Figure 1.
Signalling via the Ras-RAF arm of ERK-MAPK is required for HCMV reactivation.
A–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 C) 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).
Figure 2.
The binding of phosphorylated CREB to the MIEP correlates with reactivation.
A) 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) 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). C) 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.
Figure 3.
Deletion of the creb response elements from the MIEP abrogates reactivation in DCs.
A) Monocytes infected with wild type (WT), Revertant (Rev) or CRE deletion virus (ΔCRE) were analysed 5 days post infection for UL138, UL123 and GAPDH gene expression by qRT-PCR. Viral gene expression (UL138 & UL123) was expressed as a ratio to GADPH. A further no RT control is shown for UL123 (4–6) to discriminate between RNA and low level DNA contamination. B) DNA from 10∧5 latently infected cells was harvested and viral DNA quantified by qPCR. After normalisation to GAPDH, viral genome copy number per cell was calculated. C) immature DCs derived from HCMV infected monocytes were stimulated with LPS (1–3) or IL-6 (4–6) 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,B). D) Chromatin immunopreciptations on immature DCs (iDC) derived from monocytes infected with wild type (WT), Revertant (Rev) or CRE deletion virus (ΔCRE) were performed alongside LPS (2,5,8) and IL-6 (3,6,9) stimulated DCs (3 hours post stimulation) for histone H3-K42Me binding. DNA was amplified in an MIEP PCR and expressed as ratio to input. S.D. of n = 2.
Figure 4.
Inhibition of mitogen and stress activated kinase activity is a potent inhibitor of HCMV reactivation.
A) RNA isolated from DCs stimulated with IL-6 (24 hours) following incubation DMSO (1) or ERK (2), MSK (3) and RSK (4) inhibitors was amplified by qRT-PCR. IE expression was standardised to GAPDH (2ΔCT) and the fold change in gene expression (2ΔΔCT) was expressed relative to the DMSO sample. B) Western blot analysis for phosphor- and total MSK was performed on iDCs or iDCs stimulated with IL6 following pre-treatment with RSK, ERK, MSK inhibitors or DMSO control. C) RNA isolated from DCs pre-treated with mock (1), DMSO (2) or MSK inhibitor (3) then infected for 8 hours with HCMV (HFF MOI = 5) was analysed for IE expression by qRT-PCR. D) DCs pre-treated with mock (1), DMSO (2) or MSK inhibitors (3) were infected with HCMV (HFF MOI = 5). Twenty four hours post infection the percentage of IE positive cells was calculated by indirect immunofluorescence and Hoechst nuclear counterstaining. S.D. shown from n = 3 (A–C).
Figure 5.
Inhibition of mitogen and stress kinase activity blocks CREB and histone phosphorylation at the MIEP.
A) 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) 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-S10P (S10) and histone H3-K93Me (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. C) 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.
Figure 6.
Phosphorylation of histone H3 is dependent on CREB binding to the MIEP.
A) 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-S10P 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–C) 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-S10P (S10P), anti-histone H3-K93Me (K9M) or isoptype control (IgG) antibodies and then amplified in an MIEP qPCR and signal expressed as a ratio of the Input. C) 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-S10P 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-K93Me (5,6) were amplified in an MIEP qPCR and expressed as a ratio of Input. S.D. of n = 2. D) 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.
Figure 7.
A dominant negative MEK1 blocks IE gene expression and CREB and histone H3 phosphorylation at the MIEP.
A) 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) 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. C) 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.
Figure 8.
Okadaic acid promotes histone phosphorylation at the MIEP and partially rescuing the CRE deletion virus.
A) 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 (2ΔCT) and the fold change in gene expression (2ΔΔCT) following okadaic acid addition was expressed relative to the DMSO control. S.D of n = 3 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-S10P 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.
Figure 9.
Model for the induction of IE gene expression in DCs.
The expression of IE gene expression is restricted in CD14+ monocytes and is inherently unresponsive to inflammatory IL-6 signalling. However, cellular differentiation to a permissive phenotype renders latently infected cells responsive to stimuli that support IE gene expression and, ultimately, reactivation.