Abstract

Aims

Coxsackievirus B3 (CVB3) is known to cause a variety of human diseases including acute and chronic myocarditis as well as dilated cardiomyopathy (DCM). However, the mechanisms by which CVB3 causes diseases are not well understood.

Methods and results

Studies identifying protein–protein interactions during CVB3 infection are useful in delineating the pathogenesis of acute or chronic myocarditis. Screening a human heart cDNA library revealed a yet unknown interaction partner of the proapoptotic protein Siva. We demonstrate that Siva specifically interacts with the heart and skeletal muscle protein telethonin. The expression of Siva is increased in heart tissue of CVB3-infected mice and the proteins colocalize in cardiomyocytes.

Conclusion

telethonin might be involved in CVB3-mediated cell damage and in the resulting cardiac dysfunction due to the interaction with Siva. We suggest a molecular mechanism through which coxsackieviral infection contributes to the pathogenesis of chronic myocarditis and in particular of acquired forms of DCM.

Introduction

Coxsackievirus B3 (CVB3), an enterovirus of the picornavirus family, has been implicated in a variety of serious diseases including acute and chronic myocarditis, dilated cardiomyopathy (DCM), type I insulin-dependent diabetes mellitus and pancreatitis.1–3 CVB3 is the serotype most frequently associated with myocarditis,4 and its pathogenesis has been studied extensively in mice. A majority of symptomatic patients recover well from acute myocarditis. However, 10–20% of patients will develop chronic disease, and a subset progresses over time to DCM.5–7 During DCM one or both ventricles dilate and decompensate, leading to progressive heart failure and significantly increased mortality.8 The mechanism by which CVB3 causes acute or chronic myocarditis, in particular leading to DCM, is not well characterized.4,9

Some forms of DCM involve the cytoskeleton. Mutations in genes, encoding sarcomeric proteins such as actin and titin are the cause of different forms of cardiomyopathy. Dystrophin was found to be responsible for Duchenne and Becker muscular dystrophy as well as for X-linked DCM.10–12 CVB3 induces DCM by protease 2A-mediated cleavage and inactivation of Dystrophin as well as dissociation of Dystrophin-associated glycoproteins from the sarcolemma essential for myocyte and cardiomyocyte integrity, due to the central role of cytoskeletal proteins in cardiac function.13

Infections of CVB3 are often accompanied by programmed cell death, and apoptotic processes were found in myocardial tissue of patients with DCM.14 Apoptotic cells are detectable in inflammatory lesions and outside of inflamed areas in the myocardial tissue of CVB3-infected mice.15 Previously, we identified the interaction between the CVB3 capsid protein VP2 and the proapoptotic host protein Siva,16 which is known to be involved in CD27- or GITR-induced apoptosis by providing the missing death domain or contact sites for downstream located proteins of different cell death pathways.17,18 Furthermore, transcription of the mouse equivalent of Siva (mSiva) is induced in pancreas and heart tissue after CVB3H3 infection.16 Efficient binding of VP2 to amino acids 118–136 of Siva is important for the activity of the host protein, yielding severe destruction and apoptosis in tissue. A comparison between CVB3H3 and the mutant CVB3H310A1, in which asparagine at position 165 in VP2 is changed into aspartate,19 shows differences in the apoptotic potential in vivo and in vitro as verified by viral spread and virus progeny production, Siva mRNA expression or DNA (deoxyribonucleic acid) fragmentation. Therefore, the results indicate a mechanism by which apoptosis contributes to coxsackievirus-dependent pathogenesis.20,21

To study more thoroughly the pathological processes caused by CVB3, we performed a two-hybrid screening of a heart cDNA (complementary DNA) library using Siva as bait and identified the cytoskeletal protein telethonin as a new binding partner. The interaction was confirmed in vivo and in vitro. We found that the binding site of telethonin to Siva is located in the N-terminal region, whereas the C-terminus of Siva is necessary and sufficient for interaction. We demonstrate colocalization of mSiva and mouse telethonin (mTelethonin) in infected cardiomyocytes. The interaction could help to explain cell damage and cardiac dysfunction of CVB3-infected animals. These findings indicate a molecular mechanism by which CVB3 contributes to the pathogenesis of myocardial diseases, and demonstrate a potential role of cytoskeletal protein alterations in acquired DCM.

Methods

Plasmids

All plasmids used were generated by standard PCR cloning methods. The cDNA molecules were amplified by Pwo polymerase (Roche Diagnostics, Mannheim, Germany) and ligated into the target vectors (Table 1). The correct insertion was confirmed by DNA sequencing.

