First Brazilian Case Report of Unrelated Patients with Identical ISG15 Mutation.

BACKGROUND: ISG15 deficiency is a mixed syndrome of Mendelian susceptibility to mycobacterial infections (MSMD), a rare inherited condition characterized primarily by recurrent infections from low-virulence mycobacteria and monogenic type I interferonopathy. OBJECTIVE: To characterize the laboratory and molecular features of two patients from different families affected by the same ISG15 variant. METHODS: We began with clinical characterization and investigation, assessed IL-12/IFN-γ production, performed genetic characterization through WES and Sanger sequencing, conducted an in silico molecular analysis of the genetic ISG15 variant's protein impact, and utilized RNAseq for transcriptome analysis to understand pathway impacts on ISG15-deficient subjects from unrelated families. RESULTS: A mutation in the ISG15 gene was identified, affecting two patients treated in different hospitals and cities in Brazil (Fortaleza and Sao Paulo), who are also members of unrelated families. Both patients showed low IFN-γ production when stimulated with BCG or BCG + IL-12. ISG15 deficiency presented with two distinct clinical phenotypes: infectious and neurological. It was identified that both patients are homozygous for the variant (c.83 T > A). Furthermore, it was observed that the mutant protein p.L28Q results in an unstable protein with increased flexibility (ΔΔG: -2.400 kcal/mol). Transcriptome analysis revealed 1321 differentially expressed genes, with significant upregulation in interferon pathways, showing higher expression in patients compared to controls. CONCLUSION: This study describes the first reported cases in Brazil of two unrelated patients with the same ISG15 mutation c.83 T > A, exhibiting infectious features such as mycobacterial infections and systemic candidiasis, neurological findings, and skin lesions, without adverse reactions to the BCG vaccine. CLINICAL IMPLICATIONS: Reporting ISG15 gene mutations in Brazilian patients enhances understanding of genetic susceptibilities, guiding effective diagnostics and treatment. Identifying high-risk individuals aids clinical practices, genetic counseling, and influences public health policies. We have identified the first case in Brazil of the same ISG15 variant c.83 T > A that was identified in two unrelated patients with distinct clinical phenotypes, infectious and neurological.

SARS-CoV-2 variants induce increased inflammatory gene expression but reduced interferon responses and heme synthesis as compared with wild type strains.

SARS-CoV-2 variants of concern (VOC) have been associated with increased viral transmission and disease severity. We investigated the mechanisms of pathogenesis caused by variants using a host blood transcriptome profiling approach. We analysed transcriptional signatures of COVID-19 patients comparing those infected with wildtype (wt), alpha, delta or omicron strains seeking insights into infection in Asymptomatic cases.Comparison of transcriptional profiles of Symptomatic and Asymptomatic COVID-19 cases showed increased differentially regulated gene (DEGs) of inflammatory, apoptosis and blood coagulation pathways, with decreased T cell and Interferon stimulated genes (ISG) activation. Between SARS-CoV-2 strains, an increasing number of DEGs occurred in comparisons between wt and alpha (196), delta (1425) or, omicron (2313) infections. COVID-19 cases with alpha or, delta variants demonstrated suppression transcripts of innate immune pathways. EGR1 and CXCL8 were highly upregulated in those infected with VOC; heme biosynthetic pathway genes (ALAS2, HBB, HBG1, HBD9) and ISGs were downregulated. Delta and omicron infections upregulated ribosomal pathways, reflecting increased viral RNA translation. Asymptomatic COVID-19 cases infected with delta infections showed increased cytokines and ISGs expression. Overall, increased inflammation, with reduced host heme synthesis was associated with infections caused by VOC infections, with raised type I interferon in cases with less severe disease.

Overcoming Irinotecan Resistance by Targeting Its Downstream Signaling Pathways in Colon Cancer.

