The findings demonstrate that the pSFV-driven Trim-Away system is a powerful tool for viral protein degradation and provides a significant advantage against rapidly mutating viruses.
Abstract
Background
Developing effective antiviral strategies is urgently needed during global viral pandemics. Traditional approaches, including small-molecule inhibitors, neutralizing antibodies, and RNA interference (RNAi), often face challenges such as drug resistance, limited specificity, and inefficient delivery. These limitations highlight the pressing need for innovative strategies focused on the targeted degradation of viral proteins.
Methods
We developed an optimized Trim-Away system employing a receptor-Fc fusion protein strategy. This system integrates the E3 ubiquitin ligase TRIM21 with engineered receptor-Fc proteins to ensure highly specific recognition and intracellular degradation. A key innovation is the use of the Semliki Forest virus (SFV) self-amplifying replicon (pSFV). This platform enables sustained and robust expression of the Trim-Away components. Furthermore, this plasmid-based delivery eliminates the need for protein purification, thereby streamlining the process and improving delivery efficiency.
Results
The system effectively degrades diverse viral targets. Specifically, it successfully degraded the spike proteins of both wild-type SARS-CoV-2 and its Omicron variant. It also targeted adeno-associated virus (AAV) capsid proteins. In vivo assays further confirmed that the self-amplifying replicon markedly reduces AAV-encoded luciferase expression. These data demonstrate that the system maintains high potency even at low dosages.
Conclusions
Our findings demonstrate that the pSFV-driven Trim-Away system is a powerful tool for viral protein degradation. The receptor-Fc strategy provides a significant advantage against rapidly mutating viruses. This study establishes a versatile and adaptable platform for future antiviral intervention.
This work pioneers the development of CA-targeted degraders, providing a framework for next-generation anti-HIV therapies with high potency and resistance barriers, via a proteasome-driven pathway.
Mei Wang, Ze-Yu Peng, Yang Zhou et al.· Journal of Medicinal Chemist...· 0 citations
ABSTRACT Influenza A virus (IAV) poses a serious threat to public health due to its high mutability and rapid transmissibility. The viral nucleoprotein (NP), a highly conserved and essential component, has emerged as an ideal target for antiviral therapies. However, its biological function has proven challenging to modulate with conventional drugs, and no NP-targeting therapeutics have reached the market so far. Here, we report the development and application of an aptamer-based proteolysis-targeting chimera (PROTAC) for the targeted degradation of IAV NP. Utilizing the Direct-to-Biology (D2B) platform, we efficiently screened and identified NP-PROTAC#4 as a functional candidate. Subsequently, we developed a lipid nanoparticle (LNP) formulation of NP‑PROTAC#4 (LNP@NP‑PROTAC#4) for effective intracellular delivery. Importantly, LNP@NP-PROTAC#4 demonstrated potent antiviral activity both in vitro and in vivo. Mechanistically, NP-PROTAC#4 exerts its antiviral effects by targeting and degrading NP via the ubiquitin-proteasome system. In conclusion, our findings provide the first evidence that NP-targeted PROTAC degrader exhibits therapeutic effects, proposing a novel therapeutic strategy for IAV.
The COVID-19 pandemic and its protracted consequences underscore the urgent need for more effective antiviral strategies. Current antiviral strategies face a persistent challenge achieving sufficient viral suppression while minimizing off-target toxicity, particularly against such highly mutable viruses. Here, we describe a Receptor-Drug Conjugate (RDC) strategy, in which a therapeutic payload is covalently linked to a decoy receptor, enabling virus-triggered targeted intracellular delivery. Angiotensin-converting enzyme 2 (ACE2), as the essential receptor for SARS-CoV-2 entry, has been widely exploited for virus-neutralizing strategies. Targeting SARS-CoV-2 as a proof-of-concept, we conjugated peptide nucleic acids (PNAs) designed to target the viral ORF1ab region (±30 bp) to ACE2-Fc. The ACE2-PNA conjugate demonstrated superior inhibitory efficacy against multiple SARS-CoV-2 variants relative to ACE2-Fc alone. We confirmed that ACE2-PNA retains the extracellular neutralization activity of soluble ACE2, while being selectively internalized into virus-infected cells via virus-mediated endocytosis. Owing to its inherent protease resistance, the PNA component remains intact upon cytoplasmic entry and subsequently exerts antisense inhibitory activity against viral RNA. The mechanistic feasibility of ACE2-PNA was further validated in a mouse model. Collectively, RDC represents a novel virus-triggered targeted delivery platform that confers dual antiviral efficacy through extracellular virion neutralization and intracellular inhibition of viral replication. It has significant implications for reducing off-target toxicity and enhancing antiviral potency, and is furthermore readily adaptable to diverse viral pathogens and therapeutic payloads.
