THAP11-mediated K48- and K63-linked ubiquitination is essential for the degradation of porcine reproductive and respiratory syndrome virus nonstructural protein 1β

THAP11-mediated K48- and K63-linked ubiquitination is essential for the degradation of porcine reproductive and respiratory syndrome virus nonstructural protein 1β | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article THAP11-mediated K48- and K63-linked ubiquitination is essential for the degradation of porcine reproductive and respiratory syndrome virus nonstructural protein 1β Binghua Chen, Yongsheng Xie, Zhan He, Yongjie Chen, Jiecong Yan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6114224/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jun, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen in the global pig industry, causing significant economic losses. Due to its rapid mutation, effective antiviral treatments or vaccines are still lacking. Therefore, it is essential to identify potential host factors that interact with PRRSV-encoded proteins. In this study, a porcine alveolar macrophage cDNA library was used to identify host proteins interacting with PRRSV nonstructural protein 1β (Nsp1β) through a yeast two-hybrid system. A total of 34 potential host factors were identified, with thanatos-associated protein 11 (THAP11) showing a strong interaction with Nsp1β. These interactions were further analyzed using Gene Ontology and KEGG pathway analysis. Co-localization of Nsp1β with THAP11, poly(rC)-binding protein 1 (PCBP1), thioredoxin-interacting protein (TXNIP), and cathepsin D (CTSD) was observed, and Co-IP assays confirmed the Nsp1β-THAP11 interaction. Overexpression of THAP11 reduced PRRSV N protein accumulation, indicating an antiviral effect, while silencing THAP11 enhanced PRRSV replication. Furthermore, THAP11 promoted the degradation of Nsp1β by increasing K48- and K63-linked ubiquitination, thereby restricting PRRSV replication. These findings suggest that THAP11 exerts an antiviral effect by interacting with and degrading Nsp1β via the ubiquitin-proteasome system, providing insights for future PRRSV defense strategies. Yeast two-hybrid screening Virus-host interactions PRRSV Nsp1β THAP11 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases in the global pig industry, leading to significant economic losses worldwide [ 1 ]. It is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), which severely affects the reproductive health of sows and the respiratory health of piglets [ 2 ]. The disease results in reduced productivity and substantial economic burdens on pig farming. PRRSV exhibits rapid mutation and high genetic variability, which makes it particularly difficult to control with existing vaccines and antiviral therapies [ 3 – 7 ]. Consequently, new research aimed at identifying critical host-virus interactions and discovering novel antiviral targets is essential for improving PRRS management and treatment [ 8 , 9 ]. PRRSV is a single-stranded, positive-sense RNA virus, approximately 15 kilobases (kb) in length. It belongs to the family Arteriviridae , containing 10–12 open reading frames (ORFs), encoding several nonstructural (Nsp1-Nsp12) and structural proteins (GP2a, E, GP3, GP4, GP5, M and N) [ 10 , 11 ]. PRRSV is known to inhibit type I interferon responses through its nonstructural proteins (Nsp1α, Nsp1β, Nsp2, Nsp4 and Nsp11) [ 12 – 18 ]. Nonstructural protein 1β (Nsp1β) is one of the key nonstructural proteins encoded by the PRRSV. As an essential viral protein, Nsp1β plays a critical role in the pathogenesis of PRRSV by modulating the host's immune responses and assisting the virus in evading immune detection [ 19 – 21 ]. The protein is encoded by the PRRSV genome as part of the viral replicase complex, which is responsible for viral RNA replication and transcription [ 20 , 22 ]. The functional analysis of Nsp1β has primarily focused on its immune evasion mechanisms, as this is a critical factor in PRRSV pathogenesis. Early studies demonstrated that Nsp1β interferes with interferon signaling by preventing the activation of the interferon regulatory factor 3 (IRF3) pathway [ 19 , 23 ]. This inhibits the production of type I interferons, which are typically induced upon viral infection as part of the host’s early antiviral responses. Further studies have shown that Nsp1β may also directly interact with other components of the innate immune system. For example, Nsp1β has been reported to interact with the host’s cellular RNA sensing pathways, preventing the host from detecting viral RNA effectively [ 14 , 20 , 23 ]. It inhibits the JAK-STAT pathway by preventing the nuclear translocation of STAT1 and STAT2, which are essential for initiating the interferon (IFN) responses [ 24 , 25 ]. This blockade significantly hinders the host’s ability to mount an effective immune defense, allowing the virus to evade immune recognition. Furthermore, Nsp1β binds to nucleoporin 62 (Nup62), a component of the nuclear pore complex, to block the nuclear export of antiviral mRNA and proteins. This action impairs the host’s capacity to produce antiviral proteins, further contributing to the virus’s ability to replicate unchecked [ 26 ]. Additionally, Nsp1β stabilizes its own protein form by interacting with host deubiquitinating enzymes, such as USP1, preventing