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. 2012 Feb;22(2):360-71.
doi: 10.1038/cr.2011.114. Epub 2011 Jul 19.

Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function

Jerry Wong  1 , Jingchun Zhang, Bobby Yanagawa, Zongshu Luo, Xiangsheng Yang, Jiang Chang, Bruce McManus, Honglin Luo
Affiliations

Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function

Jerry Wong et al. Cell Res. 2012 Feb.

Abstract

Enteroviral infection can lead to dilated cardiomyopathy (DCM), which is a major cause of cardiovascular mortality worldwide. However, the pathogenetic mechanisms have not been fully elucidated. Serum response factor (SRF) is a cardiac-enriched transcription regulator controlling the expression of a variety of target genes, including those involved in the contractile apparatus and immediate early response, as well as microRNAs that silence the expression of cardiac regulatory factors. Knockout of SRF in the heart results in downregulation of cardiac contractile gene expression and development of DCM. The goal of this study is to understand the role of SRF in enterovirus-induced cardiac dysfunction and progression to DCM. Here we report that SRF is cleaved following enteroviral infection of mouse heart and cultured cardiomyocytes. This cleavage is accompanied by impaired cardiac function and downregulation of cardiac-specific contractile and regulatory genes. Further investigation by antibody epitope mapping and site-directed mutagenesis demonstrates that SRF cleavage occurs at the region of its transactivation domain through the action of virus-encoded protease 2A. Moreover, we demonstrate that cleavage of SRF dissociates its transactivation domain from DNA-binding domain, resulting in the disruption of SRF-mediated gene transactivation. In addition to loss of functional SRF, finally we report that the N-terminal fragment of SRF cleavage products can also act as a dominant-negative transcription factor, which likely competes with the native SRF for DNA binding. Our results suggest a mechanism by which virus infection impairs heart function and may offer a new therapeutic strategy to ameliorate myocardial damage and progression to DCM.