Among the most popular chemotherapeutic agents, irinotecan, regarded as a prodrug belonging to the camptothecin family that inhibits topoisomerase I, is widely used to treat metastatic colorectal cancer (CRC). Although immunotherapy is promising for several cancer types, only microsatellite-instable (~7%) and not microsatellite-stable CRCs are responsive to it. Therefore, it is important to investigate the mechanism of irinotecan function to identify cellular proteins and/or pathways that could be targeted for combination therapy. Here, we have determined the effect of irinotecan treatment on the expression/activation of tumor suppressor genes (including p15Ink4b, p21Cip1, p27Kip1, and p53) and oncogenes (including OPN, IL8, PD-L1, NF-κB, ISG15, Cyclin D1, and c-Myc) using qRT-PCR, Western blotting, immunofluorescence (IF), and RNA sequencing of tumor specimens. We employed stable knockdown, neutralizing antibodies (Abs), and inhibitors of OPN, p53, and NF-κB to establish downstream signaling and sensitivity/resistance to the cytotoxic activities of irinotecan. Suppression of secretory OPN and NF-κB sensitized colon cancer cells to irinotecan. p53 inhibition or knockdown was not sufficient to block or potentiate SN38-regulated signaling, suggesting p53-independent effects. Irinotecan treatment inhibited tumor growth in syngeneic mice. Analyses of allograft tumors from irinotecan-treated mice validated the cell culture results. RNA-seq data suggested that irinotecan-mediated activation of NF-κB signaling modulated immune and inflammatory genes in mice, which may compromise drug efficacy and promote resistance. In sum, these results suggest that, for CRCs, targeting OPN, NF-κB, PD-L1, and/or ISG15 signaling may provide a potential strategy to overcome resistance to irinotecan-based chemotherapy.

MDA5 ISGylation is crucial for immune signaling to control viral replication and pathogenesis.

The posttranslational modification (PTM) of innate immune sensor proteins by ubiquitin or ubiquitin-like proteins is crucial for regulating antiviral host responses. The cytoplasmic dsRNA receptor melanoma differentiation-associated protein 5 (MDA5) undergoes several PTMs including ISGylation within its first caspase activation and recruitment domain (CARD), which promotes MDA5 signaling. However, the relevance of MDA5 ISGylation for antiviral immunity in an infected organism has been elusive. Here, we generated knock-in mice (MDA5 K23R/K43R ) in which the two major ISGylation sites, K23 and K43, in MDA5 were mutated. Primary cells derived from MDA5 K23R/K43R mice exhibited abrogated endogenous MDA5 ISGylation and an impaired ability of MDA5 to form oligomeric assemblies leading to blunted cytokine responses to MDA5 RNA-agonist stimulation or infection with encephalomyocarditis virus (EMCV) or West Nile virus. Phenocopying MDA5 -/- mice, the MDA5 K23R/K43R mice infected with EMCV displayed increased mortality, elevated viral titers, and an ablated induction of cytokines and chemokines compared to WT mice. Molecular studies identified human HERC5 (and its functional murine homolog HERC6) as the primary E3 ligases responsible for MDA5 ISGylation and activation. Taken together, these findings establish the importance of CARD ISGylation for MDA5-mediated RNA virus restriction, promoting potential avenues for immunomodulatory drug design for antiviral or anti-inflammatory applications.

Interference without interferon: interferon-independent induction of interferon-stimulated genes and its role in cellular innate immunity.

Interferons (IFNs) are multifaceted proteins that play pivotal roles in orchestrating robust antiviral immune responses and modulating the intricate landscape of host immunity. The major signaling pathway activated by IFNs is the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway, which leads to the transcription of a battery of genes, collectively known as IFN-stimulated genes (ISGs). While the well-established role of IFNs in coordinating the innate immune response against viral infections is widely acknowledged, recent years have provided a more distinct comprehension of the functional significance attributed to non-canonical, IFN-independent induction of ISGs. In this review, we summarize the non-conventional signaling pathways of ISG induction. These alternative pathways offer new avenues for developing antiviral strategies or immunomodulation in various diseases.

Characterization of interferon-stimulated gene 15 from Bostrychus sinensis: Cloning, expression and functional analyses.