its degradation through the ubiquitin-proteasome pathway [ 27 ]. This stabilization not only preserves Nsp1β’s ability to suppress immune responses but also promotes viral replication. Nsp1β also stabilizes hypoxia-inducible factor 1 alpha (HIF-1α), a key regulator of inflammation, through deubiquitination. This process enhances viral replication by promoting inflammation and modulating immune responses to favor the virus’s survival [ 28 ]. Moreover, Nsp1β inhibits NLRP3 inflammasome activation, a crucial player in triggering inflammatory responses. This inhibition further suppresses the host’s immune responses, facilitating viral persistence [ 29 ]. Nsp1β also interacts with host proteins like DEAD-box RNA helicase DDX21 and GTPase-activating protein SH3 domain–binding protein 1 (G3BP1), which regulate viral replication and stress granule dynamics [ 30 , 31 ]. These interactions help modulate the host cellular environment to favor viral replication while avoiding immune detection. Lastly, Nsp1β induces the degradation of karyopherin α1 (KPNA1), a key molecule in the nuclear import of immune signaling complexes like Interferon-stimulated gene factor 3 (ISGF3) [ 24 ]. By disrupting the nuclear translocation of these complexes, Nsp1β effectively inhibits the host immune responses, further enhancing viral replication. In summary, Nsp1β uses multiple mechanisms, including the disruption of immune signaling pathways, the stabilization of key host factors, and interference with antiviral mRNA export, to create a favorable environment for PRRSV replication and immune evasion. Thanatos-associated protein 11 (THAP11) is a transcriptional regulator containing a highly conserved THAP domain [ 32 ]. THAP11 has been implicated in regulating apoptosis, oxidative stress, and immune responses, making it a potential factor in viral infections. The THAP domain is found in a variety of proteins involved in cellular stress responses and DNA repair, and THAP11 itself is known to interact with key host proteins to control cellular processes such as cell survival and inflammation [ 33 – 37 ]. While THAP11's role in cellular processes is well-documented, its involvement in viral infections, particularly in PRRSV infection, has not been thoroughly studied. Given THAP11’s ability to modulate immune responses and its role in cellular stress pathways, it is possible that it plays a role in the host’s defense against viral infections, including PRRSV. In this study, we utilized a porcine alveolar macrophages (PAMs) cDNA library and a yeast two-hybrid system to screen for host proteins interacting with PRRSV Nsp1β. A total of 34 potential interacting host proteins were identified. Among these, THAP11 demonstrated a particularly strong interaction with Nsp1β, making it a prime candidate for further investigation. Co-localization experiments, as well as Co-IP assays, confirmed the interaction between Nsp1β and THAP11. Furthermore, overexpression of THAP11 resulted in a significant reduction in PRRSV N protein accumulation, suggesting that THAP11 exerts an antiviral effect by inhibiting PRRSV replication. On the other hand, silencing THAP11 enhanced PRRSV replication, indicating that THAP11 plays a critical role in controlling viral replication. Further mechanistic studies revealed that THAP11 promotes the degradation of Nsp1β through the ubiquitin-proteasome system, specifically by enhancing K48- and K63-linked ubiquitination. This finding suggests that THAP11 functions not only as a regulatory protein but also as an important factor in the degradation of Nsp1β, thereby limiting the virus's ability to evade immune detection and replicate efficiently. These results highlight THAP11 as a novel antiviral host factor in PRRSV infection, providing a potential target for developing new antiviral strategies. This study contributes to the growing body of knowledge on host-virus interactions in PRRSV infection and provides new insights into the role of THAP11 in modulating viral replication through the ubiquitin-proteasome system. Understanding how PRRSV manipulates host proteins, such as THAP11, to evade immune detection and promote replication could pave the way for the development of more effective antiviral therapies and vaccines. Materials and methods Plasmids and cell lines The bait plasmid for the yeast library screen was constructed using a traditional method. The cDNA encoding Nsp1β from the classical CH-1a strain of PRRSV-2 subtype 2 was amplified by PCR with specific primers listed in Supplemental Table S1 . The amplified product was then cloned into the yeast pGBKT7 vector using EcoRI and BamHI restriction sites to express the Gal4 DNA-binding domain fusion protein. The cDNA library for PAMs was constructed using SMART technology and cloned into the pGADT7-Rec vector through gene recombination techniques. The full-length cDNA sequence of the gene of interest was synthesized via reverse transcription PCR (RT-PCR) and then cloned into the pGADT7 yeast vector with EcoRI and XhoI restriction sites added to the primers listed in Supplemental Table S1 . Marc-145 and HEK293T cells were obtained from previous laboratory stocks and cultured in Dulbecco’s Modified Eagle Medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a 5% CO 2 incubator. All experiments were performed in the