Proteasomal Protein Degradation: Adaptation of Cellular Proteolysis With Impact on Virus—and Cytokine-Mediated Damage of Heart Tissue During Myocarditis

Viral myocarditis is an inflammation of the heart muscle triggered by direct virus-induced cytolysis and immune response mechanisms with most severe consequences during early childhood. Acute and long-term manifestation of damaged heart tissue and disturbances of cardiac performance involve virus-triggered adverse activation of the immune response and both immunopathology, as well as, autoimmunity account for such immune-destructive processes. It is a matter of ongoing debate to what extent subclinical virus infection contributes to the debilitating sequela of the acute disease. In this review, we conceptualize the many functions of the proteasome in viral myocarditis and discuss the adaptation of this multi-catalytic protease complex together with its implications on the course of disease. Inhibition of proteasome function is already highly relevant as a strategy in treating various malignancies. However, cardiotoxicity and immune-related adverse effects have proven significant hurdles, representative of the target's wide-ranging functions. Thus, we further discuss the molecular details of proteasome-mediated activity of the immune response for virus-mediated inflammatory heart disease. We summarize how the spatiotemporal flexibility of the proteasome might be tackled for therapeutic purposes aiming to mitigate virus-mediated adverse activation of the immune response in the heart

ONX 0914 Lacks Selectivity for the Cardiac Immunoproteasome in CoxsackievirusB3 Myocarditis of NMRI Mice and Promotes Virus-Mediated Tissue Damage

Inhibition of proteasome function by small molecules is highly efficacious in cancer treatment. Other than non-selective proteasome inhibitors, immunoproteasome-specific inhibitors allow for specific targeting of the proteasome in immune cells and the profound anti-inflammatory potential of such compounds revealed implications for inflammatory scenarios. For pathogen-triggered inflammation, however, the efficacy of immunoproteasome inhibitors is controversial. In this study, we investigated how ONX 0914, an immunoproteasome-selective inhibitor, influences CoxsackievirusB3 infection in NMRI mice, resulting in the development of acute and chronic myocarditis, which is accompanied by formation of the immunoproteasome in heart tissue. In groups in which ONX 0914 treatment was initiated once viral cytotoxicity had emerged in the heart, ONX 0914 had no anti-inflammatory effect in the acute or chronic stages. ONX 0914 treatment initiated prior to infection, however, increased viral cytotoxicity in cardiomyocytes, promoting infiltration of myeloid immune cells into the heart. At this stage, ONX 0914 completely inhibited the β5 subunit of the standard cardiac proteasome and less efficiently blocked its immunoproteasome counterpart LMP7. In conclusion, ONX 0914 unselectively perturbs cardiac proteasome function in viral myocarditis of NMRI mice, reduces the capacity of the host to control the viral burden and promotes cardiac inflammation

A cytosolic disulfide bridge‐supported dimerization is crucial for stability and cellular distribution of Coxsackievirus B3 protein 3A

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity

Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming

Protein modification with ISG15 (ISGylation) represents a major type I IFN–induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies

Silencing the CSF-1 Axis Using Nanoparticle Encapsulated siRNA Mitigates Viral and Autoimmune Myocarditis

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms

Coxsackievirus B3 Exploits the Ubiquitin-Proteasome System to Facilitate Viral Replication

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly

PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection.

The function of the proteasome is modulated at the level of subunit expression and by association with its regulatory complexes. During coxsackievirus B3 (CVB3) myocarditis, IFN-induced formation of immunoproteasomes (ip) is known to be critical for regulating immune modulating molecules. The function of the IFN-γ-inducible proteasome regulator subunits PA28 α and β, however, in this context was unknown. During viral myocarditis, we found an increased abundance of PA28β subunits in heart tissue. PA28α/β exists in PA28-20S-PA28 and PA700-20S-PA28 hybrid proteasome complexes in cells both with either predominant ip and standard proteasome (sp) expression. Being in line with reduced proteasome activity in PA28α/β-deficient cells, we observed increased levels of oxidized and poly-ubiquitinated proteins upon TLR3-activation in these cells. Moreover, PA28α/β is capable to interfere directly with viral replication of CVB3 and facilitates the generation of CVB3-derived MHC class I epitopes by the proteasome. In contrast to a distinct function of PA28α/β in vitro, gene ablation of PA28α/β in mice being on a genetic background with resistance towards the development of severe infection had no significant impact on disease progression. Other than reported for the ip, in this host PA28α/β is dispensable to meet the demand of increased peptide hydrolysis capacity by the proteasome during viral myocarditis

Investigation of PA28αβ^-/- mice during CVB3 myocarditis.