In this study, the interferon-stimulated gene 15 (referred to as BsISG15) was sequenced and characterized in Bostrychus sinensis. BsISG15 encodes a 155-amino-acid protein weighing ∼17 kDa, featuring two conserved ubiquitin-like domains and an LRGG conjugation motif at the C-terminal. The real-time PCR assays revealed constitutive expression of the BsISG15 gene in all examined organs of healthy B. sinensis, with the peripheral blood showing the highest level of expression. The expression levels of the BsISG15 gene in the head kidney, liver, spleen, and peripheral blood of B. sinensis were significantly altered by both poly (I:C) stimulation and Vibrio parahaemolyticus infection. Western blot analyses showed that the expression of the BsISG15 protein was induced in both the liver and spleen of B. sinensis infected with either poly (I:C) or bacteria, with a concomitant increase in the levels of protein ISGylation, particularly evident in the bacterial-infected liver tissues. Besides, Western blot analyses have demonstrated that head kidney lymphocytes of B. sinensis are capable of secreting the free BsISG15 protein. The recombinant BsISG15 protein significantly increased the production of reactive oxygen species, synthesis of NO, and phagocytosis in macrophages from B. sinensis and also upregulated the expression of proinflammatory cytokine genes (IFNg, IL-1ß, IL-6, and IL-8) in these cells. Knockdown of endogenous BsISG15 elevated the expression levels of proinflammatory cytokines IL-1ß, IL-6, and IL-8, suggesting a negative regulation of BsISG15 on the inflammatory response in macrophages. The results indicate that BsISG15 plays a significant role in the innate antiviral and antibacterial immunity of B. sinensis.

Chlamydia-driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation.

Excessive inflammation upon Chlamydia trachomatis infection can cause severe damages in the female genital tract. This obligate intracellular bacterium develops mainly in epithelial cells, whose innate response contributes to the overall inflammatory response to infection. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) stimulates interferon γ (IFNγ) production and is required for bacterial clearance in several infectious contexts. Here, we describe and investigate the consequences of the increase in ISG15 expression by epithelial cells infected with C. trachomatis. Infection of HeLa cells and primary ecto-cervical epithelial cells resulted in a transcriptional upregulation of ISG15 expression. This did not involve the canonical type I interferon (IFN-I) signaling pathway and depended instead on the activation of the STING/TBK1/IRF3 pathway. The absence or reduction of ISG15 synthesis led to increased production of several cytokines and chemokines, including interleukin (IL) 6 and IL8. This implicates that ISG15 normally dampens the immune response induced by C. trachomatis infection in epithelial cells. ISG15 exerted its control from an intracellular location, but without involving ISGylation. Finally, higher levels of inflammation and delayed bacterial clearance were observed in the genital tracts of ISG15-KO mice infected by C. trachomatis compared with wild-type animals; however, IFNγ production was unchanged. Altogether, our data show that ISG15 expression acts as a brake on the immune response to C. trachomatis infection in epithelial cells and limits bacterial burden and inflammation in mice.IMPORTANCEInfection of epithelial cells by Chlamydia trachomatis elicits an innate immune response by these cells. The signaling pathways involved, and their outcomes, are still very poorly understood. In this paper, we described how Chlamydia infection triggered the expression of ISG15, a small molecule normally associated to type I interferon (IFN-I) signaling and control of INF-γ production. ISG15 synthesis by epithelial cells attenuated their immune response to Chlamydia infection. In mice, we observed that ISG15 displayed a marginal role in modulating the production of IFN-γ, a key component of the host immune response to infection, but facilitated bacterial clearance. Overall, our study strengthens the importance of ISG15 not only in the resolution of viral but also of bacterial infection and document its role of "immune brake" in the context of Chlamydia infection.

Cellular stress increases DRIP production and MHC Class I antigen presentation.

