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. 2017;6(1):13-25.

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

Verity Kew  1 , Mark Wills  1 , Matthew Reeves  2
Affiliations

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

Verity Kew et al. J Mol Biochem. 2017.

Abstract

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.

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Conflict of interest statement

Conflicts of interest The authors declare no conflicts of interest associated with this work.

Figures

Figure 1
Figure 1. HCMV infection blocks cisplatin A mediated activation of Bak.
(A) Western blot analysis of CD34+ cells either mock (M), HCMV infected (V) or cisplatin A treated (CspA) for Bak, MCL-1, BCL-XL, Bcl-2 or GAPDH expression 2 hours post-infection. (B) qRT-PCR analysis of RNA isolated from mock, HCMV infected or cisplatin A treated cells for Bak or MCL-1 expression 2 hours post-infection. Changes in gene expression were identified using GAPDH and 2-ΔΔCT method. n=3 (C) CD34+ cells were mock or HCMV infected and then incubated with DMSO or cisplatin A 3 hours post-infection. Cells were then permeabilised and stained with an antibody that specifically recognises an N terminal peptide exposed in activated Bak protein or with an isotype matched control. Fluorescent staining was achieved using a FITC-Goat anti-mouse antibody and then cells analysed by flow cytometry.
Figure 2
Figure 2. HCMV infection induces a transient activation of Bak which is not reversed when ERK responses are inhibited.
(A) CD34+ cells were infected with HCMV and then at times 0 to 3 hours post infection cells were permeabilised and stained for evidence of Bak activation by flow cytometry. (B-C) CD34+ cells were either pre-treated with an ERK inhibitor (B) or subjected to ELK1 or control siRNA knock-down (C) and then either mock of HCMV infected. Cells were then permeabilised and stained for evidence of Bak activation by flow cytometry.
Figure 3
Figure 3. HCMV infection promotes down-regulation of pro-apoptotic proteins in an ERK dependent manner.
(A,B) Western blot analysis of CD34+ cells either mock (M), HCMV infected (HCMV) or HCMV infected after 1 hour of pre-incubation with and ERK-MAPK inhibitor (ERK) for expression of PUMA (A) or BIM isoforms. (B) protein expression was performed at 2 hours post-infection. GAPDH served as loading control. Densitometry was used to measure relative levels of protein expression (C) qRT-PCR analysis of RNA isolated from mock or HCMV infected cells for PUMA, BIM or MCL-1 expression was performed 2 hours post-infection. Changes in gene expression were identified using GAPDH and 2-ΔΔCT method, n=3.
Figure 4
Figure 4. HCMV targets ELK1 for phosphorylation in an ERK dependent manner.
(A) Western blot analysis of CD34+ cells, either mock (M) or HCMV infected (V) for ELK1 and ELK1 phosphorylation, 2 hours post-infection. Actin served as loading control. (B) Western blot analysis of CD34+ cells, either mock (M) or HCMV infected (HCMV) cells, with or without prior incubation with ERK-MAPK inhibitor (ERK) or DMSO control for 1 hour at 2 hours post-infection. Actin served as loading control. (C) Chromatin immuno-precipitation assays were performed on CD34+ cells with isotype (IgG), ELK1 or phosphor-ELK1 (ELK-1p) antibodies. ChIPs were performed on CD34+ cells, HCMV infected CD34+ cells or the equivalent but with prior incubation with U0126 ERK inhibitor for 1 hour. Cells were analysed at 2 hours post infection, n=3. Students t-test was used to test for significance at p<0.05.
Figure 5
Figure 5. Depletion of ELK1 from CD34+ cells abrogates the HCMV survival response.
(A) Western blot analysis of CD34+ cells 48 hours post-transfection with a control (Con), ELK1-specific (ELK) siRNA for ELK1 and GAPDH expression. (B) qRT-PCR analysis of RNA isolated from mock or HCMV infected cells 2 hours post-infection that have first been treated with either mock, control (Scr KD) or ELK1 (ELK KD) siRNAs. Changes in gene expression were identified using GAPDH and 2-ΔΔCT method, n=3. Students t-test was used to test for significance at p<0.05. (C) CD34+ cells were either mock or HCMV infected and then, 3 hours post-infection, incubated with cisplatin A or DMSO control. Alternatively, CD34+ cells were transfected with control or ELK-1 specific siRNAs and then either mock or HCMV-infected. In all experiments cell viability was measured 24 hours post-infection. Graph is the average of 2 independent experiments analysed in triplicate.
Figure 6
Figure 6. ELK-1 is required for the reversal of Bak activation and the establishment of HCMV latency.
(A,B) CD34+ cells were subjected to ELK1 or control siRNA knockdown and then either mock of HCMV-infected. Cells were then either permeabilised and stained for evidence of Bak activation by flow cytometry at 3hpi (A) or analysed 3 days post infection for evidence of viral latent gene expression (B).
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