Table 1

Summary of all constructs used in the yeast two-hybrid and binding experiments

Name Vector Brief description aa Sequence 
(A) Two-hybrid experiments 
 Siva pAS2-1 Gal4-DNA-BD 1–175 
 Siva-1 pAS2-1 Gal4-DNA-BD 1–157 
 Siva-2 pAS2-1 Gal4-DNA-BD 1–150 
 telethonin-E51A/E51C pACT2 Gal4-AD 5–167 
 telethonin pACT2 Gal4-AD 1–167 
 telethonin-1 pACT2 Gal4-AD 1–84, 142–167 
 telethonin-2 pACT2 Gal4-AD 1–82 
(B) Binding assays 
 Siva-DD-C pGEX-4T-3 GST fusion 48–175 
 Siva-C1 pGEX-4T-3 GST fusion 150–175 
 Siva-C2 pGEX-4T-3 GST fusion 114–175 
 Siva-DD-ΔC pGEX-4T-3 GST fusion 49–150 
 Siva-ΔDD pGEX-4T-3 GST fusion 49–105 
 His6–telethonin pET-32b His6 fusion 1–167 
Name Vector Brief description aa Sequence 
(A) Two-hybrid experiments 
 Siva pAS2-1 Gal4-DNA-BD 1–175 
 Siva-1 pAS2-1 Gal4-DNA-BD 1–157 
 Siva-2 pAS2-1 Gal4-DNA-BD 1–150 
 telethonin-E51A/E51C pACT2 Gal4-AD 5–167 
 telethonin pACT2 Gal4-AD 1–167 
 telethonin-1 pACT2 Gal4-AD 1–84, 142–167 
 telethonin-2 pACT2 Gal4-AD 1–82 
(B) Binding assays 
 Siva-DD-C pGEX-4T-3 GST fusion 48–175 
 Siva-C1 pGEX-4T-3 GST fusion 150–175 
 Siva-C2 pGEX-4T-3 GST fusion 114–175 
 Siva-DD-ΔC pGEX-4T-3 GST fusion 49–150 
 Siva-ΔDD pGEX-4T-3 GST fusion 49–105 
 His6–telethonin pET-32b His6 fusion 1–167 

Yeast two-hybrid experiments

The yeast strain KFY3 (MATa, ura3–52, his3–200, ade2–101, lys2–801, trp1–901, leu2–3, -112, gal4Δ, gal80Δ, cyh2r, LYS2::GAL1UAS-HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA-lacZ, ADE2::ADE2, leu2::PGAL1-yEGFP3-loxP-kanMX-loxP)22 was used. For directed two-hybrid experiments, cDNA molecules were cloned into the bait vector pAS2-1, or the prey vector pACT2 (BD Biosciences Clontech, Heidelberg, Germany), respectively, to yield fusions with the DNA-binding domain (BD) or activation domain (AD) of the yeast Gal4 transcription factor. Yeast cells were transformed as described,23 plated onto selective agar dishes and incubated at 30°C for 3 days.

A human heart cDNA library (BD Biosciences Clontech) was screened for proteins that interact with Siva. Agar plates with cotransformants were probed for LacZ activity by a filter lift assay according to Breeden and Nasmyth.24 Plasmid DNA from positive clones was rescued from yeast cells, transformed into Escherichia coli (E. coli) strain HB10125 and isolated with standard procedures. In a second two-hybrid assay, three single colonies of KFY3 transformants containing the bait plasmid and one of the obtained library plasmids were analysed. Positive clones were selected and the inserts of library plasmids sequenced. These sequences were compared by BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/).

Verification of interactions and identification of the human Siva and telethonin BDs were investigated with full length and truncated fusion proteins. Plasmids containing the Gal4-DNA-BD or -AD fusion proteins were transformed into KFY3 cells. Three single colonies of KFY3 transformants were picked and grown in liquid minimal medium at 30°C overnight. The activity of β-galactosidase was determined using a quantitative liquid assay according to Breeden and Nasmyth.26

Protein expression and binding assays

Telethonin was cloned into pET-32b (Novagen, Merck, Darmstadt, Germany). Standard protocols were used for His6 fusion protein expression and purification with a cobalt-based immobilized metal affinity chromatography resin (BD Biosciences Clontech).27 All subfragments of Siva were cloned into pGEX-4T-3. Fusion proteins were purified from E. coli lysates by using Glutathione Sepharose® 4B (GE Healthcare, Munich, Germany). Eluate purity was assessed by SDS–PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis).28

Interaction between human Siva and telethonin was verified by in vitro pull-down assays. Briefly, 30 µg of fusion proteins, bound to their solid matrix (cobalt-based resin or Glutathione Sepharose, respectively), were incubated with 45 µg of eluted fusion protein of the interaction partner in a total volume of 150 µL binding buffer (50 mM Tris–HCl pH 7.5; 150 mM NaCl; 0.1% NP40; 1 mM EDTA). Subsequent steps were performed as previously described.22

Slot blot assays were performed while 1.5 µg of glutathione S-transferase (GST) fusion proteins, GST and BSA (bovine serum albumin) were blotted on nitrocellulose membranes. The membranes were blocked with 2.5% BSA in PBS (phosphate buffered saline) at 4°C overnight and incubated at room temperature for 2 h in presence or absence of human recombinant telethonin at a concentration of 15 µg. After washing with PBS and 0.05% Tween, the bound telethonin was visualized using anti-telethonin antibody (BD Biosciences Clontech).

Immunochemistry

Human embryonic kidney 293 (HEK293) cells were transfected with plasmid pVITRO2–mcs (Invivogen, San Diego, CA, USA) containing Siva and telethonin sequences using Effectene Transfection Reagent (Qiagen, Hilden, Germany). Transfection efficiency of HEK293 cells was determined with EGFP by fluorescence microscopy after 48 h. The cells were treated for immunofluorescence detection as described later.

Adult (8–10 weeks of age) male inbred BALB/c (H-2dd) mice were inoculated by intraperitoneal injection of 0.2 mL saline containing the stated amount of virus (infectious dose 0.5 LD50 of CVB3H3, virus titer: 4.7 × 104pfu/100 mg tissue).19 All procedures conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). For each group (infected or non-infected), four mice were treated. For indirect immunofluorescence, frozen 10 µm thick sections were prepared from heart tissue of non-infected and of CVB3-infected mice 7 days p.i. These experiments have been repeated three times. The sections were fixed with 3.8% paraformaldehyde in PBS for 12 min and permeabilized with 0.5% saponine in PBS for 11 min at room temperature. After washing in 0.1% saponine/PBS, sections were blocked with 1% FCS in 0.1% saponine/PBS at 4°C overnight. The sections were washed and then incubated with the primary anti-Siva antibody from goat (1/25 dilution) and the anti-telethonin antibody from rabbit (1/50 dilution, Santa Cruz, Heidelberg, Germany). Detection of the primary antibody was performed using indocarbocyanine-conjugated donkey anti-goat and fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin (Jackson Immunoresearch, West Grove, PA, USA). An LSM 510 laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany) was used for confocal microscopy at ×63 magnification.

