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

During early encounters between a pathogen and a cell, receptors located on the cell surface, in the cytosol or within endosomal (storage) compartments engage with the pathogen's nucleic acid (DNA/RNA) or pathogen-associated molecular patterns (PAMPs), as a host response to combat infection. The host response can initiate specific gene expression patterns and posttranscriptional (prior to translation of a gene into a protein product) control mechanisms at multiple levels and stages during disease development. The outcomes cause cells to produce type I interferons (IFNs) as a first line of defense to orchestrate a complex defense network in both infected and noninfected cells. As a classic example, the ubiquitin family protein IFN-stimulated gene of 15 kDa (known as ISG15) and its conjugation machinery including the enzyme Ube1L represents an IFN-induced broad spectrum antimicrobial system.

Protein modification with ISG15 known as ISGlyation is a major antimicrobial system; commonly perceived as a key to lock the cell gates and prevent the spread of threats—but scientists have yet to identify its common mechanism of action and the viral species-specific aspects of the process. In a new report in Science Advances, Meike Kespohl and an international research team in microbiology, biomolecular medicine, medical biotechnology and healthcare in Germany, U.S. and Belgium used a multiphase coxsackievirus B3 (CV) infection model to identify the host response. During CV infection, the first wave resulted in hepatic injury of the liver, followed by a second wave culminating in cardiac damage.

The scientists showed that ISGlyation activated ISG15 proteins to act on antiviral proteins, causing nonhematopoietic cells (which include airway epithelial cells or AECs; critical players in the inflammatory process initiated during airway infection) vital for CV control—into a resistant antiviral state. Due to altered energy demands, ISG15 also adapted liver metabolism during infection, which the scientists demonstrated using shotgun proteomics combined with metabolic network engineering to reveal how ISG15 promoted gluconeogenesis (generation of glucose) in liver cells. In the absence of a protease or a protein enzyme known as the ubiquitin specific protein (USP18) that breaks down ISG15, the cells showed increased resistance to clinically relevant CV strains. The results therefore suggest inhibiting USP18 to stabilize ISGlyation and investigate treatments during CV-associated human disease.

CV disease is highly prevalent among newborn infants and young children, causing substantial medical and socioeconomic impact, with an etiology of hepatitis, myocarditis, encephalomyelitis and coagulopathy for multisystem sepsis. The disease can be mimicked in mice with similarities to humans, where mice show a robust systemic response on early CV infection. The biosynthesis of molecules required for an efficient antiviral response against CV consumes large portions of cellular energy packets or energy transfer molecules known as adenosine triphosphate (ATP). As a result, IFNs also activate the uptake and turnover of glucose within infected cells. IFN signaling can concurrently trigger cardioprotective effects but its molecular operation during CV infection remains elusive. To study the cellular response, Kespohl et al. used animal models in the lab to understand ISG15-mediated protection from viral toxicity.

Inhibiting CV burden via ISGlyation

The team investigated the viral burden during CV infection in gene knockout mice that lacked protein expression for ISG15 (ISG15^-/-) and Ube1L (Ube1L^-/-) proteins. Deleting Ube1L prevented ISGlyation but did not affect the function of freely available ISG15—allowing the scientists to distinguish between ISGlyation-dependent functions and those mediated by the free form of the protein. Approximately 36 hours after CV infection, they noted the formation of ISG15 conjugates in the liver, pancreas, spleen and heart tissue, and increased protein ISGlyation during disease progression. ISGlyation accelerated CV clearance from the liver and spleen due to higher CV titers in the knockout animal models compared to the wild-type (regular) controls. When they inactivated the protease (protein enzyme) USP18 specific to ISG15 breakdown, they saw increased cellular resistance toward CV infection.

ISGlyation within nonhematopoietic cells can protect from CV pathology.

The team hypothesized that ISG15 protein offered protection from CV through nonhematopoietic cell types and tissues. Kespohl et al. tested the hypothesis using a genetically modified mouse model that did not express the Ube1L protein (Ube1L^-/-) so as to prevent ISGlyation and compared the results with wild-type bone marrow chimeras. They observed an increased CV load in the gene knockout mice, causing high-grade inflammation and tissue destruction as well as increased chemokine expression. The results demonstrated the protective role of ISGlyation to control the CV-triggered disease. They then reconstituted the compromised mice with wild-type bone marrow cells with functional ISGlyation machinery, but their condition did not improve. The work highlighted the role of non-bone-marrow-derived somatic cells to prevent viral cytotoxicity and inflammatory tissue damage during CV compared to bone marrow-derived immune cells.

ISGlyation increased the expression levels of antiviral proteins and ISG15 reprogrammed central liver metabolism during CV infection.

The scientists then studied and identified molecular mechanisms of protein ISGlyation that suppressed the virus in cells targeted by CV infection. They profiled the proteins inside infected liver tissue using mass-spectrometry (MS)-based proteomics (the study of proteins). The analysis showed the upregulation of antiviral vectors; IFN-induced proteins with tetratricopeptide repeats (IFIT) 1 and 3, alongside the ISG15 protein. Extensive findings proved that the ISG15 system also regulated protein expression levels of antiviral proteins (IFIT 1/3) post-transcriptionally, i.e., between transcription and translation—at the RNA level.

When viral infections activate the host defense pathways, cellular demands for ATP (adenosine triphosphate) will alter and remodel central metabolic processes. In this study, IFN treatment (precursor pathway to ISG15) reduced CV replication in cell cultures and decreased cellular glucose consumption back to control levels. CV infection in whole organisms lead to elevated glucose uptake by infected cells, while impairing the function of the exocrine pancreas—demanding metabolic reprogramming for recovery. The scientists observed infection-triggered hypoglycemia, increased energy demand, malnutrition and lower glucose storage in the liver of CV infected mouse models. ISG15 influenced the central liver metabolism at multiple stages of infection by increasing the capacity of liver tissue to produce endogenous glucose and conduct efficient glycolysis during early and late stages of disease. Using metabolic models, Kespohl et al. illustrated how ISG15 reprogrammed the central liver metabolism during infection for efficient glucose production and storage.

Identifying the antiviral capacity of human ISG15

The team then examined the concept of stabilizing ISGlyation by inhibiting the protease USP18 for therapeutic applications during CV infection. Kespohl et al. investigated if the antiviral capacity in the mouse also applied to humans to show there was no barrier between the two (in human cell culture or mouse models) during ISGlyation to effectively counteract CV infection. While the results were based on a laboratory CV strain, little was known of the impact of ISGlyation on a clinical viral counterpart. The researchers tested clinical viral variants from patients to determine viral sensitivity to ISG15 in cell culture and noted that improving ISGlyation could inhibit viral replication for all clinical CV isolates tested in the work.

In this way, Meike Kespohl and colleagues precisely understood the functions of the ISG15 system during antiviral and metabolic rewiring to combat CV infection. Despite the impressive antiviral activity observed for CV, ISG15 is not effective against all viruses since viral resistances evolved due to a constant battle of immune evasion mechanisms of the pathogen and the corresponding host immune response. However, inactivating the ISG15-degradation-specific protease USP18 can enhance antiviral capacity of the ISG15 system against clinical CV serotypes with relatively minimal side-effects. The data support inhibiting USP18 protease as a host-directed antiviral approach to combat CV pathology in man.

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