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Figures

Figure 1
Figure 1
Cardiac function and cardiac gene expression following CVB3 infection. (A) Cardiac function following CVB3 infection by echocardiography. At day 0 (before infection), and 3, 9, and 30 days following CVB3 or sham infection of A/J mice, systolic and diastolic parasternal long and short axis measurements were obtained. Left ventricular posterior wall (LVPW) thickening and ejection fraction are presented as percentage changes (mean ± SD, n = 4 for each group). pi, post infection. (B) CVB3- or sham-infected mouse hearts (n = 4 for each group, pooled) at 9 days pi were collected for Affymetrix array analysis. Gene changes in intensity (averages of two replicates of the microarray analysis) were plotted as a ratio of CVB3- to sham-infected hearts for visualization. Only genes that exhibited a 1.8-fold change (log2) or greater were included. MHC, myosin heavy chain; MLC, myosin light chain. (C) Murine HL-1 cardiomyocytes were sham- or CVB3-infected for 24 h. Real-time quantitative RT-PCR was performed to examine the expression of indicated genes. The gene expression was normalized to GAPDH mRNA, and then displayed as fold changes compared to sham infection (mean ± SD, n = 3). ANP, atrial natriuretic peptide; MEF2, myocyte enhancer factor 2; NFATc, nuclear factor of activated T cells.
Figure 2
Figure 2
Protein and gene expression of SRF following CVB3 infection. (A) Protein expression of SRF following CVB3 infection of murine HL-1 cardiomyocytes. HL-1 cells were either sham infected or infected with CVB3 for various times as indicated. Western blotting was performed to examine protein expression of SRF (top, using anti-C-terminal SRF antibody; bottom, using anti-N-terminal SRF antibody), viral protein VP1 and β-actin (loading control). (B) Protein distribution of SRF following CVB3 infection of HL-1 cells. HL-1 cells were infected with CVB3 for 20 h, double-immunocytochemical staining was then performed using anti-C-terminal SRF antibody and anti-VP1 antibody to examine the expression and localization of SRF (red) and viral protein VP1 (green), respectively. The nucleus was counterstained with DAPI (blue). (C) Gene expression of SRF after CVB3 infection. RNA samples were extracted from HL-1 cardiomyocytes at 24 h following CVB3 infection. Gene levels of SRF were measured by real-time quantitative RT-PCR and normalized to GAPDH mRNA (mean ± SD, n = 3). (D) SRF expression in A/J mouse heart following 3 and 9 days of CVB3 infection. Heart extracts were used for western blot analysis of protein expression of SRF (using an anti-C-terminal SRF antibody) and GAPDH (loading control). Left panels, levels of SRF were quantitated by densitometric analysis using NIH ImageJ V1.43, normalized to GAPDH, and presented as mean ± SD. Right panels, representative western blot of SRF expression in mouse heart at 9 days post infection.
Figure 3
Figure 3
Cleavage of SRF following CVB3 infection. HeLa cells were transiently transfected with a plasmid expressing 3×FLAG-SRF for 48 h, followed by CVB3 infection for 7 h. (A) Schematic diagram of the 3×FLAG-SRF construct and western blot detection of SRF with the antibodies described in B and C. Western blotting was performed to examine protein expression of viral protein VP1, β-actin (loading control), and SRF using an anti-FLAG antibody that recognizes the N-terminus of SRF (B) or using an anti-C-terminal SRF antibody (C). NS, nonspecific bands.
Figure 4
Figure 4
Effect of caspase inhibition on CVB3-induced cleavage of SRF. HeLa cells were transiently transfected with a plasmid expressing 3×FLAG-SRF or an empty vector for 48 h, followed by CVB3 infection for 7 h in the presence or absence of z-VAD-fmk (50 μM), a pan caspase inhibitor. Western blotting was performed to examine protein expression of SRF (top, using anti-FLAG antibody; middle, using anti-C-terminal SRF antibody), viral protein VP1, and β-actin (loading control).
Figure 5
Figure 5
Cleavage of SRF by overexpressing viral protease 2A. (A) SRF prediction cleavage site for viral protease 2A. The SRF protein sequence was searched for putative cleavage sites of picornavirus proteases using NetPicoRNA V1.0 algorithm. *Cleavage prediction score above 0.500 are predicted as potential cleavage sites. #Surface exposure scores above 0.500 are predicted as surface exposed sites. (B, C) HeLa cells were transiently transfected with 3×FLAG-tagged SRF construct for 48 h, followed by additional transfection with either empty vector (control) or viral protease 2A plasmid for another 48 h. Western blotting was performed to examine protein expression of SRF using anti-FLAG antibody (B) that recognizes the N-terminus of SRF, or by anti-C-terminal SRF antibody (C). The 2A proteolytic activity was confirmed by the cleavage of eIF4γ. Expression of β-actin was shown as a loading control. NS, nonspecific bands.
Figure 6
Figure 6
Cleavage of SRF after T326 following CVB3 infection. (A) Diagram of point mutation constructs of SRF. (B) HeLa cells were transiently transfected with 3×FLAG-tagged SRF-wild-type (SRF-WT) or SRF mutants (SRF-G349E or SRF-G327E as indicated) for 48 h, followed by 7 h CVB3 infection. Transfection of an empty vector (pcDNA) was used as negative controls. Western blotting was performed to examine protein expression of SRF using anti-FLAG antibody and viral protein VP1. Expression of β-actin was shown as a loading control. NS, nonspecific bands. (C) Identification of protease 2A substrate cleavage site. Substrate recognition by 2A depends on a degenerate amino acid pattern upstream of the cleavage site. The cleavage recognition site usually contains a T, or S at position P2 and an L, I, or M at position P4. A glycine residue at the P1′ C-terminal side of the scissile bond of the cleavage site occurs in all known substrates of the 2A protease.
Figure 7
Figure 7
Cellular localization and transcriptional activity of SRF mutants. (A, B) HeLa cells (A) or HL-1 cardiomyocytes (B) were transiently transfected with 3×FLAG-tagged SRF-N or SRF-C fragments of the cleaved SRF products for 48 h using Lipofectamine 2000. Immunocytochemical staining for transfected SRF was performed using anti-FLAG antibody. Nucleus was counterstained with DAPI. (C, D) HeLa cells (C) or HL-1 cardiomyocytes (D) were transiently co-transfected with cardiac α-actin luciferase reporter plasmid and constructs expressing SRF-WT or empty vector and/or SRF-G327E, SRF-N, and SRF-C mutants as indicated for 48 h. Luciferase assay was performed and the values are presented as folder changes (mean ± SD, n = 3) over those of control reporter (LacZ), which were arbitrarily set as 1.
Figure 8
Figure 8
Effects of SRF dysregulation on CVB3 replication. (A) Effects of knockdown of SRF on CVB3 replication. The gene expression of SRF was knocked down by siRNA. siCON, scramble siRNA control. Upper, western blot analysis was carried out to examine protein level of viral protein VP1, SRF (with anti-FLAG antibody), and β-actin (loading control). Lower, plaque assay was performed to examine virus titers. The results are shown as mean ± SD (n = 3). (B) Effect of overexpression of SRF-wild-type (SRF-WT) and two truncated SRF mutants on CVB3 replication. HeLa cells were transiently transfected with SRF-WT or truncated SRF mutants (SRF-N or SRF-C as indicated) for 48 h, followed by 7 h CVB3 infection. Western blot analysis was carried out to examine protein level of viral protein VP1, SRF (with anti-FLAG antibody) and β-actin (loading control).
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