ISG15 modification of PCNA inhibits porcine deltacoronavirus replication via HERC5 and USP18

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus responsible for substantial economic losses in the livestock industry, primarily causing acute diarrhea in piglets. Beyond its impact on swine, PDCoV also presents a potential zoonotic risk, highlighting the urgent need for effective antiviral strategies. Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is well-established for its critical roles in antiviral immunity and immune regulation through its interactions with various cellular and viral proteins. Despite this, the precise molecular mechanisms by which ISG15 exerts its effects on PDCoV infection, particularly regarding specific host targets and regulatory pathways, remain largely uncharacterized. This knowledge gap hinders the development of targeted interventions.

Researchers utilized CRISPR-Cas9 technology to generate ISG15 knockout IPEC-J2 cell lines and also constructed stable ISG15-overexpressed IPEC-J2 cell lines to study its role in PDCoV infection. They employed tandem affinity purification followed by mass spectrometry with ISG15 to identify novel candidate ISGylation proteins. Further experiments investigated the interactions between HERC5, USP18, and PCNA during PDCoV infection, with the primary endpoint being the assessment of PDCoV replication levels under various ISG15 and ISGylation conditions.

The study initially demonstrated that genetic knockout of ISG15 using CRISPR-Cas9 technology significantly facilitated PDCoV replication in IPEC-J2 cells, while ISGylation deficiency (mutation of the ISG15 C-terminal LRLRGG motif) attenuated the protein's antiviral capacity. To identify novel ISGylation targets, tandem affinity purification coupled with mass spectrometry was performed, leading to the identification of 538 known and novel candidate ISGylation proteins. Among these, proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair, was highlighted.

Crucially, ISGylation of PCNA was found to significantly inhibit PDCoV replication. Further mechanistic investigation revealed that during PDCoV infection, HERC5, an ISG15 E3 ligase, directly interacted with PCNA and actively promoted its ISGylation. Conversely, USP18, a de-ISGylase, was identified as the enzyme responsible for de-ISGylating PCNA, indicating a dynamic and tightly regulated post-translational modification process. These findings collectively establish ISGylation as a novel regulatory modification of PCNA that plays a pivotal role in the host's antiviral response to PDCoV.