HCMV activation of ERK-MAPK drives a multi-factorial response promoting the survival of infected myeloid progenitors

Viral binding and entry provides the first trigger of a cell death response and thus how human cytomegalovirus (HCMV) evades this – particularly during latent infection where a very limited pattern of gene expression is observed – is less well understood. It has been demonstrated that the activation of cellular signalling pathways upon virus binding promotes the survival of latently infected cells by the activation of cell encoded anti-apoptotic responses. In CD34+ cells, a major site of HCMV latency, ERK signalling is important for survival and we now show that the activation of this pathway impacts on multiple aspects of cell death pathways. The data illustrate that HCMV infection triggers activation of pro-apoptotic Bak which is then countered through multiple ERK-dependent functions. Specifically, ERK promotes ELK1 mediated transcription of the key survival molecule MCL-1, along with a concomitant decrease of the pro-apoptotic BIM and PUMA proteins. Finally, we show that the elimination of ELK-1 from CD34+ cells results in elevated Bak activation in response to viral infection, resulting in cell death. Taken together, these data begin to shed light on the poly-functional response elicited by HCMV via ERK-MAPK to promote cell survival

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.The research participants were enrolled in the Biomedical Research Centres/Units Inherited Diseases Genetic Evaluation (BRIDGE) Bleeding and Platelet Disorders (BPD) study (UK REC10/H0304/66). We are grateful to all the donors who allowed us to use their samples for this study. We thank Sofia Papadia from the NIHR BioResource for organizing the recalls of BRIDGE-BPD participants. The genome sequencing of the BRIDGE-BPD participants was supported by the NIHR BioResource–Rare Diseases (to ET, KD, and WHO). The NIHR BioResource–Rare Diseases is responsible for the delivery of the rare diseases pilot phase of the 100,000 Genomes Project and is funded by the National Institute for Health Research (NIHR; http://www.nihr.ac.uk). Research in the Ouwehand laboratory also receives funding support from the European Commission, NIHR, Wellcome Trust, Medical Research Council (MRC), and British Heart Foundation under numbers RP-PG-0310-1002 and RG/09/12/28096. SKW is supported by an MRC Clinical Training Fellowship (MR/K023489/1). ADM receives support from the Bristol NIHR Biomedical Research Unit for Cardiovascular Disease. This work was supported by a Project Grant (no. 575535), a Program Grant (no. 1016647), a Fellowship (1063008 to BTK and 1058344 to WSA), Project Grants (to PWG and ECH), and an Independent Research Institutes Infrastructure Support Scheme Grant (no. 361646) from the Australian National Health and Medical Research Council; a fellowship from the Sylvia and Charles Viertel Foundation (to BTK); a start-up grant, a fellowship, and a grant from the German Research Foundation (SFB 688, PL707/1-1 and PL707/2-1 to IP); the Kids’ Cancer Project (to PWG); a Fellowship from the European Hematology Association (to MRT) and the British Heart Foundation (PG/13/77/30375 to MRT); NHS Blood and Transplant (to WHO and MRT); the Australian Cancer Research Fund; and a Victorian State Government Operational Infrastructure Support Grant

Mitogen and stress activated kinases act co-operatively with CREB during the induction of human cytomegalovirus immediate-early gene expression from latency.

The devastating clinical consequences associated with human cytomegalovirus (HCMV) infection and reactivation underscores the importance of understanding triggers of HCMV reactivation in dendritic cells (DC). Here we show that ERK-mediated reactivation is dependent on the mitogen and stress activated kinase (MSK) family. Furthermore, this MSK mediated response is dependent on CREB binding to the viral major immediate early promoter (MIEP). Specifically, CREB binding to the MIEP provides the target for MSK recruitment. Importantly, MSK mediated phosphorylation of histone H3 is required to promote histone de-methylation and the subsequent exit of HCMV from latency. Taken together, these data suggest that CREB binding to the MIEP is necessary for the recruitment of the kinase activity of MSKs to initiate the chromatin remodelling at the MIEP required for reactivation. Thus the importance of CREB during HCMV reactivation is to promote chromatin modifications conducive for viral gene expression as well as acting as a classical transcription factor. Clearly, specific inhibition of this interaction between CREB and MSKs could provide a strategy for therapeutic intervention

Inhibition of mitogen and stress kinase activity blocks CREB and histone phosphorylation at the MIEP.

<p><b>A</b>) Western blot for phosphor and total CREB, phosphor and total ERK1/2, phosphor and total MSK and GAPDH was performed on immature DCs or DCs stimulated with IL-6 (30 mins) after incubation with DMSO or MSK inhibitor (2 hours). <b>B</b>) Chromatin immunopreciptations on immature DCs (iDC) derived from monocytes infected with HCMV (1–3) were performed alongside IL-6 (4–12) stimulated DCs for phosphor-CREB (CR), histone H3-S10^P (S10) and histone H3-K9^3Me (K9) binding at 2 hours post stimulation. DNA was amplified in an MIEP PCR and expressed as ratio of the Input sample. S.D. of n = 2. <b>C</b>) Chromatin immunopreciptations on immature DCs (iDC) stimulated with IL-6 were performed at 15 mins to 3 hours post stimulation with an anti-MSK antibody or an isotype matched control. DNA was amplified in an MIEP PCR and expressed as ratio of the Input sample. S.D. of n = 2.</p

The binding of phosphorylated CREB to the MIEP correlates with reactivation.