Results

Interaction between human Siva and telethonin

Screening for novel interaction partners of Siva

To identify interaction partners involved in CVB3 pathogenesis and apoptosis induced by Siva, a human heart cDNA two-hybrid library was screened using Siva as bait. A total of 210 000 independent yeast colonies were analysed. After retransformation of isolated cDNA clones into yeast cells, the cytoskeletal protein telethonin was identified as a potential binding partner. Results of LacZ filter assays with corresponding clones are shown in Figure 1A.

Figure 1

Interaction between Siva and telethonin. (A) Screening of a human heart cDNA (complementary deoxyribonucleic acid) library for interaction partners of Siva. 210 000 clones were screened for LacZ activity by filter lift assay. Plasmids from clones showing a positive signal were rescued and tested again. Two clones encoding the cDNA of the cytoskeletal protein telethonin were identified as interaction partners. To exclude artificial transactivation and DNA-binding by one of the interaction partners itself, the corresponding clones or telethonin were analysed by transformation with Gal4-BD (binding domain) or Gal4-AD (activation domain) alone. For positive Control the homodimerization of the regulatory subunit of the yeast adenosine 3′, 5′-cyclic monophosphate-dependent protein kinase A encoded by the BCY1 gene was used. (B) and (C) Verification of Siva and telethonin interaction by full-length proteins and determination of the particular binding domains of both Siva and telethonin by truncated fusion proteins. Plasmids carrying Siva, Siva-1 or Siva-2 and telethonin, telethonin-1 or telethonin-2 (Table 1A), alternatively Gal4-BD and Gal4-AD alone were transformed into yeast reporter strain KFY3. The resulting cells were analysed for β-galactosidase activity with liquid assays. Only the interactions of Siva and telethonin, Siva and telethonin-1 or telethonin-2, respectively reconstituted an active Gal4 transcription factor, demonstrated by β-galactosidase activity measured after 2 h of incubation with ortho-nitrophenyl-beta-D-galactopyranoside. Data represent the mean ± SE of triplicate samples from three independent experiments. The statistical significance of β-galactosidase activity between the indicated groups was calculated using the Student’s t-test: *P < 0.05.

Figure 1

Interaction between Siva and telethonin. (A) Screening of a human heart cDNA (complementary deoxyribonucleic acid) library for interaction partners of Siva. 210 000 clones were screened for LacZ activity by filter lift assay. Plasmids from clones showing a positive signal were rescued and tested again. Two clones encoding the cDNA of the cytoskeletal protein telethonin were identified as interaction partners. To exclude artificial transactivation and DNA-binding by one of the interaction partners itself, the corresponding clones or telethonin were analysed by transformation with Gal4-BD (binding domain) or Gal4-AD (activation domain) alone. For positive Control the homodimerization of the regulatory subunit of the yeast adenosine 3′, 5′-cyclic monophosphate-dependent protein kinase A encoded by the BCY1 gene was used. (B) and (C) Verification of Siva and telethonin interaction by full-length proteins and determination of the particular binding domains of both Siva and telethonin by truncated fusion proteins. Plasmids carrying Siva, Siva-1 or Siva-2 and telethonin, telethonin-1 or telethonin-2 (Table 1A), alternatively Gal4-BD and Gal4-AD alone were transformed into yeast reporter strain KFY3. The resulting cells were analysed for β-galactosidase activity with liquid assays. Only the interactions of Siva and telethonin, Siva and telethonin-1 or telethonin-2, respectively reconstituted an active Gal4 transcription factor, demonstrated by β-galactosidase activity measured after 2 h of incubation with ortho-nitrophenyl-beta-D-galactopyranoside. Data represent the mean ± SE of triplicate samples from three independent experiments. The statistical significance of β-galactosidase activity between the indicated groups was calculated using the Student’s t-test: *P < 0.05.

Verification of interaction and determination of Siva and telethonin binding domains

DNA sequencing revealed that the encoded fusion protein of the original two-hybrid clone lacked the first four residues of telethonin. To reduce the probability of a false positive interaction, the entire telethonin sequence was fused with Gal4-AD and probed with full-length Siva using a yeast two-hybrid liquid assay (Table 1A). Both fusion proteins interacted, as determined by the β-galactosidase activity of the plasmid containing cells (Figure 1B and C). The data revealed that the first four amino acids of telethonin were not necessary for Siva binding. To identify the binding regions of Siva and telethonin, two-hybrid liquid assays with full-length proteins and truncated fragments were performed. As shown in Figure 1B, only full-length Siva interacted with telethonin, whereas fragments thereof, i.e. Siva-1 and Siva-2 did not. We concluded that the C-terminus of Siva is necessary and sufficient for telethonin binding. Furthermore, the binding region of telethonin was restricted to the N-terminus (Figure 1C). The cells containing the plasmids telethonin-1 or telethonin-2 showed less β-galactosidase activity compared with cells expressing the full-length protein suggesting that these telethonin fragments lack amino acids required for Siva binding.

