Discover how FMDV's irreversible inactivation of ISG15 creates unique detection opportunities
Imagine a skilled thief who not only steals your valuables but also permanently disables your security system on the way out. This is precisely the strategy employed by the foot-and-mouth disease virus (FMDV), a highly contagious animal pathogen that causes annual economic losses estimated between $6.5 and $21 billion globally 1 .
For years, this virus has successfully evaded detection by dismantling a crucial part of the host's immune defense. But now, scientists have discovered that the very mechanism FMDV uses to evade immunity creates a unique fingerprint that could revolutionize how we detect viral infections.
Recent groundbreaking research has revealed that FMDV's leader protease (Lbpro) disables a key immune protein called ISG15 in a previously unknown way—one that leaves behind conclusive evidence of its activity 2 . This discovery opens up exciting possibilities for developing novel detection strategies that could potentially distinguish infected from vaccinated animals, offering new hope for controlling this devastating livestock disease.
To appreciate the significance of this discovery, we first need to understand the players involved. Our cells are not defenseless against viral invaders; they come equipped with a sophisticated security system known as the innate immune response. When a virus is detected, cells release signaling proteins called interferons, which trigger the production of hundreds of antiviral proteins known as interferon-stimulated genes (ISGs) 3 .
One of the most important of these ISGs is ISG15, a ubiquitin-like protein that acts as a multifunctional defense weapon 3 . Structurally, ISG15 resembles two ubiquitin molecules joined together, giving it unique properties 4 .
Attachment to host and viral proteins disrupts viral replication
Signaling molecule that modulates immune responses
During viral infection, ISG15 becomes one of the most highly upregulated proteins, making it a critical component of our antiviral defense system 4 . Not surprisingly, successful viruses have evolved sophisticated countermeasures to neutralize this threat.
Many viruses deploy special proteins called proteases that specifically target ISG15, effectively sabotaging the host's defense network. Typically, these viral proteases function as deISGylases—enzymes that carefully remove ISG15 from proteins it has modified, essentially reversing the ISGylation process 2 . This careful removal allows both the ISG15 and the protein it was attached to be recycled and used again, causing minimal collateral damage.
Coronaviruses like SARS-CoV-2, for instance, encode a papain-like protease (PLpro) that expertly cleaves ISG15 from modified proteins, helping the virus evade immune detection 5 . These conventional deISGylases target the isopeptide bond that connects the C-terminal glycine-glycine (GlyGly) motif of ISG15 to substrate proteins 2 . It's a precise, surgical strike that neutralizes the defense while leaving the components intact for potential future use.
The foot-and-mouth disease virus employs a different and far more destructive strategy. Instead of carefully reversing the ISGylation process, its leader protease (Lbpro) permanently disables the ISG15 system through irreversible cleavage 2 .
Unlike conventional deISGylases that cut after the GlyGly motif, Lbpro cuts ISG15 at the peptide bond preceding this motif—specifically between Arg155 and Gly156 2 6 . This unusual cleavage site has profound consequences:
| Viral Protease | Virus Family | Cleavage Site | Result | Reversibility |
|---|---|---|---|---|
| FMDV Lbpro | Picornavirus | Before GlyGly motif | Permanent ISG15 inactivation | Irreversible |
| SARS-CoV-2 PLpro | Coronavirus | After GlyGly motif | ISG15 removal | Reversible |
| CCHFV vOTU | Bunyavirus | After GlyGly motif | ISG15 removal | Reversible |
This unconventional strategy initially appears to give FMDV a significant advantage—by permanently disabling the ISG15 system, the virus ensures this defense cannot be rapidly reactivated. However, this very mechanism creates a unique opportunity for detection.
The discovery of Lbpro's unique mechanism emerged from a series of elegant experiments that combined structural biology, biochemistry, and virology. When researchers initially characterized Lbpro's activity, they noticed something unusual: while it targeted ISG15 with high efficiency, it lacked robust activity against standard assay substrates 6 .
Mass spectrometry analysis of Lbpro-treated ISG15 revealed a surprising result—instead of the expected cleavage after the GlyGly motif, the protein was cut between Arg155 and Gly156, leaving the GlyGly dipeptide attached to the substrate 6 .