level 2 Biosafety Laboratory at the College of Veterinary Medicine, South China Agricultural University. Yeast two hybrid (Y2H) assays In Y2H assays, a PAMs cDNA library for Sus scrofa was constructed to screen for proteins interacting with PRRSV Nsp1β using the GAL4 system as described in the BD Matchmaker Library Construction and Screening Kits User Manual (Clontech, Palo Alto, CA). Nsp1β was cloned into the yeast pGBKT7 vector and used as the bait by co-transformation into Y2H Gold yeast cells with PAMs cDNA library. Transformants were grown on SD/-Leu/-Trp/-His medium (TDO), positive clones were selected on the Ade/Leu/Trp/His-deficient medium and then confirmed by 5-Br-4-Cl-3-indoxyl α-D-galactoside (X-α-Gal) and Aureobasidin A (AbA)/(QDO/X/A) assays. To confirm the interaction between proteins, full length host proteins were fused into the pGADT7 vector (Clontech, Palo Alto, CA) as a prey. The corresponding proteins were in in Y2H assays. Transformants were plated on synthetic defined medium lacking Leu and Trp (SD/-Leu/-Trp, DDO), grown for three days, and then transferred to synthetic defined medium lacking Ade, Leu, Trp and His, supplemented with 70 µg/ml X-α-Gal and AbA (SD/-Ade/-Leu/-Trp/-His/X-α-Gal, QDO/X/A). Positive control experiments were performed by co-transforming pGBK-53 and pGAD-T, while negative controls were performed with pGBK-Lam and pGAD-T. Three independent experiments were conducted to confirm the results. Detection of auto-activating and toxicity BD-Nsp1β and the blank pGADT7 vector were co-transformed into Y2H cells. The transformants were then plated onto DDO/X, TDO/X, and QDO/X/AbA selective media for screening. Confocal assays HEK293T cells were cultured in 12-well plates containing 20 mm polylysine-coated coverslips. When the cells reached 70–80% confluence, they were co-transfected with plasmids expressing Nsp1β and THAP11 or appropriate controls. At 24 hours post-transfection (hpt), the cells were processed for immunofluorescence (IF) staining according to standard procedures. Finally, the polylysine-coated coverslips were mounted onto glass slides using nail polish. Fluorescent images were captured using a confocal fluorescence microscope (Leica, Germany). Western blot analysis Cell samples cultured in 6-well or 12-well plates were lysed with RIPA buffer (high) (Solarbio) as per the manufacturer's protocol. The lysates underwent ultrasonic disruption and were then centrifuged at 12,000 rpm for 10 minutes (min). The resulting supernatant was collected into 500 µL sterile tubes, combined with 5×SDS-PAGE loading buffer, heated at 100°C for 10 min, and cooled on ice for 2 min. Protein separation was performed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), utilizing the One-Step PAGE Gel Fast Preparation Kit (Vazyme Biotechnology, China). The proteins were subsequently transferred to a PVDF membrane via semi-dry transfer. After blocking the membrane with a solution containing 5% nonfat dry milk and 0.1% Tween 20 for 1 hour (h) at room temperature, it was incubated with specific primary or endogenous antibodies overnight at 4°C. Following this, the membrane was incubated with HRP-conjugated anti-mouse/rabbit IgG secondary antibody for 1 h at room temperature. Finally, protein bands were visualized using the Amersham ImageQuant 800 (Cytiva, Washington, DC, USA). Co-immunoprecipitation (Co-IP) assays Magnetic beads conjugated with anti-Myc or anti-mCherry antibodies were prepared following the instructions provided in the Protein A/G immunoprecipitation Kit (Selleck). In the Co-IP assay, cells cultured on 6-well plates were lysed for 30 min on ice with 250 µL of cell lysis buffer (Beyotime), supplemented with 1 mM PMSF protease inhibitor. The lysates were disrupted using ultrasonic treatment and centrifuged at 12,000 rpm for 15 min. A small aliquot of the supernatant was collected for input detection, while the remainder was incubated with protein A/G magnetic beads (Selleck) for antibody binding, according to the manufacturer’s protocol. The antigen-antibody complexes were incubated for 2 h at room temperature, and the beads were washed five times with a washing buffer (50 mM Tris, 150 mM NaCl, 0.5% detergent, pH 7.5). To elute the proteins, 40 µL of 1×SDS-PAGE loading buffer was added to the beads, followed by denaturation at 100°C for 5 min. The proteins were then separated by SDS-PAGE for detection. Immunofluorescence assays (IFA) Cells were fixed in 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% Triton X-100 for 5 min. After blocking with 1% BSA for 1 h at room temperature, cells were incubated with primary antibodies overnight. Following washes, the cells were exposed to secondary antibodies conjugated with Alexa Fluor 488 or 555 fluorochromes (Cell Signaling Technology) for 1 h at room temperature. After further washing, the cells were stained with DAPI (Beyotime, China) for 5 min. Finally, fluorescence images were captured using an inverted microscope (Nikon ECLIPSE Ti2). Quantitative RT-PCR (qPCR) Total RNA was extracted from Marc-145 cells using TRizol reagent. The first strand of cDNA was synthesized via reverse transcription polymerase chain reaction (RT-PCR). Quantitative PCR (qPCR) was subsequently performed using ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Vazyme, Nanjing, China), following