In vitro binding between Siva and telethonin

The interaction between Siva and telethonin in vitro was confirmed by pull-down experiments. Siva variants used in these studies are shown in Table 1B and Figure 2A. We found that His6–telethonin was able to bind Siva-DD-C, Siva-C1 and Siva-C2, but did not show any interaction with Siva-DD-ΔC and Siva-ΔDD (Figure 2B and C). GST and full-length GST–TopBP1 [GST–topoisomerase ii (beta)-binding protein 1] served as negative Controls. Slot blot experiments revealed interaction between telethonin and Siva-DD-C or Siva-C2 only (Figure 2D). In brief, the results show the C-terminus of Siva is necessary and sufficient for telethonin binding.

Figure 2

Siva and telethonin interact biochemically. (A) Topology of Siva fragments that were used during these studies according to protein domains of recent publications.17,22,30 (B) A pull-down assay demonstrates that His6–telethonin bound Siva-DD-C, Siva-C1 and Siva-C2, but not Siva-DD-ΔC and Siva-ΔDD (Table 1B). Glutathione S-transferase (GST) and GST fusion proteins of Siva [lane: (1) Siva-DD-C, (2) Siva-C1, (3) Siva-C2, (4) GST, (5) Siva-DD-ΔC, (6) Siva-ΔDD, and (7) His6–telethonin] attached to Glutathione Sepharose were mixed with His6–telethonin. The resins were washed, subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotted using S-protein alkaline phosphatase (AP) conjugate (1/5000; Novagen) against telethonin. (C) A GST pull-down assay demonstrates that Siva-DD-C, Siva-C1 and Siva-C2 bound to His6–telethonin. The His6–telethonin attached to cobalt-based immobilized metal affinity chromatography resins were mixed with GST and GST fusion proteins {lane: (1) GST, (2) GST–TopBP1 [Topoisomerase II (beta)-Binding Protein 1], (3) Siva-DD-C, (4) Siva-DD-ΔC, (5) Siva-C1, (6) Siva-C2, and (7) Siva-ΔDD}. The resins were washed, subjected to SDS–PAGE, immunoblotted using anti-GST antibody (rabbit, 1/2000; Sigma Aldrich, Taufkirchen Germany) and anti-rabbit AP (1/8000). (D) Binding of telethonin and several Siva fragments in a slot blot experiment. 1.5 µg of GST, BSA (bovine serum albumin) and GST fusion proteins bound to nitrocellulose membrane, were incubated with 15 µg of eluted His6–telethonin. After washing, bound telethonin was detected by using anti-telethonin antibody (mouse, 1/1000; BD Biosciences Clontech) and anti-mouse horseradish peroxidase conjugate (1/1000). A representative result of three independent experiments is shown in each case.

Figure 2

Siva and telethonin interact biochemically. (A) Topology of Siva fragments that were used during these studies according to protein domains of recent publications.17,22,30 (B) A pull-down assay demonstrates that His6–telethonin bound Siva-DD-C, Siva-C1 and Siva-C2, but not Siva-DD-ΔC and Siva-ΔDD (Table 1B). Glutathione S-transferase (GST) and GST fusion proteins of Siva [lane: (1) Siva-DD-C, (2) Siva-C1, (3) Siva-C2, (4) GST, (5) Siva-DD-ΔC, (6) Siva-ΔDD, and (7) His6–telethonin] attached to Glutathione Sepharose were mixed with His6–telethonin. The resins were washed, subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotted using S-protein alkaline phosphatase (AP) conjugate (1/5000; Novagen) against telethonin. (C) A GST pull-down assay demonstrates that Siva-DD-C, Siva-C1 and Siva-C2 bound to His6–telethonin. The His6–telethonin attached to cobalt-based immobilized metal affinity chromatography resins were mixed with GST and GST fusion proteins {lane: (1) GST, (2) GST–TopBP1 [Topoisomerase II (beta)-Binding Protein 1], (3) Siva-DD-C, (4) Siva-DD-ΔC, (5) Siva-C1, (6) Siva-C2, and (7) Siva-ΔDD}. The resins were washed, subjected to SDS–PAGE, immunoblotted using anti-GST antibody (rabbit, 1/2000; Sigma Aldrich, Taufkirchen Germany) and anti-rabbit AP (1/8000). (D) Binding of telethonin and several Siva fragments in a slot blot experiment. 1.5 µg of GST, BSA (bovine serum albumin) and GST fusion proteins bound to nitrocellulose membrane, were incubated with 15 µg of eluted His6–telethonin. After washing, bound telethonin was detected by using anti-telethonin antibody (mouse, 1/1000; BD Biosciences Clontech) and anti-mouse horseradish peroxidase conjugate (1/1000). A representative result of three independent experiments is shown in each case.

Siva and telethonin localization in transfected HEK293 cells

Immunofluorescence experiments demonstrated colocalization of Siva and telethonin in the cytoplasm of transfected HEK293 cells. A remarkably higher Siva expression was found in cells transfected with pVITRO2–mcs–Siva–telethonin compared with the Control (Figure 3A and B) suggesting a low basal expression of Siva in HEK293 cells per se. Therefore, a HEK293 lysate was tested for Siva by dot blot experiments. The HEK293 cells contained a small amount of Siva proteins (data not shown).

Figure 3

Colocalization of Siva and telethonin in transfected HEK293 cells. Siva and telethonin expression were monitored by indirect immunofluorescence using a goat polyclonal anti-Siva and a rabbit polyclonal anti-telethonin as first antibodies and Cy3(indocarbocyanine)-conjugated anti-goat and FITC(fluorescein isothiocyanate)-conjugated anti-rabbit as secondary antibodies. DNA (deoxyribonucleic acid) was stained with 4′, 6-diamidino-2-phenylindole-dihydrochloride. Representative photographs were obtained. (A) Colocalization of Siva and telethonin was detected in transfected HEK293 cells. The cells were transfected with 0.3 µg of the expression plasmid pVITRO2–mcs–Siva–telethonin using Effectene Transfection Reagent for 48 h and treated for indirect immunofluorescence detection. (B) HEK293 cells were transfected only with the expression plasmid pVITRO2–mcs as Control.