Background: Defective ribosomal products (DRiPs) are non-functional proteins rapidly degraded during or after translation being an essential source for MHC class I ligands. DRiPs are characterized to derive from a substantial subset of nascent gene products that degrade more rapidly than their corresponding native retiree pool. So far, mass spectrometry analysis revealed that a large number of HLA class I peptides derive from DRiPs. However, a specific viral DRiP on protein level was not described. In this study, we aimed to characterize and identify DRiPs derived from a viral protein. Methods: Using the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) which is conjugated N-terminally to ubiquitin, or the ubiquitin-like modifiers FAT10 or ISG15 the occurrence of DRiPs was studied. The formation and degradation of DRiPs was monitored by western blot with the help of a FLAG tag. Flow cytometry and cytotoxic T cells were used to study antigen presentation. Results: We identified several short lived DRiPs derived from LCMV-NP. Of note, these DRiPs could only be observed when the LCMV-NP was modified with ubiquitin or ubiquitin-like modifiers, but not in the wild type form. Using proteasome inhibitors, we could show that degradation of LCMV-NP derived DRiPs were proteasome dependent. Interestingly, the synthesis of DRiPs could be enhanced when cells were stressed with the help of FCS starvation. An enhanced NP118-126 presentation was observed when the LCMV-NP was modified with ubiquitin or ubiquitin-like modifiers, or under FCS starvation. Conclusion: Taken together, we visualize for the first time DRiPs derived from a viral protein. Furthermore, DRiPs formation, and therefore MHC-I presentation, is enhanced under cellular stress conditions. Our investigations on DRiPs in MHC class I antigen presentation open up new approaches for the development of vaccination strategies.

ISG15 is involved in chondrogenic differentiation through activation of IFN-γ signaling.

Interferon-gamma (IFN-γ) was found to increase in the synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). However, few studies have been conducted to elucidate the role of IFN-γ in cartilage metabolism and regeneration. In this study, we investigated whether cartilage regeneration is driven by interferon-stimulated gene 15 (ISG15) under the control of IFN-γ. IFN-γ significantly increased ITS-induced chondrogenic differentiation of ATDC5 cells. Knockdown of IFN-γ receptor (IFN-γR) inhibited IFN-γ-induced chondrogenic differentiation and reduced ACAN and Col II expression. In addition, ISG15 expression was highly elevated in response to IFN-γ, whereas its expression was downregulated by knockdown of IFN-γR, indicating that ISG15 is closely related to IFN-γ signaling. Furthermore, chondrogenic differentiation and expression of ACAN and Col II were significantly reduced following knockdown of ISG15 in ATDC5 cells despite the presence of IFN-γ. ISGylation of cellular proteins found in chondrogenic differentiated cells was related to activation of IFN-γ signaling. In addition, ISG15/ISGylation was significantly observed in the regenerated cartilage tissue 7 days after FTCI of young mice compared with sham control. Our findings showed that upregulation of ISG15 and/or ISGylation of cellular proteins may play a critical role in cartilage regeneration through activation of IFN-γ signaling.

HSV-1-induced N6-methyladenosine reprogramming via ICP0-mediated suppression of METTL14 potentiates oncolytic activity in glioma.

Upon infection with herpes simplex virus 1 (HSV-1), the virus deploys multiple strategies to evade the host's innate immune response. However, the mechanisms governing this phenomenon remain elusive. Here, we find that HSV-1 leads to a decrease in overall m6A levels by selectively reducing METTL14 protein during early infection in glioma cells. Specifically, the HSV-1-encoded immediate-early protein ICP0 interacts with METTL14 within ND10 bodies and serves as an E3 ubiquitin protein ligase, targeting and ubiquitinating METTL14 at the lysine 156 and 162 sites. Subsequently, METTL14 undergoes proteasomal degradation. Furthermore, METTL14 stabilizes ISG15 mRNA mediated by IGF2BP3 to promote antiviral effects. Notably, METTL14 suppression significantly enhances the anti-tumor effect of oncolytic HSV-1 (oHSV-1) in mice bearing glioma xenografts. Collectively, these findings establish that ICP0-guided m6A modification controls the antiviral immune response and suggest that targeting METTL14/ISG15 represents a potential strategy to enhance the oncolytic activity of oHSV-1 in glioma treatment.

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis.

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host (immune) proteins such as MDA5 and IRF3 in a process called ISGylation, thereby limiting the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.

SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

STAT3 Increases CVB3 Replication and Acute Pancreatitis and Myocarditis Pathology via Impeding Nuclear Translocation of STAT1 and Interferon-Stimulated Gene Expression.