the manufacturer's instructions. Plasmid transfection and RNA interference (RNAi) HEK293T or Marc-145 cells were grown in 6/12-well plates with 80% confluence, the different doze recombinant plasmids were separately transfected or co-transfected into the cells via transfection reagents polyethylenimine (PEI) or Lipofectamine 2000. Two small interfering RNAs (siRNAs) targeting THAP11 gene coding sequence (CDS) region was designed and ordered by SYNBIO Technologies, Suzhou, China (Supplemental Table S1 ). siTHAP11-1 and siTHAP11-2 or negative control (siNC) was respectively transfected into Marc-145 cells using the Lipofectamine 2000 transfection reagent. At 24 hpt, cells were harvested for the analysis of THAP11-knockdown efficiency through real-time PCR and western blot. Functional enrichment analysis of interacting protein GO enrichment and KEGG pathway enrichment analysis against screening interacted proteins were conducted using online the Metascape website ( https://metascape.org/gp/index.html ), and visualization of enriched data was analyzed via bioinformatics website ( https://www.bioinformatics.com.cn/ ). Statistical analysis In this study, assays were conducted with at least three independent replications as per the instruction manual. The qPCR experiments were performed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher, USA), and data were analyzed using GraphPad Prism software (version 8.0). Statistical analyses were carried out using Student's t-test and one-way ANOVA. A difference with p-values less than 0.05 was considered statistically significant. Results PRRSV Nsp1β does not exhibit self-activation in Y2H screening assays Nsp1β serves as a critical interferon inhibitor during PRRSV infection. To identify host factors interacting with Nsp1β, the full-length sequence of Nsp1β was inserted into the yeast library bait vector pGBKT7. The pGBKT7-Nsp1β plasmid was co-transformed with the pGADT7-T plasmid into Y2H Gold cells and cultured on selective media with varying nutritional deficiencies. The co-transformed cells produced only a few clones on the TDO selective medium supplemented with X-α-gal (TDO/X), but no clones were observed on the QDO/X medium supplemented with AbA (QDO/X/A) (Fig. 1 A). Under these conditions, Y2H screening assays using pGBKT7-Nsp1β as bait were performed on the most stringent QDO/X/A medium, which presents the highest level of nutritional deficiency (Fig. 1 B). Thirty-four potential host proteins interacting with Nsp1β were identified To screen for host proteins that interact strongly with Nsp1β, bait plasmids and prey (cDNA library) were co-transformed into Y2H Gold cells, which were then cultured on the stringent TDO/X medium plates. A total of 74 clones grew on the QDO/X/A medium plates, which were further tested for interaction. Positive transformants were identified through sequencing, revealing 34 potential binding partners of Nsp1β, as listed in Table 1 , including THAP11, proteasome subunit beta type-10 (PSMB10), poly(rC)-binding protein 1 (PCBP1), thioredoxin-interacting protein (TXNIP), and cathepsin D (CSTD). Table 1 Information for potential host factors interacting with PRRSV Nsp1β No ID Name Gene symbol 1 NP_001038079.1 thioredoxin-interacting protein TXNIP 2 NP_001038030.1 proteasome subunit beta type-10 PSMB10 3 AAY42145.2 cathepsin D CTSD 4 XP_020922421.1 probable ATP-dependent RNA helicase DDX5 isoform X3 DDX5 5 XP_013843801.1 L-lactate dehydrogenase B chain isoform X1 LDHB 6 XP_005655974.2 persulfide dioxygenase ETHE1, mitochondrial ETHE1 7 NP_001229990.1 alpha-actinin-1 ACTN1 8 XP_003125105.1 poly(rC)-binding protein 1 PCBP1 9 NP_001090927.1 cathepsin B precursor CTSB 10 NP_001233111.1 THAP domain-containing protein 11 THAP11 11 XP_003124995.1 macrophage-capping protein CAPG 12 XP_003127414.1 ER membrane protein complex subunit 10 isoform X1 EMC10 13 XM_021090073.1 sortilin 1, transcript variant X4, mRNA SORT1 14 XP_013854598.1 folate receptor 1 isoform X2 FOLR1 15 XP_020936382.1 filamin-A isoform X1 FLNA 16 NP_001072147.1 CD81 molecule CD81 17 XP_020922821.1 acyl-CoA synthetase family member 2, mitochondrial isoform X2 ACSF2 18 ABC17921.1 MHC class I antigen SLA 19 XP_003123039.1 ATP synthase subunit delta, mitochondrial ATP5D 20 NP_001278705.1 macrosialin precursor CD68 21 XP_020936879.1 apical endosomal glycoprotein isoform X7 MAMDC7 22 XP_005660860.1 ferritin heavy chain isoform X1 FTH1 23 XP_005669190.1 myb-binding protein 1A isoform X2 MYBBP1A 24 XP_001927370.3 beta-1,4-galactosyltransferase 3 B4GALT3 25 NP_001027548.1 hypoxanthine-guanine phosphoribosyltransferase HPRT1 26 XP_020936398.1 filamin-A isoform X11 FLNA 27 NP_999066.1 propionyl-CoA carboxylase beta chain, mitochondrial precursor PCCB 28 XP_001929153.1 TOX high mobility group box family member 4 TOX4 29 XP_020940051.1 docking protein 3 DOK3 30 XP_020942955.1 SH2 domain-containing protein 6 SH2D6 31 XM_005660642.3 lysine acetyltransferase 5 KAT5 32 XP_020939148.1 microtubule-associated serine/threonine-protein kinase 3 MAST3 33 XM_021080945.1 putative GTP-binding protein 6 GTPBP6 34 NP_001230636.1 cytosolic non-specific dipeptidase 2 CNDP2 GO enrichment and KEGG pathway enrichment analysis To investigate the role of Nsp1β in regulating host cell metabolism, immune responses, and self-replication processes