Figure 3

Colocalization of Siva and telethonin in transfected HEK293 cells. Siva and telethonin expression were monitored by indirect immunofluorescence using a goat polyclonal anti-Siva and a rabbit polyclonal anti-telethonin as first antibodies and Cy3(indocarbocyanine)-conjugated anti-goat and FITC(fluorescein isothiocyanate)-conjugated anti-rabbit as secondary antibodies. DNA (deoxyribonucleic acid) was stained with 4′, 6-diamidino-2-phenylindole-dihydrochloride. Representative photographs were obtained. (A) Colocalization of Siva and telethonin was detected in transfected HEK293 cells. The cells were transfected with 0.3 µg of the expression plasmid pVITRO2–mcs–Siva–telethonin using Effectene Transfection Reagent for 48 h and treated for indirect immunofluorescence detection. (B) HEK293 cells were transfected only with the expression plasmid pVITRO2–mcs as Control.

Localization of Siva and telethonin in cardiomyocytes of mice

The mouse proteins of Siva and telethonin are highly homologue to the human gene products.29,30 Two-hybrid and pull-down experiments verified the interaction between the mouse equivalents of both proteins (data not shown). To describe the protein interaction in a natural environment, frozen heart sections of non-infected and CVB3-infected mice were stained for mSiva and mTelethonin and the proteins were subsequently visualized by confocal microscopy. For secondary antibody examination several sections of CVB3-infected mice were not incubated with primary antibodies (Figure 4B). The two proteins colocalized in distinct areas of infected cardiomyocytes (Figure 4A). Non-infected cardiomyocytes also expressed both proteins but Siva was found in a considerable lower amount than in the infected cells and no colocalization could be observed (Figure 4C). Transcription of mSiva, CVB3-VP2, and β-actin in tissue of individually non-infected or CVB3-infected mice was analysed by reverse transcription polymerase chain reaction (RT–PCR) as previously described (data not shown).16

Figure 4

Localization of mSiva (mouse equivalent of Siva) and mTelethonin (mouse telethonin) in frozen heart tissue sections of non-infected and CVB3 (coxsackievirus B3)-infected mice. Sections were immunolabelled with polyclonal antibodies to Siva (goat, 1/25) and telethonin (rabbit, 1/50) as well as Cy3(indocarbocyanine)-conjugated anti-goat (1/200) and FITC(fluorescein isothiocyanate)-conjugated anti-rabbit (1/100) as secondary antibodies. DNA (deoxyribonucleic acid) was stained with 4′, 6-diamidino-2-phenylindole-dihydrochloride. Siva and telethonin expression was monitored by indirect immunofluorescence with confocal microscopy and representative photographs were obtained. (A) Colocalization of mSiva and mTelethonin can be seen in distinct areas of CVB3-infected cardiomyocytes (white arrows). There is a dramatic increase in Siva expression following CVB3 infection. (B) For antibody examination several infected sections were not incubated with primary antibodies. (C) Localization of mSiva and mTelethonin in non-infected mouse cardiomyocytes.

Figure 4

Localization of mSiva (mouse equivalent of Siva) and mTelethonin (mouse telethonin) in frozen heart tissue sections of non-infected and CVB3 (coxsackievirus B3)-infected mice. Sections were immunolabelled with polyclonal antibodies to Siva (goat, 1/25) and telethonin (rabbit, 1/50) as well as Cy3(indocarbocyanine)-conjugated anti-goat (1/200) and FITC(fluorescein isothiocyanate)-conjugated anti-rabbit (1/100) as secondary antibodies. DNA (deoxyribonucleic acid) was stained with 4′, 6-diamidino-2-phenylindole-dihydrochloride. Siva and telethonin expression was monitored by indirect immunofluorescence with confocal microscopy and representative photographs were obtained. (A) Colocalization of mSiva and mTelethonin can be seen in distinct areas of CVB3-infected cardiomyocytes (white arrows). There is a dramatic increase in Siva expression following CVB3 infection. (B) For antibody examination several infected sections were not incubated with primary antibodies. (C) Localization of mSiva and mTelethonin in non-infected mouse cardiomyocytes.

Discussion

Coxsackievirus infections have been shown to cause acute or chronic myocarditis in humans. Mouse models were established to study the complexity of CVB3-caused pathogenesis, in which apoptosis plays a considerable role. Several apoptosis-regulating proteins were found to be involved in host cell fate regulation after viral infection. Previously, we demonstrated that the structural protein VP2 of CVB3H3 binds specifically to the proapoptotic protein Siva. The interaction is required for induction of caspase-catalysed apoptosis linked to severe tissue destruction and accelerated viral spread.16,20,21 Binding of Siva to the antiapoptotic members Bcl-XL and Bcl-2 of the Bcl-2-family results in a loss of protective function and in sensitization of cells to apoptosis.31 Proapoptotic activities of the BNip proteins were also observed during interactions with Bcl-XL and Bcl-2. Zhang et al.32 showed that murine Nip21 overexpression promotes CVB3-induced apoptosis via a caspase-dependent mitochondrial pathway.

