Investigating RIPK2 as an Essential Pan-Enterovirus Host Factor: Unveiling a Promising Target for Novel Antiviral Therapeutics

Abstract

Enteroviruses are widespread pathogens implicated in acute illness and increasingly linked to chronic diseases such as type 1 diabetes. Despite their impact, no FDA-approved antiviral therapies exist for enterovirus infections. Resistance to direct-acting antivirals underscores the need for alternative strategies. Given enteroviruses’ dependence on host machinery, targeting host factors, particularly kinases involved in virus-host interactions, represents a promising approach.

This study aimed to investigate RIPK2 as an essential pan-enterovirus host dependency factor and evaluated its potential as a broad-spectrum host-directed antiviral target. By employing a comprehensive strategy, we combined selective pharmacological inhibition using the RIPK2 inhibitor GSK583, RNA interference, CRISPR-Cas9-mediated gene editing, viral replication assays and gene expression profiling in both immortalized human cell lines and primary human pancreatic islets infected with a panel of clinically relevant EV-B strains.

RIPK2 Inhibition with GSK583 elicited potent, dose-dependent suppression of a genetically diverse panel of enterovirus B species with a high selectivity index (>100). Treatment also preserved islet viability and mitigated infection-associated cytotoxicity. Genetic silencing and knockout of RIPK2 significantly impaired viral RNA synthesis and viral particle production without compromising cell viability. Time-of-drug addition assay pinpointed RIPK2’s role during early post-entry stages, likely facilitating viral genome replication and translation. Gene expression profiling further revealed that RIPK2 inhibition concurrently attenuated expression of innate immune sensors, pro-inflammatory cytokines, and antigen presentation machinery in primary islets. Interestingly, a pronounced tissue-specific divergence in antiviral gene induction was observed between primary islets and Hep-2 cells, emphasizing the importance of physiologically relevant models in antiviral research.

Our findings establish RIPK2 as a critical host dependency factor in enterovirus replication and support its inhibition as a dual-action strategy that simultaneously impairs viral propagation and dampens virus-induced immunopathology. Repurposing RIPK2 inhibitors, originally developed for inflammatory diseases, offers a promising host-directed strategy for broad-spectrum antiviral intervention. Future work should evaluate in vivo efficacy and explore applications in both acute and chronic enterovirus-associated conditions.

Popular Abstract

Target discovery for Host-Directed Broad-Spectrum Antivirals

Enteroviruses (EVs), which infect over one billion people annually, are responsible for a wide range of acute and chronic diseases, including type 1 diabetes. Despite their high prevalence and clinical relevance, no approved antiviral treatments exist. There is a critical need to identify host factors that can be therapeutically targeted for broad-spectrum antiviral strategies. Recent evidence suggests that receptor-interacting protein kinase 2 (RIPK2), a key regulator of innate immunity, may play an unrecognized role in EV replication.

This study aimed to determine whether RIPK2 is essential for enterovirus replication and to evaluate the therapeutic potential of RIPK2 inhibition in suppressing viral replication and its associated inflammation. To address these aims, we employed an interdisciplinary approach combining pharmacological inhibition using GSK583, gene editing, viral replication assays, and gene expression profiling in both immortalized human cell lines and primary human pancreatic islets infected with a panel of clinically relevant EV serotypes.

RIPK2 inhibition with GSK583 elicited potent, dose-dependent suppression of viral replication across multiple enterovirus strains. Inhibiting RIPK2 also preserved the integrity of host cells and alleviated infection-induced inflammatory responses. Furthermore, the viral replication assays suggest that RIPK2 plays a crucial role early in the viral life cycle, particularly in facilitating viral genome replication and protein production, and supports the notion of its potential as a possible broad-spectrum antiviral target. In primary islets, RIPK2 inhibition significantly attenuated virus-induced expression of innate immune sensors, pro-inflammatory cytokines, and MHC class I molecules, indicating a dual antiviral and anti-inflammatory effect. These findings establish RIPK2 as a critical host dependency factor for enterovirus replication and innate immune activation. Intriguingly, the study also uncovered tissue-specific differences in antiviral responses, underscoring the importance of using physiologically relevant human models to accurately assess host-pathogen interactions and therapeutic impact. Given that RIPK2 inhibitors are already under development for autoimmune and inflammatory diseases, repurposing them as antivirals could offer a promising and potentially expedited route to clinical translation. This dual-action strategy, limiting viral replication and mitigating harmful inflammation, may not only improve treatment outcomes for acute enteroviral infections but also help reduce the long-term burden of chronic enterovirus-associated diseases.

• author: Laghzaoui, Oussama
• supervisor: Luis Sarmiento-Pérez
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9214685
• date added to LUP: 2025-10-30 12:43:04
• date last changed: 2025-10-30 12:43:04