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNß as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Interferon-stimulated genes and their antiviral activity against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic remains an international health problem caused by the recent emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of May 2024, SARS-CoV-2 has caused more than 775 million cases and over 7 million deaths globally. Despite current vaccination programs, infections are still rapidly increasing, mainly due to the appearance and spread of new variants, variations in immunization rates, and limitations of current vaccines in preventing transmission. This underscores the need for pan-variant antivirals and treatments. The interferon (IFN) system is a critical element of the innate immune response and serves as a frontline defense against viruses. It induces a generalized antiviral state by transiently upregulating hundreds of IFN-stimulated genes (ISGs). To gain a deeper comprehension of the innate immune response to SARS-CoV-2, its connection to COVID-19 pathogenesis, and the potential therapeutic implications, this review provides a detailed overview of fundamental aspects of the diverse ISGs identified for their antiviral properties against SARS-CoV-2. It emphasizes the importance of these proteins in controlling viral replication and spread. Furthermore, we explore methodological approaches for the identification of ISGs and conduct a comparative analysis with other viruses. Deciphering the roles of ISGs and their interactions with viral pathogens can help identify novel targets for antiviral therapies and enhance our preparedness to confront current and future viral threats.

An ISG15-Based High-Throughput Screening Assay for Identification and Characterization of SARS-CoV-2 Inhibitors Targeting Papain-like Protease.

Emergence of newer variants of SARS-CoV-2 underscores the need for effective antivirals to complement the vaccination program in managing COVID-19. The multi-functional papain-like protease (PLpro) of SARS-CoV-2 is an essential viral protein that not only regulates the viral replication but also modulates the host immune system, making it a promising therapeutic target. To this end, we developed an in vitro interferon stimulating gene 15 (ISG15)-based Förster resonance energy transfer (FRET) assay and screened the National Cancer Institute (NCI) Diversity Set VI compound library, which comprises 1584 small molecules. Subsequently, we assessed the PLpro enzymatic activity in the presence of screened molecules. We identified three potential PLpro inhibitors, namely, NSC338106, 651084, and 679525, with IC50 values in the range from 3.3 to 6.0 µM. These molecules demonstrated in vitro inhibition of the enzyme activity and exhibited antiviral activity against SARS-CoV-2, with EC50 values ranging from 0.4 to 4.6 µM. The molecular docking of all three small molecules to PLpro suggested their specificity towards the enzyme's active site. Overall, our study contributes promising prospects for further developing potential antivirals to combat SARS-CoV-2 infection.

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis.

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I IFN induction to limit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through deISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387, and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.IMPORTANCEThe role of protein ISGylation in regulating host cellular processes has been studied extensively; however, how ISG15 conjugation influences the activity of viral proteins, particularly coronaviral proteins, is largely unknown. Our study uncovered that the nucleocapsid (N) protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation machinery and that this modification impedes the functional assembly of N into oligomers ultimately inhibiting viral RNA synthesis. This antiviral restriction mechanism is antagonized by the PLpro deISGylation activity of SARS-CoV-2 NSP3. This study deepens our understanding of SARS-CoV-2 protein regulation by posttranslational modifications and may open new avenues for designing antiviral strategies for COVID-19.

ISG15 accelerates acute kidney injury and the subsequent AKI-to-CKD transition by promoting TGFßR1 ISGylation.

