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

Protein modification with ISG15 blocks coxsackievirus pathology via antiviral and metabolic reprogramming
ISGylation suppresses CV titers at different phases of infection. Wild-type (wt), ISG15−/−, and Ube1L−/− mice were infected with 1 × 105 pfu of CV Nancy and sacrificed at the indicated points in time post infection (p.i.). Tissue from (A) liver and (B) heart of wild-type mice was subjected to Western blot analysis using an ISG15-specific antibody. Each lane represents tissue homogenates obtained from a different animal. (C and D) Infectious viral particles were quantified in the respective organs obtained from wild-type, ISG15−/−, and Ube1L−/− mice by plaque assay during CV infection. Each dot represents a different animal; data are summarized as median values. Student’s t tests were conducted. P values of <0.05 are indicated in the graph. (E to J) USP18C61A and wild-type littermate controls were infected with 1 × 105 pfu of CV and sacrificed at the indicated points in time. (E) Heart tissue was homogenized and subjected to Western blot analysis for detection of ISG15. Each lane represents tissue obtained from a different animal, and the shown example for each group and point in time is representative for n = 3 mice. (F) At day 6 after infection, infectious viral particles were determined in heart by TaqMan qPCR and plaque assay. Each dot represents a different animal. Data are summarized as means ± SEM; t tests were performed, and P values of <0.05 are depicted. (G) Cardiomyocytes derived from USP18C61A ISG15−/− and ISG15−/− embryos were transduced with Ad5 vectors encoding murine ISG15 and stimulated with poly(I:C). Cellular lysates were subjected to Western blot analysis. (H to J) ISG15-rescued cardiomyocytes from USP18C61A ISG15−/− and ISG15−/− embryos were infected with CV at an MOI of 0.1 for 24 hours. (H) Total RNA was isolated to determine CV genome copy numbers by TaqMan qPCR; ΔΔCt values obtained from duplicates are shown for two independent experiments. Data are summarized as means ± SEM. (I) Viral load was determined in cellular lysates by Western blot analysis of CV VP1. Densitometric analysis of VP1 and the GAPDH loading control was performed to calculate the mean (± SEM) relative VP1 expression in three independent experiments. (J) Release of infectious viral particles was assessed by plaque assay. One representative out of three independent experiments yielding the same result is depicted. Unpaired t test (plaque assay) and one-sample t tests (VP1 and CV RNA) were performed, and P values are depicted. Credit: Science Advances, doi: 10.1126/sciadv.aay1109

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 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.

Protein modification with ISG15 blocks coxsackievirus pathology via antiviral and metabolic reprogramming
Protein modification with ISG15 acts cooperatively with IFIT proteins and preserves glucose homeostasis. CV infection is a bona fide example for multiphasic state infectious disease with primary injury of liver and pancreas followed by a second viremia culminating in cardiac damage and chronic tissue damage. Early upon infection, IFNs trigger the ubiquitin-like modifier ISG15, which, in a three-step enzymatic cascade, forms covalent linkages with proteins in both infected and noninfected cells. In non–bone marrow–derived somatic cells and tissues, ISGylation inhibits viral replication, and this involves augmented protein expression levels of antiviral effectors such as IFIT1 and IFIT3. ISG15 ensures efficient storage of glucose in liver tissue of healthy mice and reprograms liver metabolism toward improved glucose production early after CV infection. Cells lacking activity of the ISG15-specific protease USP18 show a marked increased resistance against CV infection, thus providing a rationale that USP18 inhibition could be a novel host-directed approach countered to CV-associated human pathology. Credit: Science Advances, doi: 10.1126/sciadv.aay1109

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.

Protein modification with ISG15 blocks coxsackievirus pathology via antiviral and metabolic reprogramming
Protection from CV pathology requires ISGylation in nonhematopoietic cells. (A and B) ISG15−/− (CD45.2) mice were reconstituted with bone marrow cells from either ISG15−/− (CD45.2) or wild-type (wt, CD45.1) donors before CV infection, and mice were sacrificed 3 days after infection. (A) Infectious viral particles were quantified by plaque assay (wild-type ➔ ISG15−/−, n = 6; ISG15−/− ➔ ISG15−/−, n = 4). Data are summarized as median. (B) Splenic mRNA expression of the indicated cytokines and chemokines was determined by TaqMan qPCR. (C to F) Chimeric wild-type and Ube1L−/− mice were generated upon transfer of wild-type or Ube1L−/− bone marrow cells into lethally irradiated wild-type or Ube1L−/− recipients, respectively. Mice were infected with CV and sacrificed after 8 days (n = 7 in all four groups). (C) Infectious viral particles were quantified in heart tissue by plaque assay. Data are summarized as means ± SEM. (D) Myocarditis was scored microscopically by a blinded pathologist based on cardiac hematoxylin and eosin staining. (E) Representative histopathologic stains of heart tissue of each group are shown. (F) mRNA levels of the indicated genes in heart tissue were determined by TaqMan qPCR. Unequal variance versions of two-way ANOVA were performed, followed by a Sidak-Holm’s multiple comparison test. Data were summarized as means ± SEM if applicable. Credit: Science Advances, doi: 10.1126/sciadv.aay1109

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.