through interactions with host factors, Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the 34 identified interacting proteins. GO cellular component analysis revealed enrichment in the lytic vacuole, mitochondrial matrix, and melanosome (Fig. 2 A). GO molecular function analysis identified that proteins interacting with Nsp1β were associated with cell adhesion molecule binding, ligase activity, and integrin binding, while some were involved in regulating DNA binding, receptor activity, and enzyme activity (Fig. 2 B). GO biological process analysis indicated enrichment in the enzyme-linked receptor protein signaling pathway, cellular metabolism, regulation of biosynthetic processes, and antiviral immune responses (Fig. 2 C). KEGG pathway analysis highlighted that the interacting host proteins were primarily involved in cell growth and death, transport and catabolism, the host immune system, and signal transduction (Fig. 2 D). In conclusion, PRRSV Nsp1β may evade host immune responses by interacting with various cellular signaling molecules and pathways. Co-localization of Nsp1β with multiple host proteins To better visualize the interaction between Nsp1β and PCBP1, TXNIP, and CSTD in vivo, the full-length sequence of Nsp1β was inserted into the pCAGGS expression vector, with a C-terminal fusion to the mCherry tag. The open reading frames of PCBP1, TXNIP, and CSTD were separately cloned into the pcDNA3.1 vector, with a C-terminal fusion to the Myc tag. At 24 hpt of Nsp1β-mCherry with host proteins, and using pcDNA3.1-Myc as a negative control, an indirect IFA was performed to assess the co-localization between Nsp1β and the selected host proteins. Confocal fluorescence microscopy was used to observe the subcellular localization patterns. The results showed that PRRSV Nsp1β was predominantly expressed in the nucleus. Co-transfection of Nsp1β-mCherry with each of the four host proteins resulted in merged yellow fluorescence, indicating co-localization, while co-transfection of the empty vector with Nsp1β-mCherry exhibited only single fluorescence (Fig. 3 A). These findings suggest that Nsp1β co-localizes with the selected host proteins. THAP domain-containing protein 11 interacts with Nsp1β in vivo and in vitro Among the screened proteins, we selected genes with a nucleotide length of less than 2000 bp as potential study targets. In the yeast assay, strong interaction between Nsp1β and THAP11 was observed on QDO selective medium supplemented with X-α-gal and AbA (QDO/X/A) (Fig. 4 A). Next, the full-length THAP11 sequence from Chlorocebus sabaeus was cloned into the pcDNA3.1 expression vector, fused with a C-terminal Myc tag. The Nsp1β sequence from the classical PRRSV CH-1a strain was constructed into the pCAGGS-Myc expression vector, with a C-terminal fusion to the mCherry tag. Co-transfection of THAP11-Myc and Nsp1β-mCherry into HEK293T cells for 24 h was followed by incubation with either an mCherry antibody or a control IgG antibody. Co-IP assay results showed that THAP11 could be detected in the immunoprecipitate (Fig. 4 B), indicating an interaction between THAP11 and Nsp1β. Conversely, anti-Myc antibody also detected strong Nsp1β-mCherry protein in the immunoprecipitate (Fig. 4 C). Furthermore, endogenous THAP11 could be immunoprecipitated with Nsp1β in PRRSV-infected Marc-145 cells using anti-THAP11 antibody in a Co-IP assay (Fig. 4 D). Confocal fluorescence microscopy revealed overlapping yellow fluorescence in the cell nucleus when Nsp1β and THAP11 were co-expressed in HEK293T cells, demonstrating co-localization of Nsp1β with THAP11 (Fig. 4 E). Collectively, these results confirm that Nsp1β interacts with THAP11 both in vivo and in vitro. THAP11 is a transcriptional regulator containing a THAP (THAP-domain) domain. According to the AlphaFold3 prediction, the structure of THAP11 displays the characteristic THAP domain, which exhibits highly conserved secondary structural features, including β-sheets and α-helices, consistent with known structures of THAP family proteins. The protein features a tightly folded core, which is likely crucial for its roles in DNA and RNA binding, as well as transcriptional regulation (Fig. 4 F). Nsp1β is a key protein of PRRSV, known for its function in disrupting host immune responses. The AlphaFold3 prediction indicates that Nsp1β adopts a highly folded structure, containing multiple conserved α-helices and β-sheets (Fig. 4 G). Through AlphaFold3 simulation of their interaction, the results suggest that THAP11 and Nsp1β likely bind through a network of hydrogen bonds and hydrophobic interactions, as indicated by a negative change in Gibbs free energy (∆iG < 0). The THAP domain of THAP11 interacts with specific regions of Nsp1β, forming a stable binding interface (Fig. 4 H), which could potentially influence the immune evasion mechanisms of PRRSV. The predicted interaction structure provides molecular-level insights into the THAP11-Nsp1β binding, contributing to a better understanding of the role of THAP11 in PRRSV infection and its impact on viral immune evasion. THAP11 negatively regulates PRRSV replication To investigate the relationship between THAP11 and PRRSV and to understand the role of THAP11 during PRRSV infection, we first analyzed endogenous THAP11 expression in PRRSV-infected Marc-145 cells at 0, 6, 