Rationale: Acute kidney injury (AKI) has substantial rates of mortality and morbidity, coupled with an absence of efficacious treatment options. AKI commonly transits into chronic kidney disease (CKD) and ultimately culminates in end-stage renal failure. The interferon-stimulated gene 15 (ISG15) level was upregulated in the kidneys of mice injured by ischemia-reperfusion injury (IRI), cisplatin, or unilateral ureteral obstruction (UUO), however, its role in AKI development and subsequent AKI-to-CKD transition remains unknown. Methods: Isg15 knockout (Isg15 KO) mice challenged with bilateral or unilateral IRI, cisplatin, or UUO were used to investigate its role in AKI. We established cellular models with overexpression or knockout of ISG15 and subjected them to hypoxia-reoxygenation, cisplatin, or transforming growth factor- ß1 (TGF-ß1) stimulation. Renal RNA-seq data obtained from AKI models sourced from public databases and our studies, were utilized to examine the expression profiles of ISG15 and its associated genes. Additionally, published single cell RNA-seq data from human kidney allograft biopsies and mouse IRI model were analyzed to investigate the expression patterns of ISG15 and the type I TGF-ß receptor (TGFßR1). Western blotting, qPCR, co-immunoprecipitation, and immunohistochemical staining assays were performed to validate our findings. Results: Alleviated pathological injury and renal function were observed in Isg15 KO mice with IRI-, cisplatin-, or UUO-induced AKI and the following AKI-to-CKD transition. In hypoxia-reoxygenation, cisplatin or TGF-ß1 treated HK-2 cells, knockout ISG15 reduced stimulus-induced cell fibrosis, while overexpression of ISG15 with modification capacity exacerbated cell fibrosis. Immunoprecipitation assays demonstrated that ISG15 promoted ISGylation of TGFßR1, and inhibited its ubiquitination. Moreover, knockout of TGFßR1 blocked ISG15's fibrosis-exacerbating effect in HK-2 cells, while overexpression of TGFßR1 abolished the renal protective effect of ISG15 knockout during IRI-induced kidney injury. Conclusions: ISG15 plays an important role in the development of AKI and subsequent AKI-to-CKD transition by promoting TGFßR1 ISGylation.

Human Immunodeficiency Virus-Induced Interferon-Stimulated Gene Expression Is Associated With Monocyte Activation and Predicts Viral Load.

Background: Chronic immune activation is one of the hallmarks of human immunodeficiency virus (HIV) pathogenesis. Persistent upregulation of interferons (IFNs) and interferon-stimulated genes (ISGs) has previously been associated with chronic immune activation and HIV progression. Here a longitudinal analysis of the IFN and ISG response during HIV infection was performed to gain insights into the ongoing immune activation during HIV infection. Methods: IFN and ISG levels were determined using quantitative polymerase chain reaction in peripheral blood mononuclear cells of people with HIV at pre-seroconversion, during acute and chronic HIV infection, and during suppressive antiretroviral therapy (ART). Results: HIV infection induced the expression of a set of 4 ISGs-RSAD2, ISG15, IFI44L, and IFI27-which remained upregulated during chronic infection. This set of ISGs showed no clear correlations with T-cell activation as determined by co-expression of CD38 and HLA-DR. However, a strong correlation with monocyte activation marker soluble CD163 in serum was found. Furthermore, the expression of this ISG cluster was predictive of viral load before ART initiation and, on ART, expression levels normalized to pre-seroconversion levels. Conclusions: The results presented here suggests that ISG expression is linked to monocyte activation, possibly driven by viral replication.

Evaluation of the effect of sustained-release progesterone injection on the expression of interferon-related genes in repeat-breeder dairy cows.

BACKGROUND: Repeat-breeder cows repeatedly fail to conceive after at least three attempts and return to oestrus at apparently normal intervals. Repeat-breeder cows cause economic losses in dairy farms in different ways. OBJECTIVE: In the present study, we investigated the effect of sustained-release progesterone injection in two different doses on the expression of interferon-related genes in repeat-breeder dairy cows. METHODS: A total of 96 repeat-breeder primiparous and multiparous cows were assigned among three groups: control group, inseminated and do not receive progesterone treatment; P400 and P600 groups, inseminated and received a single-intramuscular injection of 400 and 600 mg slow-release progesterone 5 days after insemination, respectively. Blood sampling was carried out on Day 20 after AI for progesterone measurement and evaluation of gene expression for ISG15, MX1 and MX2 genes. RESULTS: One injection of sustained-release progesterone increased the expression of ISG15, MX1 and MX2 genes with differences between two different progesterone concentrations. For all three genes, the level of gene expression was higher in progesterone-supplemented group than in control group, when P400 and P600 groups considered together. The level of MX2 gene expression was significantly higher in pregnant cows than non-pregnant cows. There was a significant positive correlation between expression level of all three genes and blood progesterone concentration. The expression level of ISG15 gene showed a significant positive correlation with MX1 and MX2 gene expression. CONCLUSION: The use of this sustained-release progesterone is simple and can be used in repeat-breeder cows to improve fertility.