To get more insights into cell damage processes and heart failure of CVB3-infected humans, we screened a human heart cDNA library for binding partners of Siva. The cytoskeletal protein telethonin was found to interact with Siva (Figure 1A). Telethonin is located inside the sarcomeres of striated muscles, as a part of the Z-disc.33 The Z-disc is the main anchoring point of the molecular machinery that underlies muscle contraction and plays a pivotal role in muscle structure and function,34 including sarcomeric assembly and organization. Defects in the components of the Z-disc can trigger pathological pathways. Telethonin is associated with the autosomal recessive disorder limb girdle muscular dystrophy 2G.35

The present study demonstrated an interaction between Siva and telethonin using yeast two-hybrid analysis, in vitro GST pull-down and slot-blot experiments as well as colocalization studies. The domain relevant for the interaction with Siva is located within the N-terminus of telethonin. Moreover, we showed that adjacent amino acids are also involved in Siva binding (Figure 1C). There is no difference in enzyme expression between cells which contain the two fragments of telethonin. Hence, the functional C-terminus is not necessary for interaction.

The N-terminus of telethonin (aa 1–90) interacts specifically with the N-terminal immunoglobulin domains Z1 and Z2 of the giant muscle protein titin.36 Titin plays a role in elasticity and muscle assembly by providing a scaffold for other sarcomeric proteins. The interaction between titin and telethonin is required for the structural integrity associated with muscle function of sarcomeres.29,37,38 Another protein that associates with telethonin (aa 53–81) is the muscle LIM protein (MLP) which stabilizes the telethonin–titin complex. A human MLP mutation (W4R) results in a marked defect in telethonin interaction/localization. Knöll et al.39 propose that the complex contributes to the regulation of cardiac mechanical stress sensing and cardiac hypertrophy. Malfunction of this control machinery can lead to human DCM and associated heart failure. Numerous mutations in the genes of titin and telethonin are associated with DCM and hypertrophic cardiomyopathy.40,41

Furthermore, the characterization of the Siva BD revealed that the C-terminus (aa 151–175) is necessary and sufficient for the interaction (Figures 1B and 2B and C). The high number of cysteine residues and potential zinc-binding motifs at the C-terminus suggest a complex three-dimensional structure.22 Little is known about the signalling pathway leading to Siva-induced apoptosis and contradictory results exist about the concerned protein domains. Siva was found to be involved via death domain-like structures in the CD27-transduced apoptotic pathway. In contrast with previous reports, Py et al.42 show that the death domain-like structure is dispensable for induction of apoptosis in lymphoid cells; instead, the main determinants are located in both the N- and C-terminal regions. Siva is an ubiquitous protein that could participate in various apoptotic pathways in a cell-type-dependent manner.17

The transcription of mouse SIVA is increased in pancreas and heart tissue in the presence of infectious virus particles as shown by RT–PCR and TUNEL assays.16 Expression of Siva leads to programmed cell death17 and may contribute to cell damage in CVB3-infected cells and organs.20 In this report we compared the expression levels of Siva and telethonin in hearts of non-infected and CVB3-infected mice by immunofluorescence. Only infected hearts showed a high level of Siva expression in distinct areas (Figure 4A and C), whereas the expression level of telethonin could not be quantified in more detail in the current study. At the beginning only a few cells are infected with CVB3 but during the course of an infection the number of distinct areas will increase.43 The outcome of a CVB3 infection is determined by complex interactions among several variables, such as the virus genotype,44,45 or the age,3,46 and immune status47 of the host.9 Furthermore, we were able to detect colocalization of Siva and telethonin in transfected HEK293 cells as well as in distinct areas of hearts of CVB3-infected mice only (Figures 3A and 4A). Therefore, we assume that an increase in Siva expression during a CVB3 infection leads to Siva association with telethonin.

It is conceivable that Siva translocates in CVB3-infected cardiomyocytes to the Z-disc by an unknown mechanism and competes with the binding of MLP and titin to telethonin. Defects in the stabilization of the complex which is required for sarcomeric function can lead to human DCM and associated heart failure. Our results indicate a new molecular mechanism through which coxsackieviral infection could contribute to the pathogenesis of chronic myocarditis and in particular of acquired forms of DCM via apoptosis-regulating proteins. We propose that the development of an inhibitory constraint of the Siva–telethonin association will support the treatment of these deleterious diseases.

Funding

This work was supported by an internal project of the Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute.

Acknowledgements

We thank Andrea Hartmann for performing confocal microscopy of mice heart sections. We acknowledge Uta Schmidt and Frank Hänel for providing GST-TopBP1 protein and for helpful advice.

Conflict of interest: none declared.

