Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1β to promote viral replication by regulating K48 ubiquitination

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro . Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense. IMPORTANCE Deubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (Nsp1β) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1β, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1β, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Effects of the commensal microbiota on spleen and mesenteric lymph node immune function: investigation in a germ-free piglet model

“…We had obtained some highly upregulated genes, such as TNF receptor superfamily (TNFRSF) was found in spleen and MLNs upregulated genes, namely TNFRSF19 and TNFRSF17, which function to activate signaling pathways for development, organogenesis, cell death, and survival (Zhao et al, 2018). In addition, USP1 associated with innate immunity (Zhai et al, 2024), was also found in the upregulated genes, and IRF7 expressed in lymphocytes consistented with the above results (Li et al, 2021). Gene Ontology (GO) enrichment showed that many immune-related pathways, such as cellular response to external stimulus and defense response to virus were enriched in the spleen, while adaptive immune response and antiviral innate immune response were enriched in MLNs (Figures 3D, E and Supplementary Table 4).…”

Table_5.XLSX

“…We had obtained some highly upregulated genes, such as TNF receptor superfamily (TNFRSF) was found in spleen and MLNs upregulated genes, namely TNFRSF19 and TNFRSF17, which function to activate signaling pathways for development, organogenesis, cell death, and survival (Zhao et al, 2018). In addition, USP1 associated with innate immunity (Zhai et al, 2024), was also found in the upregulated genes, and IRF7 expressed in lymphocytes consistented with the above results (Li et al, 2021). Gene Ontology (GO) enrichment showed that many immune-related pathways, such as cellular response to external stimulus and defense response to virus were enriched in the spleen, while adaptive immune response and antiviral innate immune response were enriched in MLNs (Figures 3D, E and Supplementary Table 4).…”

Data_Sheet_1.PDF

“…We had obtained some highly upregulated genes, such as TNF receptor superfamily (TNFRSF) was found in spleen and MLNs upregulated genes, namely TNFRSF19 and TNFRSF17, which function to activate signaling pathways for development, organogenesis, cell death, and survival (Zhao et al, 2018). In addition, USP1 associated with innate immunity (Zhai et al, 2024), was also found in the upregulated genes, and IRF7 expressed in lymphocytes consistented with the above results (Li et al, 2021). Gene Ontology (GO) enrichment showed that many immune-related pathways, such as cellular response to external stimulus and defense response to virus were enriched in the spleen, while adaptive immune response and antiviral innate immune response were enriched in MLNs (Figures 3D, E and Supplementary Table 4).…”