Seasonal influenza causes up to 650,000 deaths annually and remains a persistent pandemic threat due to zoonotic strains crossing the species barrier. Current antivirals, which target neuraminidase or the viral polymerase, have limited efficacy and rapidly select for resistant variants, highlighting the urgent need for new therapeutic strategies. The heterotrimeric influenza polymerase (FluPol), comprising PA, PB1, and PB2 subunits, is essential for viral replication and harbors virus-specific protein-protein interfaces that are potential drug targets. We focused on the highly conserved PA-PB1 interface, where the N-terminal peptide of PB1 binds the PA C-terminal domain with high affinity. Disrupting this interaction abrogates polymerase function, halting viral replication. Using phage display, we identified PB1-derived peptides with enhanced affinity for PA and characterized their binding via biophysical methods and X-ray crystallography. Lead peptides efficiently disrupted the PB1-PA interaction and inhibited polymerase activity in cell-based assays. To address peptide delivery challenges, we expressed these inhibitors intracellularly from synthetic mRNA formulated in lipid nanoparticles, achieving robust inhibition of viral replication in cultured cells. This work establishes intracellularly expressed peptide inhibitors as a viable antiviral strategy and provides a generalizable framework for targeting essential protein-protein interactions of influenza and other RNA viruses.
Alberto Florez Prada, Chloé Sturmach, Nabila Laroui et al.· bioRxiv· 0 citations
While gasdermin (GSDM)-mediated pyroptosis is a potent immune effector, its antiviral potential remains largely untapped. Here, we introduce viral protease-initiated lytic cell death (VID), a universal mRNA therapeutic platform inspired by the modular architecture of GSDM and the clinical success of mRNA vaccines. By engineering gasdermin-D (GSDMD) to harbor viral protease-specific cleavage motifs, we generated VID activators (VIDAs) that selectively trigger lytic cell death in virus-infected cells. Using hepatitis A virus (HAV) as a model, lipid nanoparticle (LNP)-encapsulated VIDA mRNA abolished viral replication and shedding in vivo and mitigated liver injury through a coordinated "kill-and-alert" mechanism that primes bystander immunity. The platform's versatility was further demonstrated against Zika virus (ZIKV) and SARS-CoV-2. Leveraging a generative artificial intelligence (AI) framework, we designed de novo cleavage motifs for the SARS-CoV-2 main protease, yielding optimized VIDAs with superior antiviral potency. Collectively, our study establishes VIDA mRNA as a versatile, broadly applicable strategy for combating diverse viral threats.
Lin Li, Xiu-Li Yan, Hao-Yang Wang et al.· Cell· 1 citation
ABSTRACT Diseases caused by picornaviruses pose a serious threat to society due to their high contagiousness and widespread prevalence. Beyond the poliovirus vaccine, antiviral therapeutics remain unavailable for most picornaviral infections. Moreover, existing inhibitors under development generally exhibit narrow‐spectrum activity and low barriers to resistance owing to the high mutability of RNA viral proteins. To address these issues, this study constructed a proteolysis‐targeting chimera (PROTAC) targeting the 3C protease (3CPro) — a key viral enzyme essential for picornavirus protein processing, host shutoff, and immune suppression. In enterovirus 71 (EV71), a representative member of the picornavirus family, experimental results showed that PROTAC molecule effectively inhibits viral replication through a dual mechanism of action: directly inhibiting the catalytic function of the 3CPro and inducing its degradation via the ubiquitin‐proteasome pathway. Moreover, this PROTAC molecule not only demonstrated effective degradation of previously reported and AI‐predicted potential drug‐resistant EV71 mutants, but also exhibited degradation activity against 3CPro from multiple picornaviruses, indicating its potential for broad‐spectrum antiviral activity. By integrating multidisciplinary approaches, this work demonstrates that targeted protein degradation can effectively induce the degradation of 3CPro in picornaviruses, supporting the development of resistance‐resistant, broad‐spectrum antiviral agents and highlighting their potential to address evolving viral threats.
Weilong Deng, Jun-Yu Chen, Yingyue Pang et al.· Advancement of science· 0 citations