Characterization of the cardiac ISGylome and its influence on protein function during Coxsackievirus B3 infection

Infection with cardiotropic Coxsackievirus B3 (CVB3) leads to activation of the innate immune system and production of type I Interferons (IFNs). Upon detection of IFNs, cells upregulate the expression of hundreds of downstream effector proteins called Interferon Stimulated Genes (ISGs) to establish the host cellular antiviral state. Among the ISGs, the small ubiquitin-like modifier ISG15 has pleiotropic antiviral function acting in its free form or covalently conjugated to other proteins. This so called ISGylation is mediated by an E1/E2/E3 enzyme cascade, peaking around day 3 of infection in cardiac tissue. Subsequent infiltration of innate and adaptive immune cells into the myocardium results in both, viral elimination as well as inflammation-induced cell death and thus causes acute viral myocarditis. Inefficient virus clearance and persistence of viral RNA in cardiac cells can then cause chronic cardiac inflammation. Previous work showed that ISGylation suppresses CVB3-mediated pathology, and enrichment of ISGylation by deactivating the deconjugating activity of the Ubiquitin-specific protease 18 (USP18) further enhances the antiviral host response. However, the manner in which ISGylation orchestrates resistance against CVB3 remains unclear. To investigate this, the cardiac proteome and ISGylome of infected wildtype, ISG15 / , and USP18C61A/C61A mice was analyzed by LC MS/MS. On the one hand, proteome analysis showed an infection-induced upregulation of the host-cellular immune response to establish a cardiac antiviral state. Additionally, IFIT1 and IFIT3, proteins with known antiviral effect against CVB3, were identified as ISG15-target proteins and ISGylation positively influenced their expression levels. On the other hand, infection led to a downregulation of proteins associated with metabolic pathways. Nonetheless, the cardiac mitochondrial energy production was upregulated in an ISGylation-dependent manner, indicating infection-induced increased energy demand. Concordantly, analysis of the cardiac ISGylome identified a variety of metabolic enzymes to be covalently modified by ISG15. Upregulation of glycolytic levels in primary cardiomyocytes was restricted by ISG15, which might be at least partly due to an attenuated activity of ISGylated hexokinase-2 and phosphofructokinase. Taken together, this study highlights the regulation of two key response pathways in viral infection by the ISG15 system. While the direct antiviral response might be supported by ISGylation-mediated stabilization of IFIT1/3, ISG15/ISGylation further ensures the efficient regulation of metabolic energy production during infection

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

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

The in vivo ISGylome links ISG15 to metabolic pathways and autophagy upon Listeria monocytogenes infection

ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity. Here, we map the endogenous in vivo ISGylome in the liver following Listeria mono-cytogenes infection by combining murine models of reduced or enhanced ISGylation with quantitative proteomics. Our method identifies 930 ISG15 sites in 434 proteins and also detects changes in the host ubiquitylome. The ISGylated targets are enriched in proteins which alter cellular metabolic processes, including upstream modulators of the catabolic and antibacterial pathway of autophagy. Computational analysis of substrate structures reveals that a number of ISG15 modifications occur at catalytic sites or dimerization interfaces of enzymes. Finally, we demonstrate that animals and cells with enhanced ISGylation have increased basal and infection-induced autophagy through the modification of mTOR, WIPI2, AMBRA1, and RAB7. Taken together, these findings ascribe a role of ISGylation to temporally reprogram organismal metabolism following infection through direct modification of a subset of enzymes in the liver

Conceptual Readings into the Cold War: Towards Transnational Approaches from the Perspective of Latin American Studies in Eastern and Western Europe

This bibliographical and conceptual essay summarizes recent research in Cold War Studies in Europe and the Americas, especially on smaller states in historiographical studies. Against the background of an increasing connectedness and globalization of research about the Cold War, the authors highlight the importance of the full-scale integration of countries and regions of the 'Global South' into Cold War Studies. Critical readings of the newly available resources reveal the existence of important decentralizing perspectives resulting from Cold War entanglements of the 'Global South' with the 'Global North.' As a result, the idea that these state actors from the former 'periphery' of the Cold War should be considered as passive recipients of superpower politics seems rather troubled. The evidence shows (at least partially) autonomous and active multiple actors