Prime Editing–Mediated Nectin‑1 Functional Domain Modification Confers PRV Resistance in Porcine Cells: Functional Knock‑out Architecture for Blocking Host‑Virus Docking and a Next‑Generation Computational Genetic Engineering Framework for Livestock
-
Physiological collapse caused by complete removal of the host receptor and bottleneck in designing genetically antiviral livestock breeds. Pseudorabies virus (PRV), a double‑stranded DNA enveloped virus of the Alphaherpesvirinae, is a major threat to swine productivity. The virus uses the cell‑surface adhesion molecule Nectin‑1 as its primary host receptor to trigger endocytosis and membrane‑fusion pathways. Conventional genome‑engineering guidelines have relied on traditional knockout (KO) strategies that delete the entire Nectin‑1 coding sequence to block infection at the source. However, this blunt approach disrupts Nectin‑1’s native roles in cell‑cell adhesion and epithelial tissue formation, leading to embryonic lethality, developmental defects, or false‑positive phenotypes—a critical blind spot. The lack of reversible micro‑domain engineering that preserves the protein’s endogenous function while selectively filtering viral entry has been a longstanding barrier to sustainable disease‑resistant livestock breeding pipelines.
-
Prime‑editing‑driven four‑amino‑acid reprogramming of the Nectin‑1 gD‑binding interface. Recent advances in genome engineering have deployed next‑generation prime editing (PE) systems that enable precise base substitution, insertion, or deletion without double‑strand breaks (DSBs), thereby mitigating cytotoxicity associated with complete receptor loss. The research team performed in silico structural‑mechanical modeling of the interface where PRV glycoprotein D (gD) contacts Nectin‑1. Four key amino‑acid residues governing binding energy were identified, and pegRNAs were designed to introduce monoallelic and homozygous micro‑mutations with high efficiency.
-
Demonstration of the topological mechanism of viral entry inhibition and establishment of a functional knockout. Dynamic tracking of PRV infection susceptibility in the PE‑modified porcine cell lines revealed a definitive "functional knockout" phenotype. The engineered Nectin‑1 architecture allowed normal initial attachment of PRV particles to the cell surface but prevented allosteric matching with gD, thereby clamping the downstream, rate‑limiting internalization step. Notably, the quadruple‑mutant fusion model maintained normal cell viability and proliferation while achieving virus‑resistance levels statistically indistinguishable from conventional Nectin‑1 KO cells.
-
Standardization of programmable smart breeding infrastructure and establishment of agri‑biosecurity standards. This integrated data‑package for agricultural genetics and synthetic biology redefines livestock disease‑control governance from reactive feed additives and quarantine to a "host‑genome‑code‑scan‑based programmable infection‑interference infrastructure." Computational optimization matrices were developed to suppress off‑target genotoxic noise below baseline levels. The derived Nectin‑1 prime‑editing correction coefficients and structural‑mechanical parameters will serve as a backbone for CMC safety assessments in IND filings for next‑generation transgenic livestock, dramatically compressing regulatory timelines for sustainable, environmentally friendly antiviral breeding programs.
BACKGROUND: The continuous evolution and cross-species transmission risk of pseudorabies virus (PRV) poses a substantial threat to the swine industry and public health. Nectin-1, an essential host receptor for PRV entry, represents a promising antiviral target; however, direct gene knockout (KO) raises biosafety concerns, and conventional genome-editing approaches remain inefficient for precise multiplex modification of critical functional residues. RESULTS: Here, we employed prime editing (PE) to systematically re-engineer the PRV entry receptor Nectin-1, introducing single and combinatorial point mutations at four structurally defined amino acids within the gD-binding interface. PE enable efficient homozygous multiplex editing of these functionally critical residues in porcine cells. Functional analyses showed that these targeted mutations specifically impaired viral internalization without affecting attachment and exhibited combinatorial effects, resulting in reductions in viral replication and release. Notably, the quadruple-site mutant conferred a level of resistance comparable to that observed in Nectin-1 KO cells. CONCLUSION: Collectively, this study establishes a framework for "functional knockout," in which precise editing of essential microdomains confers resistance to virus infection while preserving protein integrity. These findings, derived from in vitro cellular models, suggest a potentially safer and more controllable strategy for antiviral genome-edited breeding, pending further validation in vivo.
Why it matters: The genomic‑engineering discoveries reported here extend beyond theoretical method development to direct activation of global livestock‑biotech supply chains and B2B animal‑genetic‑resource business lines. In the event of a PRV epidemic that causes catastrophic herd loss in swine operations, precisely edited Nectin‑1 domain stem cells can be employed to eradicate the viral replication circuit from the earliest developmental stages, thereby preserving the animal’s innate immune homeostasis. Simultaneously, coupling the high‑efficiency single‑base correction kinetics of prime editing enables virtual simulation of false‑positive chromosomal instability noise that arises from multiplex allele‑complex mutations, and provides a platform interface to fine‑tune the integrity of cell‑line transformation. Moreover, during large‑scale validation trials of resistant livestock by multinational agribusinesses, computational filtering of genetic background variation among test animals eliminates false‑positive batch errors in mass‑production specifications, functioning as a backbone infrastructure that maximizes the probability of IND approval and FDA food‑safety clearance.