<p>PA28^+/+ and PA28^-/- mice were infected with CVB3. (A) Body weight was monitored daily and mice were sacrificed at day 8 post infection (means ± SEM). (B) Heart, lung, spleen and thymus weight was determined and normalized to the respective tibia length (means + SEM). (C) Representative haematoxylin/eosin (HE) staining of cardiac tissue sections obtained from CVB3-infected PA28^+/+ (n = 10) and PA28^−/− (n = 7) mice are shown. (D) Myocardial damage comprising cardiac cell necrosis, inflammation, and scarring was quantified applying a myocarditis score from 0 to 4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173259#pone.0173259.ref036" target="_blank">36</a>]. (E) The severity of pancreas tissue destruction upon CVB3 infection was rated on a scale from 0% to 100% on HE stains of pancreatic tissue sections.</p

Characterization of PA28-proteasome complexes in wild-type and LMP7^−/− macrophages.

<p>(A) Cellular extracts were generated from wild-type, LMP7^−/− and PA28α/β^−/− BMMs. Proteins were separated by native PAGE and subjected to Western blotting of proteasome core (α-α4) and PA28 complex (α-PA28β) co-stained for PA700 complex (α-Rpt6), respectively. 20S proteasome purified from mouse B8 fibroblasts was applied for comparison. Fluorescence dye-coupled secondary antibodies were used for detection. *1 –unspecific signal, *2 –PA700-20S and PA700-20S-PA28, *3 –PA700 intermediate complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173259#pone.0173259.ref051" target="_blank">51</a>], 4*–PA200-20S-PA200 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173259#pone.0173259.ref052" target="_blank">52</a>], 5*–PA28α/β not bound to a proteasome core. (B) Magnified section of the α-α4/α-Rpt6 and α-PA28β/α-Rpt6 double-staining from (A) depicting the high molecular mass proteasome complexes PA700-20S and PA700-20S-PA28. (C) Expression and accessibility of catalytically active proteasome subunits within different proteasome-proteasome regulator complexes was investigated with MV151 pan-reactive ABP binding to all active sites and analyzed by densitometry (means + SEM of n = 3). Amido black staining indicates equal protein loading. (D) Total BMM protein extracts were subjected to glycerol density gradient centrifugation to separate distinct proteasome complexes. Proteasome-dependent peptide hydrolysis was determined in the obtained fractions by cleavage of the short fluorogenic peptide Suc-LLVY-AMC. Glycerol gradient fractions 3–13 (29%– 37.5% glycerol) containing high and low molecular mass proteasome complexes were concentrated and subjected to SDS-PAGE and Western blot analysis of proteasome subunits. Densitometric analysis of immune-blots is represented as relative intensities normalized to the most intense signal, respectively (means ± SEM, n = 4). Dotted boxes depict glycerol gradient fractions with the highest abundance of PA700-20S-PA28 complexes as determined by native PAGE (E) and immuno-blotting of PA28β (D).</p

Influence of PA28α/β on MHC class I antigen processing of CVB3 epitopes.

<p>(A) Murine embryonic fibroblasts were treated with IFN-β for 24 hours. For assessing MHC class I expression, cells were stained with anti-H-2K^b mAb and analyzed by flow cytometry (mean + SEM, n = 3). (B) For <i>in vitro</i> peptide processing studies PA28 from human erythrocytes, 20S sp from human lymphoblasts (T2 cells) and 20S ip from stably LMP2/LMP7-transfected T2 cells (T27 cells) were purified. The purity of the preparations was examined by Coomassie staining of a SDS-PAGE gel. Two epitope-harboring CVB3 peptides—the 28-mer polypeptide P3D_2158-2185 (C) and the 31-mer polypeptide VP2_272-302 (D)–were processed in the presence of T2-derived sp (filled black circles) and T27-derived ip (filled green circles) for the indicated incubation periods. Open circles represent results of proteasome fragmentation studies in the presence of PA28 for sp (black) and ip (green). The graphs in the upper panel summarize the generation of the respective CVB3 epitopes (C) P3D_2170-2177 and (D) VP2_285-293 detected by ESI–MS/MS. The lower graphs depict the substrate degradation as fold changes of the ion counts at point in time = zero. All ion counts of peptide substrates were normalized to the amount of the respective substrate detected in the sp assay at point in time = zero. All data are means ± SEM of two technical replicates. Unpaired t-tests were performed at each point in time to compare ion counts for P3D_2170-2177 and VP2_285-293 in the absence (filled circles) or presence of PA28 (open circles). All experiments shown are representative for at least two independent experiments.</p