12, 24, 36 hours post-infection (hpi) with a multiplicity of infection (MOI) of 0.5. The results showed that the accumulation of THAP11 protein did not significantly change at different time points following PRRSV infection (Fig. 5 A). Next, Marc-145 cells were transfected with either THAP11-Myc or an empty pcDNA3.1 vector for 12 h, after which PRRSV at MOI of 0.5 was inoculated into the cells for 12, 24, and 36 h. The accumulation of the PRRSV N protein in the THAP11-Myc-expressing group was significantly lower than in the control group (Fig. 5 B), indicating that exogenous expression of THAP11 inhibits PRRSV replication. To further confirm the function of THAP11 during PRRSV infection, we performed siRNA-mediated RNA interference to knock down endogenous THAP11 expression. Two siRNAs targeting the THAP11 coding region (siTHAP11-1 and siTHAP11-2) were transfected into Marc-145 cells, with siNC (non-targeting control) as a negative control. At 12 hpt, PRRSV was added to the cells and cultured for an additional 36 h. The expression of THAP11 was significantly reduced in the siTHAP11 groups compared to the siNC control, and the accumulation of the PRRSV ORF7 gene was upregulated more than twofold (Fig. 5 C). Additionally, THAP11 protein levels were lower in the siRNA-silenced cells compared to the siNC control, while the accumulation of the PRRSV N protein was significantly higher in the siTHAP11 groups (Fig. 5 D), demonstrating that knockdown of THAP11 facilitates PRRSV replication. Furthermore, IFA revealed that overexpression of THAP11 markedly reduced the fluorescence accumulation of the PRRSV N protein compared to cells expressing the empty vector, at both 12 and 24 hpi (Fig. 6 A & B). In conclusion, these results suggest that THAP11 negatively regulates PRRSV replication. THAP11 degrades Nsp1β via the ubiquitin-proteasome system THAP11 has been reported to interact with PCBP1 to regulate transcription, suggesting that THAP11 may be a multifunctional protein. To explore the biological significance of its interactions, we constructed a plasmid pCAGGS-Nsp1β-HA, in which Nsp1β was fused with a C-terminal HA tag. Co-transfection of Nsp1β-HA and dose-independent THAP11-Myc plasmids into HEK293T cells was performed. Protein analysis revealed that the accumulation of Nsp1β gradually decreased as THAP11 expression increased (Fig. 7 A). To clarify the degradation pathway, HEK293T cells pre-transfected with Nsp1β-HA and THAP11-Myc plasmids for 12 h were treated with the proteasome inhibitor MG132, the autophagy inhibitor 3-MA, or BafA1, with DMSO as the control. Western blot analysis showed that only the MG132-treated group retained Nsp1β protein accumulation, indicating that MG132 inhibited Nsp1β degradation (Fig. 7 B). This suggests that THAP11 promotes Nsp1β degradation through the proteasome pathway. Since proteasomal degradation requires ubiquitination, we next investigated whether Nsp1β could be modified by ubiquitination. HEK293T cells were transfected with either Nsp1β-mCherry alone or in combination with Ub-HA for 24 h. Co-immunoprecipitation (Co-IP) assays analyzed the ubiquitination levels of Nsp1β. Immunoprecipitation with a mCherry antibody revealed a distinct ubiquitinated band in the co-transfection group, while the single transfection did not show any modified bands (Fig. 7 C), confirming that Nsp1β can be ubiquitinated. To determine if THAP11 promotes Nsp1β ubiquitination and degradation, HEK293T cells were co-transfected with Nsp1β-mCherry, Ub-HA, and either an empty pcDNA3.1 vector or THAP11-Myc plasmids. At 24 hpt, Co-IP results showed that the ubiquitination levels of Nsp1β were significantly increased in cells expressing THAP11-Myc compared to the control group (Fig. 7 D). Additionally, the ubiquitination levels increased in a dose-dependent manner (Fig. 7 E). Collectively, these results indicate that THAP11 enhances the ubiquitination of Nsp1β, leading to its degradation via the proteasome. THAP11 promotes K48 and K63 conjugated ubiquitination of Nsp1β for degradation To further elucidate the type of ubiquitin conjugation involved in THAP11-mediated Nsp1β degradation, we focused on the ubiquitin-proteasome system, where K48 and K63-linked ubiquitin chains are generally associated with the degradation of misfolded proteins or pathogens. We constructed ubiquitin mutants retaining either the K48 or K63 lysine site (K48 and K63), as well as mutants where K48 or K63 were replaced with arginine (K48R or K63R), all in an HA-tagged vector. Co-transfection of Nsp1β-mCherry, Ub(K48)-HA, and THAP11-Myc (or empty vector), as well as Nsp1β-mCherry, Ub(K63)-HA, and THAP11-Myc (or empty vector), into HEK293T cells was performed. Co-immunoprecipitation (Co-IP) analysis revealed that Nsp1β underwent significant ubiquitination modification when either the K48 or K63 lysine sites were retained in the ubiquitin molecule (Fig. 8 A). Moreover, the expression of THAP11 further enhanced the levels of K48 and K63 ubiquitination of Nsp1β (Fig. 8 B). These findings suggest that THAP11 promotes both K48- and K63-conjugated ubiquitination of Nsp1β, leading to its degradation. Discussion This study used a yeast two-hybrid system to screen for host proteins interacting with PRRSV Nsp1β, identifying 34 potential interacting proteins. Among these, THAP11 showed a