References

1
Baboonian
C
Davies
MJ
Booth
JC
McKenna
WJ
Coxsackie B viruses and human heart disease
Curr Top Microbiol Immunol
 , 
1997
, vol. 
223
 (pg. 
31
-
52
)
2
Clements
GB
Galbraith
DN
Taylor
KW
Coxsackie B virus infection and onset of childhood diabetes
Lancet
 , 
1995
, vol. 
346
 (pg. 
221
-
223
)
3
Drescher
KM
Kono
K
Bopegamage
S
Carson
SD
Tracy
S
Coxsackievirus B3 infection and type I diabetes development in NOD mice: insulitis determines susceptibility of pancreatic islets to virus infection
Virology
 , 
2004
, vol. 
329
 (pg. 
381
-
394
)
4
Bowles
NE
Towbin
JA
Molecular aspects of myocarditis
Curr Opin Cardiol
 , 
1998
, vol. 
13
 (pg. 
179
-
184
)
5
Kereiakes
DJ
Parmley
WW
Myocarditis and cardiomyopathy
Am Heart J
 , 
1984
, vol. 
108
 (pg. 
1318
-
1326
)
6
O’Connell
JB
The role of myocarditis in end-stage dilated cardiomyopathy
Tex Heart Inst J
 , 
1987
, vol. 
14
 (pg. 
268
-
275
)
7
Sole
MJ
Liu
P
Viral myocarditis: a paradigm for understanding the pathogenesis and treatment of dilated cardiomyopathy
J Am Coll Cardiol
 , 
1993
, vol. 
22
 (pg. 
99A
-
105A
)
8
Whitton
JL
Feuer
R
Myocarditis, microbes and autoimmunity
Autoimmunity
 , 
2004
, vol. 
37
 (pg. 
375
-
386
)
9
Esfandiarei
M
McManus
BM
Molecular biology and pathogenesis of viral myocarditis
Annu Rev Pathol Mech Dis
 , 
2008
, vol. 
3
 (pg. 
127
-
135
)
10
Itoh-Satoh
M
Hayashi
T
Nishi
H
Koga
Y
Arimura
T
Koyanagi
T
, et al.  . 
Titin mutations as the molecular basis for dilated cardiomyopathy
Biochem Biophys Res Commun
 , 
2002
, vol. 
291
 (pg. 
385
-
393
)
11
Olson
TM
Michels
VV
Thibodeau
SN
Tai
YS
Keating
MT
Actin mutations in dilated cardiomyopathy, a heritable form of heart failure
Science
 , 
1998
, vol. 
280
 (pg. 
750
-
752
)
12
Towbin
JA
The role of cytoskeletal proteins in cardiomyopathies
Curr Opin Cell Biol
 , 
1998
, vol. 
10
 (pg. 
131
-
139
)
13
Badorff
C
Lee
GH
Lamphear
BJ
Martone
ME
Campbell
KP
Rhoads
RE
, et al.  . 
Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy
Nat Med
 , 
1999
, vol. 
5
 (pg. 
320
-
326
)
14
Olivetti
G
Abbi
R
Quaini
F
Kajstura
J
Cheng
W
Nitahara
JA
, et al.  . 
Apoptosis in the failing human heart
N Engl J Med
 , 
1997
, vol. 
336
 (pg. 
1131
-
1141
)
15
Gebhard
JR
Perry
CM
Harkins
S
Lane
T
Mena
I
Asensio
VC
, et al.  . 
Coxsackievirus B3-induced myocarditis: perforin exacerbates disease, but plays no detectable role in virus clearance
Am J Pathol
 , 
1998
, vol. 
153
 (pg. 
417
-
428
)
16
Henke
A
Launhardt
H
Klement
K
Stelzner
A
Zell
R
Munder
T
Apoptosis in coxsackievirus B3-caused diseases: interaction between the capsid protein VP2 and the proapoptotic protein Siva
J Virol
 , 
2000
, vol. 
74
 (pg. 
4284
-
4290
)
17
Prasad
KV
Ao
Z
Yoon
Y
Wu
MX
Rizk
M
Jacquot
S
, et al.  . 
CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein
Proc Natl Acad Sci USA
 , 
1997
, vol. 
94
 (pg. 
6346
-
6351
)
18
Spinicelli
S
Nocentini
G
Ronchetti
S
Krausz
LT
Bianchini
R
Riccardi
C
GITR interacts with the pro-apoptotic protein Siva and induces apoptosis
Cell Death Differ
 , 
2002
, vol. 
9
 (pg. 
1382
-
1384
)
19
Knowlton
KU
Jeon
ES
Berkley
N
Wessely
R
Huber
S
A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3
J Virol
 , 
1996
, vol. 
70
 (pg. 
7811
-
7818
)
20
Henke
A
Nestler
M
Strunze
S
Saluz
HP
Hortschansky
P
Menzel
B
, et al.  . 
The apoptotic capability of coxsackievirus B3 is influenced by the efficient interaction between the capsid protein VP2 and the proapoptotic host protein Siva
Virology
 , 
2001
, vol. 
289
 (pg. 
15
-
22
)
21
Martin
U
Nestler
M
Munder
T
Zell
R
Sigusch
HH
Henke
A
Characterization of coxsackievirus B3-caused apoptosis under in vitro conditions
Med Microbiol Immunol
 , 
2004
, vol. 
193
 (pg. 
133
-
139
)
22
Nestler
M
Martin
U
Hortschansky
P
Saluz
HP
Henke
A
Munder
T
The zinc containing pro-apoptotic protein Siva interacts with the peroxisomal membrane protein pmp22
Mol Cell Biochem
 , 
2006
, vol. 
287
 (pg. 
147
-
155
)
23
Klebe
RJ
Harriss
JV
Sharp
ZD
Douglas
MG
A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast
Gene
 , 
1983
, vol. 
25
 (pg. 
333
-
341
)
24
Breeden
L
Nasmyth
K
Regulation of the yeast HO gene
Cold Spring Harb Symp Quant Biol
 , 
1985
, vol. 
50
 (pg. 
643
-
650
)
25
Boyer
HW
Roulland-Dussoix
D
A complementation analysis of the restriction and modification of DNA in Escherichia coli
J Mol Biol
 , 
1969
, vol. 
41
 (pg. 