<p><b>A</b>) Chromatin immunopreciptations on monocytes (Mono), immature DCs (iDC), mature DCs (mDC) or mature DCs pre-treated with ERK inhibitor were performed with an anti-CREB or isotype control antibody. CREB binding is expressed relative to IgG control. <b>B</b>) Chromatin immunopreciptations on monocytes (Mono), immature DCs (iDC), mature DCs (mDC), mature DCs pre-treated with ERK or p38 inhibitors were performed with an anti-CREB, anti-phospho CREB or isotype control antibody. The relative levels of phosphorylated CREB binding was expressed a percentage of total CREB binding. S.D. shown from n = 3 (A,B). <b>C</b>) Western blot for phosphor-CREB, total CREB and GAPDH was performed on immature DCs or DCs stimulated with LPS after incubation with mock, DMSO or ERK-MAPK inhibitor.</p

Signalling via the Ras-RAF arm of ERK-MAPK is required for HCMV reactivation.

<p><b>A–B</b>) RNA isolated from immature DCs or DCs stimulated with LPS (A) or IL-6 (B) was amplified qRT-PCR (IE and GAPDH). Prior to addition of LPS or IL-6 cells were incubated with inhibitors of ERK, Raf or tpl2 signalling for 1 hour. IE RNA expression was expressed as a fold decrease compared to mock treated control <b>C</b>) immature DCs (1) were incubated with DMSO (2,3) or ERK (4,5), tpl2 (6,7) and Raf (8,9) inhibitors for 1 hour then stimulated with LPS (black) or IL-6 (grey) to promote reactivation. The percentage of IE positive cells was calculated by indirect immunofluorescence and Hoechst nuclear counterstaining. S.D. shown from n = 3 (A–C).</p

Okadaic acid promotes histone phosphorylation at the MIEP and partially rescuing the CRE deletion virus.

<p><b>A</b>) Immature DCs (iDC) derived from monocytes infected with Revertant (Rev) or CRE deletion virus (ΔCRE) were treated with DMSO (1,3) or okadaic acid (2, 4) and amplified by qRT-PCR. IE expression was standardised to GAPDH (2^ΔCT) and the fold change in gene expression (2^ΔΔCT) following okadaic acid addition was expressed relative to the DMSO control. S.D of n = 3 <b>B</b>) Immature DCs (iDC) derived from monocytes infected with Revertant (Rev) or CRE deletion virus (ΔCRE) were treated with DMSO (iDC) or okadaic acid (OKA) and subject to ChIP with an anti-histone H3-S10^P antibody or isotype control. Samples were amplified in MIEP qPCRs and the signals expressed as a ratio of the Input. S.D. of n = 2.</p

A dominant negative MEK1 blocks IE gene expression and CREB and histone H3 phosphorylation at the MIEP.

<p><b>A</b>) Western blot analysis of iDCs (1), stimulated with IL-6 (2–4) post transduction with adenoviral vectors expressing GFP (3) or dn-MEK1 (4). <b>B</b>) RNA isolated from IL-6 stimulated iDCs pre infected with AD-GFP or AD-dnMEK was analysed by RT-qPCR for IE gene expression and expressed relative to IL-6 stimulated control. <b>C</b>) ChIP analyses were performed with isotype control (IgG), anti-phospho-CREB (CR), anti-phospho-serine 10 histone H3 (S10) or anti-trimethylated lysine 9 histone H3 (K9) antibodies and DNA amplified in an MIEP specific qPCR. Values were expressed relative to the Input control.</p

Phosphorylation of histone H3 is dependent on CREB binding to the MIEP.

<p><b>A</b>) Immature DCs (iDC) derived from monocytes infected with wild type (WT), Revertant (Rev) or CRE deletion virus (ΔCRE) were left un-stimulated (1,3,5) or IL-6 treated cells (2,4,6). Then, chromatin immunoprecipitation of histone H3-S10^P was performed and samples amplified in an MIEP qPCR (2 hours post stimulation). Following normalisation to GAPDH, samples were expressed as a ratio of the Input signal. <b>B–C</b>) immature (latent;1,2) and IL-6 stimulated (reactivating, 3,4) DCs (2 hours post stimulation) were subject to chromatin immunoprecipitation with an anti-histone H3-S10^P (S10P), anti-histone H3-K9^3Me (K9M) or isoptype control (IgG) antibodies and then amplified in an MIEP qPCR and signal expressed as a ratio of the Input. <b>C</b>) The primary ChIPs from the reactivating samples (B) were then subject to a second ChIP with a phosphor CREB antibody or isotype matched control. The phosphor-CREB IP from H3-S10^P IP (1–4) was then expressed as a ratio of the Input signal for the MIEP or GAPDH qPCR. Alternatively, the phosphor-CREB or isotype control IPs from H3-K9^3Me (5,6) were amplified in an MIEP qPCR and expressed as a ratio of Input. S.D. of n = 2. <b>D</b>) IL-6 stimulated DCs (2 hours) were subject to a primary IP with anti-histone H3-serine 10 antibody then subject to second IP with an anti-MSK1 antibody or isoptype control. Samples were amplified in an MIEP or GAPDH promoter specific PCR and signal expressed as a ratio of Input. S.D. of n = 2.</p