strong interaction with Nsp1β and was selected for further investigation. Overexpression of THAP11 significantly reduced PRRSV N protein accumulation, demonstrating antiviral activity, while knockdown of THAP11 enhanced PRRSV replication. Importantly, THAP11 facilitated the degradation of Nsp1β via increasing K48- and K63-linked ubiquitination, thereby inhibiting PRRSV replication. THAP11, a transcriptional regulator, may modulate host immune responses through its interaction with PRRSV Nsp1β. By reducing Nsp1β accumulation, overexpression of THAP11 weakens the virus's immune evasion ability and limits its replication. Conversely, THAP11 knockdown promotes Nsp1β accumulation, facilitating viral replication. Additionally, THAP11 mediates Nsp1β degradation via the ubiquitin-proteasome pathway, elucidating THAP11's regulatory role during PRRSV infection. This study is the first to reveal the interaction between THAP11 and PRRSV Nsp1β, as well as its potential antiviral role. Previous studies have reported interactions between THAP11 and other host factors like PCBP1 and TXNIP, which play critical roles in transcription and cellular responses. However, the relationship between THAP11 and PRRSV has not been widely explored. This study fills this gap, suggesting that THAP11 may be involved in the host antiviral response through the ubiquitin-mediated degradation of Nsp1β. While this study identifies the potential antiviral role of THAP11, several limitations exist. First, the detailed mechanism by which THAP11 regulates Nsp1β degradation through ubiquitination remains to be fully elucidated and requires further experimental validation. Second, although the results observed in HEK293T cells are promising, whether these findings translate to in vivo pig models remains uncertain. Additionally, the roles of PCBP1, TXNIP, PSMB10, and CTSD in PRRSV infection need further confirmation through additional experiments. Among the identified interacting proteins, PCBP1 has been shown to interact with PRRSV Nsp1β and plays a key role in the lifecycle of several other viruses [ 38 – 42 ], indicating its potential importance in viral replication and immune modulation. In the context of Hepatitis C virus (HCV) infection, PCBP1 is involved in stabilizing viral RNA and promoting its replication. Research has demonstrated that PCBP1 binds to the 5' untranslated region (UTR) of the HCV RNA, stabilizing the RNA and facilitating its replication [ 40 ]. Similarly, PCBP1 has been implicated in regulating the translation of the SV40 large T antigen [ 43 ], further supporting its role as a multifunctional factor in viral processes. The influence of PCBP1 on viral RNA stability and replication is likely critical to the replication of many viruses. In the case of PRRSV, PCBP1 may influence the stability and transcription of viral RNA, potentially enhancing viral replication. Given that PRRSV relies on host cell machinery for RNA synthesis and stability, PCBP1's interactions with the viral genome could play a pivotal role in facilitating these processes. Future studies should explore the specific interactions between PCBP1 and the PRRSV genome to fully understand its contribution to viral replication. Understanding how PCBP1 regulates viral RNA could open new avenues for therapeutic intervention, particularly in modulating host-virus interactions to limit viral replication. TXNIP has been studied extensively for its involvement in oxidative stress, inflammation, and immune-related pathways [ 44 ]. While direct evidence linking TXNIP to PRRSV is scarce, its broader role in viral infections and immune responses points to its potential relevance in PRRSV pathogenesis. TXNIP is known to regulate the NLRP3 inflammasome, which plays a pivotal role in triggering inflammatory pathways and pyroptosis under oxidative stress conditions [ 45 ]. This inflammasome activation could impact viral infections by modulating immune responses, including those seen in PRRSV infections. TXNIP has also been implicated in apoptosis regulation and redox balance, both of which influence viral replication and immune evasion [ 44 ]. TXNIP has been shown to regulate the cellular response during COVID-19 infection, where it inhibits oxidative stress in T cells, facilitating viral spread. TXNIP's role in viral replication appears to be closely linked to its ability to modulate cellular redox states [ 46 ]. By maintaining the redox balance, TXNIP could create a more favorable environment for viral replication and immune evasion [ 47 ]. These insights suggest that TXNIP may help PRRSV manipulate host immune responses, offering an interesting avenue for future research on host-virus interactions. Further investigation is warranted to determine whether PRRSV influences TXNIP expression or activity, potentially contributing to its pathogenicity. PSMB10, also known as MECL-1, is an integral component of the immunoproteasome, which plays a crucial role in antigen processing for MHC class I presentation [ 48 ]. Induced by interferon-gamma, PSMB10 replaces constitutive proteasome subunits in immune cells and assists in the degradation of viral proteins for immune recognition. This process is essential for the immune response during viral infections, and the immunoproteasome, including PSMB10, is involved in modulating pathways