To understand how Lbpro achieves this unusual cleavage, researchers designed a specialized ISG15 suicide probe and determined the crystal structure of Lbpro bound to this probe 7 . The structure, resolved at 1.5 Å resolution, showed how Lbpro's acidic groove (formed by Asp49, Glu96, and Glu147) cradles ISG15's positively charged C-terminal residues, positioning the cleavage site perfectly 6 .
The critical insight came when researchers realized that the GlyGly remnant left on substrate proteins could be detected using existing anti-GlyGly antibodies originally developed for ubiquitin proteomics research 2 . This meant that Lbpro's activity during FMDV infection creates a durable molecular signature that can be identified with standard laboratory tools.
| Experimental Approach | Key Finding | Significance |
|---|---|---|
| Mass Spectrometry | Cleavage between Arg155-Gly156 | Revealed unprecedented cleavage site |
| X-ray Crystallography | Structure of Lbpro-ISG15 complex | Explained molecular basis of specificity |
| Biochemical Assays | Preference for ISG15 over ubiquitin | Established substrate preference |
| Immunological Detection | Anti-GlyGly antibodies detect cleavage products | Opened diagnostic applications |
The experimental results revealed several surprising aspects of Lbpro's activity:
Lbpro shows strong preference for ISG15 over ubiquitin, requiring approximately 1,000-fold higher concentrations to cleave ubiquitin 6 .
Lbpro can process all forms of ISG15 present in cells—precursor, mature, and substrate-bound forms 6 .
The GlyGly remnant created by Lbpro provides a consistent, host-derived epitope that doesn't depend on variable viral components 2 .
These findings were particularly significant because they suggested a detection method that would remain effective even as the virus mutated—a common challenge in viral diagnostics.
Studying the intricate battle between ISG15 and viral proteases requires specialized tools and techniques. The following table highlights key reagents and methods that have enabled these discoveries.
| Tool/Reagent | Function/Application | Example in ISG15 Research |
|---|---|---|
| ISG15 Suicide Probes | Covalently trap protease-substrate complexes | Engineered ISG15CTD-ΔC probe for Lbpro crystallization 6 |
| Anti-GlyGly Antibodies | Detect GlyGly remnants on substrates | Identification of Lbpro cleavage products during FMDV infection 2 |
| FRET-Based Assays | High-throughput screening for protease inhibitors | ISG15-based FRET assay for SARS-CoV-2 PLpro inhibitors 5 |
| Recombinant ISG15 & Proteases | In vitro biochemical characterization | Purified Lbpro and ISG15 for cleavage assays 6 |
| Crystallography | Structural determination of protein complexes | Lbpro-ISG15 structure determination at 1.5 Å resolution 7 |
The implications of this research extend far beyond FMDV. Understanding how viruses manipulate host immune systems provides fundamental insights that can be applied to multiple areas of science and medicine.
The detailed structural information about how Lbpro binds ISG15 creates opportunities for developing specific protease inhibitors 7 . Such inhibitors could potentially neutralize this virulence mechanism, providing a therapeutic approach against FMDV. Similar strategies are already being explored for other viral proteases, including SARS-CoV-2 PLpro 5 .
The strategy of detecting viral activity through the specific molecular signatures it leaves on host proteins represents a novel approach to infection diagnostics 2 . This method could be particularly valuable for:
Research on ISG15 and viral countermeasures continues to uncover surprising complexities in our immune system. Recent studies have revealed that:
The discovery that FMDV irreversibly inactivates ISG15 represents a fascinating example of how scientific curiosity can transform a viral evasion strategy into a powerful detection tool. What initially appeared to be a clever viral tactic to permanently disable host defenses has been revealed as a potential Achilles' heel—an immutable, host-derived signature of infection that persists long after the virus has moved on.
This case exemplifies how basic scientific research into molecular mechanisms can yield unexpected practical applications. By meticulously unraveling the unusual cleavage mechanism of Lbpro, scientists have not only advanced our understanding of viral immune evasion but have also opened the door to innovative diagnostic strategies that could ultimately help control one of the world's most economically significant animal diseases.
As research continues, the principles learned from studying ISG15 and viral proteases may well inspire new approaches for detecting and treating a wide range of infectious diseases, proving once again that sometimes a pathogen's greatest strength can become its most exploitable weakness.
Irreversible cleavage leaves detectable GlyGly signature
Evasion mechanism becomes detection opportunity
Could distinguish infected from vaccinated animals