459
-
472
)
26
Breeden
L
Nasmyth
K
Cell cycle control of the yeast HO gene: cis- and trans-acting regulators
Cell
 , 
1987
, vol. 
48
 (pg. 
389
-
397
)
27
Porath
J
Carlsson
J
Olsson
I
Belfrage
G
Metal chelate affinity chromatography, a new approach to protein fractionation
Nature
 , 
1975
, vol. 
258
 (pg. 
598
-
599
)
28
Laemmli
UK
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
 , 
1970
, vol. 
227
 (pg. 
680
-
685
)
29
Gregorio
CC
Trombitas
K
Centner
T
Kolmerer
B
Stier
G
Kunke
K
, et al.  . 
The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity
J Cell Biol
 , 
1998
, vol. 
143
 (pg. 
1013
-
1027
)
30
Yoon
Y
Ao
Z
Cheng
Y
Schlossman
SF
Prasad
KV
Murine Siva-1 and Siva-2, alternate splice forms of the mouse Siva gene, both bind to CD27 but differentially transduce apoptosis
Oncogene
 , 
1999
, vol. 
18
 (pg. 
7174
-
7179
)
31
Xue
L
Chu
F
Cheng
Y
Sun
X
Borthakur
A
Ramarao
M
, et al.  . 
Siva-1 binds to and inhibits BCL-X(L)-mediated protection against UV radiation-induced apoptosis
Proc Natl Acad Sci USA
 , 
2002
, vol. 
99
 (pg. 
6925
-
6930
)
32
Zhang
HM
Yanagawa
B
Cheung
P
Luo
H
Yuan
J
Chau
D
, et al.  . 
Nip21 gene expression reduces coxsackievirus B3 replication by promoting apoptotic cell death via a mitochondria-dependent pathway
Circ Res
 , 
2002
, vol. 
90
 (pg. 
1251
-
1258
)
33
Valle
G
Faulkner
G
De Antoni
A
Pacchioni
B
Pallavicini
A
Pandolfo
D
, et al.  . 
Telethonin, a novel sarcomeric protein of heart and skeletal muscle
FEBS Lett
 , 
1997
, vol. 
415
 (pg. 
163
-
168
)
34
Clark
KA
McElhinny
AS
Beckerle
MC
Gregorio
CC
Striated muscle cytoarchitecture: an intricate web of form and function
Annu Rev Cell Dev Biol
 , 
2002
, vol. 
18
 (pg. 
637
-
706
)
35
Moreira
ES
Wiltshire
TJ
Faulkner
G
Nilforoushan
A
Vainzof
M
Suzuki
OT
, et al.  . 
Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein Telethonin
Nat Genet
 , 
2000
, vol. 
24
 (pg. 
163
-
166
)
36
Mues
A
van der Ven
PF
Young
P
Fürst
DO
Gautel
M
Two immunoglobulin-like domains of the Z-disc portion of titin interact in a conformation-dependent way with Telethonin
FEBS Lett
 , 
1998
, vol. 
428
 (pg. 
111
-
114
)
37
Mayans
O
van der Ven
PF
Wilm
M
Mues
A
Young
P
Fürst
DO
, et al.  . 
Structural basis for activation of the titin kinase domain during myofibrillogenesis
Nature
 , 
1998
, vol. 
395
 (pg. 
863
-
869
)
38
Trinick
J
Tskhovrebova
L
Titin: a molecular control freak
Trends Cell Biol
 , 
1999
, vol. 
9
 (pg. 
377
-
380
)
39
Knöll
R
Hoshijima
M
Hoffman
HM
Person
V
Lorenzen-Schmidt
I
Bang
ML
, et al.  . 
The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy
Cell
 , 
2002
, vol. 
111
 (pg. 
943
-
955
)
40
Bos
JM
Poley
RN
Ny
M
Tester
DJ
Xu
X
Vatta
M
, et al.  . 
Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and Telethonin
Mol Genet Metab
 , 
2006
, vol. 
88
 (pg. 
78
-
85
)
41
Perrot
A
Posch
MG
Osterziel
KJ
Deletion of Glu at codon 13 in the TCAP gene encoding the Z-disc protein titin-cap/Telethonin is a rare non-synonymous polymorphism
Mol Genet Metab
 , 
2006
, vol. 
88
 (pg. 
199
-
200
)
42
Py
B
Slomianny
C
Auberger
P
Petit
PX
Benichou
S
Siva-1 and an alternative splice form lacking the death domain, Siva-2, similarly induce apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway
J Immunol
 , 
2004
, vol. 
172
 (pg. 
4008
-
4017
)
43
Henke
A
Huber
S
Stelzner
A
Whitton
JL
The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis
J Virol
 , 
1995
, vol. 
69
 (pg. 
6720
-
6728
)
44
Al-Hello
H
Davydova
B
Smura
T
Kaialainen
S
Ylipaasto
P
Saario
E
, et al.  . 
Phenotypic and genetic changes in coxsackievirus B5 following repeated passage in mouse pancreas in vivo
J Med Virol
 , 
2005
, vol. 
75
 (pg. 
566
-
574
)
45
Gauntt
CJ
Gomez
PT
Duffey
PS
Grant
JA
Trent
DW
Witherspoon
SM
, et al.  . 
Characterization and myocarditic capabilities of coxsackievirus B3 variants in selected mouse strains
J Virol
 , 
1984
, vol. 
52
 (pg. 
598
-
605
)
46
Khatib
R
Chason
JL
Silberberg
BK
Lerner
AM
Age-dependent pathogenicity of group B coxsackieviruses in Swiss-Webster mice: infectivity for myocardium and pancreas
J Infect Dis
 , 
1980
, vol. 
141
 (pg. 
394
-
403
)
47
Woodruff
JF
Kilbourne
ED
The influence of quantitated post-weaning undernutrition on coxsackievirus B3 infection of adult mice. I. Viral persistence and increased severity of lesions
J Infect Dis
 , 
1970
, vol. 
121
 (pg. 
137
-
163
)