Protein modification with ISG15 blocks coxsackievirus pathology via antiviral and metabolic reprogramming
ISG15 reprograms the central liver metabolism during CV infection. (A) Hepatic tissue obtained from wild-type (wt) and ISG15−/− mice (n = 3) during early (day 3) and late (day 8) state of CV infection was subjected to LC-MS/MS analysis. Heatmaps summarizing all differentially regulated hepatic proteins during infection for both strains are depicted. The relative abundance of each protein is color-coded based on the z score normalized log2-transformed LFQ intensities. Blue color indicates proteins of high abundance, and yellow color indicates proteins of low abundance as compared to row means. A hierarchical clustering resolved six distinct clusters, with annotation shown on the right. (B) Heatmap-based clusters were subjected to Gene Ontology (GO) analysis, and proteins involved in selected enriched metabolic GO terms with catabolic ATP-generating function (FA oxidation, carbohydrate catabolic process, and OXPHOS) are depicted at an early and late state of CV infection, applying the same color code as used in (A) (blue, up-regulation; yellow, down-regulation). If the GO term of interest was not found within a dataset, individual proteins were not plotted. (C) At the indicated time points of infection, liver biopsies were obtained from wild-type and ISG15−/− mice. The basal oxygen consumption (top) and extracellular acidification (bottom) rates were monitored using a Seahorse Biosciences extracellular flux analyzer. Values were normalized to protein content in the biopsies. Data of at least six mice per group were summarized as means ± SEM. A one-way ANOVA was performed followed by a Tukey’s multiple comparison test. (D and E) Liver proteome data together with HEPATOKIN1, a model of central liver metabolism , were used to assess the metabolic alterations in liver tissue of wild-type and ISG15−/− mice during viral infection. Metabolic models for the different conditions were constructed by scaling the maximal activity for each enzyme using the LFQ intensities for each protein obtained from MaxQuant analysis at the respective point in time. (D) For a standard 24-hour profile metabolite plasma profile, diurnal glucose exchange fluxes were simulated in wild-type and ISG15−/− mice at each time point of viral infection. Negative exchange fluxes indicate net release from the liver to the plasma (gluconeogenesis), while positive values indicate hepatic glucose uptake (glycolysis). (E) For each condition, experimentally determined blood glucose levels were used as model input to calculate realistic exchange fluxes and glycogen levels. Credit: Science Advances, doi: 10.1126/sciadv.aay1109

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 , 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.

Protein modification with ISG15 blocks coxsackievirus pathology via antiviral and metabolic reprogramming
Human ISG15 suppresses CV replication. (A) ISG15 expression was deleted in HeLa cells using CRISPR-Cas9 gene editing. ISG15-ko cells and wild-type cells were infected with CV (MOI 0.1) for 16 hours. Expression of CV VP1 was determined by Western blot analysis in four independent experiments, and the obtained signal normalized to GAPDH was compared to wild-type samples. Infectious virus particles were quantified in five independent experiments by plaque assay. Data are summarized as means ± SEM. (B) HeLa cells were transfected with siRNA targeting human ISG15 or a nontargeting control siRNA. Cells were subsequently infected with CV (MOI 0.01) for 16 hours. VP1 protein expression was determined and normalized to the control sample in four independent experiments. Plaque assays were performed in two independent experiments. Data are summarized as described in (A). (C) HeLa cells stably expressing FLAG-tagged human ISG15 and respective control cells were infected with CV (MOI 0.1) for 16 hours. VP1 expression and virus titer were determined as described in (A) in three independent experiments. (D) Primary embryonic cardiomyocytes obtained from ISG15−/− mice were transduced with Ad5 vectors expressing human ISG15 (hISG15) or control for 48 hours at MOI 25 before CV infection (MOI 0.1) for 24 hours. VP1 levels were determined by Western blot analysis, and infectious viral particles were quantified by plaque assay in three independent experiments. (E) Cardiomyocytes derived from USP18C61A ISG15−/− and ISG15−/− embryos were transduced with Ad5 vectors encoding hISG15 and infected with CV in three independent experiments for detection of the viral load by Western blot analysis of VP1 as well as plaque assay. One-sample t tests were performed for all summarized VP1 data. Unpaired t tests were conducted for all plaque assay data. (F and G) CVB isolates were obtained from patients presenting with neurological symptoms that may have been of infectious origin. (F) ISG15-ko HeLa cells and control cells were infected with the indicated CV serotypes (MOI 0.1), and infectious virus particles were determined after 16 hours by plaque assay. The relative increase of the viral titer in ISG15-ko HeLa cells as compared to control cells is depicted for a representative experiment. Three independent experiments demonstrated similar results. (G) The ISG15 system was induced in USP18C61A ISG15−/− and ISG15−/− cardiomyocytes by transduction of Ad5 vectors encoding hISG15. Cells were infected with CV serotypes as follows: CVB1 425, MOI 1 (2 days); CVB1 506, MOI 1 (1 day); CVB3 1072, MOI 10 (2 days); CVB3 180, MOI 10 (1 day); CVB4 686, MOI 10 (2 days); CVB4 120, MOI1 (2 days); CVB5 679, MOI 1 (1 days); CVB5 800, MOI 10 (1 day). Infectious viral particles were quantified by plaque assay. Relative reduction of the viral load in USP18C61A ISG15−/− cells as compared to ISG15−/− cardiomyocytes with restored ISG15 expression is depicted for a representative of at least three independent experiments. Data are means ± SEM; one-sample t tests were performed, and P values are depicted. Credit: Science Advances, doi: 10.1126/sciadv.aay1109

In this way, Meike Kespohl and colleagues precisely understood the functions of the ISG15 system during antiviral and metabolic rewiring to combat CV . 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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More information: Meike Kespohl et al. Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming, Science Advances (2020). DOI: 10.1126/sciadv.aay1109

Yi-Chieh Perng et al. ISG15 in antiviral immunity and beyond, Nature Reviews Microbiology (2018). DOI: 10.1038/s41579-018-0020-5

Anja Basters et al. Structural basis of the specificity of USP18 toward ISG15, Nature Structural & Molecular Biology (2017). DOI: 10.1038/nsmb.3371

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