic acid (LPA) (B, C), nocodazole (C), or Rho activator II (C) as described in Materials and Methods. After fixation, cells were stained with anti-hedls antibody (green) to visualize PBs, and Alexa 555-conjugated phalloidin (red) to visualize actin. (C and D) To quantify the effect of Rho activation on PB accretion, the number of cells with normal (approximately 1 μm in diameter)-sized PBs per each field of view was counted. After transduction with vGPCR, the number of vGPCR-positive cells with normal PBs was counted. For each treatment, 100 to 200 cells were counted from three independent experiments. The fold reduction in the number of cells with PBs was calculated as described in Materials and Methods. (D to F) After transduction with vGPCR (wild type or signaling-inactive mutant R143A) or control retroviruses, selected cells were either mock treated in serum-free medium or treated with 1 μg/ml of C3 transferase, an irreversible inhibitor of RhoA subfamily GTPases, for 6 h, followed by 1 h in normal medium. Additional wells were treated with an inhibitor of the RhoA-associated kinase, ROCK, for 1 h, or not treated. These cells were fixed and triple stained with antibodies to the PB resident hedls protein (green) and vGPCR (falsely colored blue) and Alexa 647-conjugated phalloidin (red) to visualize actin stress fibers. Images are representative of two (R143A mutant) or five (vector and wild-type vGPCR) independent experiments (scale bar = 10 μm). Insets clearly indicate the presence or absence of PBs in vGPCR-expressing cells under different conditions.
Fig 6
Fig 6
Induction of KSHV lytic gene expression in endothelial cells eliminates cytoplasmic PBs. Control iSLK cells (A) or iSLK.219 cells latently infected with rKSHV.219 (B) were either not treated or treated with Dox and/or valproic acid to induce RTA expression and reactivation from latency. Twenty-four hours after induction, cells were fixed and immunostained for PBs using anti-DDX6 antibody. Scale bars = 10 μm. GFP expression is driven by a constitutive CMV promoter and marks all infected cells, whereas RFP is driven by an RTA-responsive promoter and identifies cells supporting lytic KSHV infection (indicated by white arrows).
Fig 7
Fig 7
Model of the mechanism of vGPCR-mediated ARE-mRNA stabilization. In this working model, vGPCR stimulates two signal transduction pathways that negatively impact ARE-mRNA decay. vGPCR has been reported to promote the phosphorylation and polyubiquitination of TAK1 (9), which initiates a signal transduction pathway involving activation of MKK3 and/or MKK6 (47), p38 MAPK, and the p38 substrate MK2. MK2 phosphorylates destabilizing ARE-binding proteins like tristetraprolin (TTP), generating binding sites for cytoplasmic 14-3-3 scaffolding proteins that disrupt recruitment of a complex of endo- and exonucleases known as the exosome (15, 39). In addition, MK2 phosphorylates the heat shock protein 27 (Hsp27), which has been shown to form a complex with p115GEF to activate RhoA (27). In response to a variety of stresses, including viral infection, eukaryotic translation initiation factor 2α (eIF2α) is phosphorylated, which prevents initiation by limiting the availability of eIF2-GTP-tRNAmet. Specific RNA binding proteins bind to these stalled translation preinitiation complexes and nucleate the formation of large cytoplasmic mRNP aggregates known as stress granules (SGs), in which translationally inactive transcripts are triaged and routed to sites of reinitiation or degradation (2). TTP has been reported to transport bound ARE-mRNAs to processing bodies (PBs), sites of translational repression, and mRNA degradation (35). vGPCR activates RhoA (43, 57), and recent work has shown that RhoA activation disrupts the stress-induced rearrangement of PBs and stabilizes ARE-mRNAs (62). We provide evidence that vGPCR impacts both MK2 activity and PB dynamics, thereby potently stabilizing ARE-mRNAs and increasing the production of proteins encoded by ARE-mRNAs (e.g., Cox-2, IL-6). These studies provide the first evidence for vGPCR-mediated reprogramming of host gene expression at the posttranscriptional level, which may facilitate secretion of angiogenic growth factors from lytically infected cells.
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