like NF-κB signaling [ 49 ]. In viral infections such as classical swine fever virus, the proteasome, and specifically PSMB10, processes viral proteins, promoting their recognition by the immune system [ 50 ]. This suggests that PSMB10 may play a role in PRRSV infection, potentially contributing to the degradation of viral proteins and enhancing immune system recognition. CTSD, an aspartic protease located primarily in lysosomes, plays a significant role in various viral infections by aiding different stages of the viral lifecycle. In viral contexts, CTSD facilitates the processing of viral proteins, promotes viral entry, and triggers immune responses, such as antigen presentation [ 51 ]. For instance, CTSD has been shown to activate viral glycoproteins after endocytosis, facilitating viral fusion with the host cell membrane [ 52 , 53 ]. In infections like SARS-CoV-2, CTSD, along with other cathepsins such as B and L, is essential for cleaving the viral spike protein, which is crucial for viral entry into the host cell [ 54 ]. This highlights the importance of CTSD in viral infections and suggests that it may play a similar role in PRRSV, potentially influencing viral entry and immune modulation. By expanding the understanding of how proteins like TXNIP, PSMB10, and CTSD interact with viral proteins like Nsp1β, this research opens up new possibilities for exploring host-virus interactions in PRRSV and other viral infections. Further investigation into these pathways could lead to the development of targeted therapeutic strategies to modulate these interactions and combat PRRSV infections more effectively. This study reveals, for the first time, that THAP11 inhibits PRRSV replication by interacting with and degrading Nsp1β via the ubiquitin-proteasome pathway. By revealing the interaction between THAP11 and Nsp1β, Future studies will focus on exploring THAP11’s role in other viral infections and whether it can be considered a broad-spectrum antiviral factor. Furthermore, given the interactions between PCBP1, TXNIP, and CTSD with PRRSV in this study, future work will further investigate these proteins' roles in PRRSV infection and evaluate their potential as antiviral targets. Declarations Funding sources This work was supported by the National Key Research and Development Program of China (2023YFD1801500), the Basic and Applied Basic Research Foundation of Guangdong Province (2024A1515012991), the Science and Technology Planning Project of Guangzhou (2023B03J0947 and 2025D04J0072), and the Laboratory of Lingnan Modern Agriculture Project (NG2022003). 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Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYJACZiBO4AcSBx7AhHiI0SLZANSSQJIWgwMgkhgt8hHJBz8Xtt3JM752+CHQlrrE+TMSGB+8bWOQN8ehxfBGWrL0zLZnxWa30wyAWg4nbriRwGw4t43BcGcDDi0zcsyYedsOJ267nQDSciBxg0QCmzRvG9Sp+LRsnp3+AeYw9t/4tMhLQLVskM4B2cKc2HAjgY0ZnxYDnmfJ0jznDifOuJ1TcCDB4LDxhjMPmyXnnJMw3IDLlnZgiPGUHU7sn52++cOHijrZ+UCRD2/KbORx2oIqbsDg2MDA2ABkSWBXD7KlAU3AHqfSUTAKRsEoGLEAAE0aYf3C8mniAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7859-1985","institution":"South China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Chunhe","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2025-02-26 14:50:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6114224/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6114224/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05760-3","type":"published","date":"2025-06-23T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77869684,"identity":"389e7c42-7ac9-40fa-a7ad-bf01a70d053b","added_by":"auto","created_at":"2025-03-06 10:04:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1473715,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of self-activation against PRRSV Nsp1β protein in Y2H screening assays. \u003cstrong\u003e(A)\u003c/strong\u003e The pGBKT7-Nsp1β and pGADT7-T plasmids were co-transformed into Y2H Gold cells, which were then plated onto deficient culture media lacking Leu and Trp (DDO/X), Leu, Trp, and His (TDO/X), and Leu, Trp, His, and Ade (QDO/X/A), supplemented with X-α-gal and AbA. \u003cstrong\u003e(B)\u003c/strong\u003e Potential positive clones interacting with Nsp1β were screened on QDO/X/A medium. Blue colonies indicate a positive interaction, with \"+\" representing a positive result and \"−\" indicating a negative result.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/d21a7bef5bb2e4a4dbba6c13.png"},{"id":77870761,"identity":"17ed791a-b422-4eda-b9dc-20c923a9ed53","added_by":"auto","created_at":"2025-03-06 10:12:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":848012,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG enrichment analysis. \u003cstrong\u003e(A)\u003c/strong\u003e GO molecular function analysis of proteins interacting with Nsp1β, as identified in the bait screening. This analysis highlights the molecular functions associated with the interaction partners. \u003cstrong\u003e(B)\u003c/strong\u003e GO biological process analysis of the proteins interacting with Nsp1β, providing insights into the biological processes they are involved in. \u003cstrong\u003e(C)\u003c/strong\u003e GO cellular component analysis of the interacting proteins from Nsp1β bait screening, revealing the cellular locations where these proteins are likely to function. \u003cstrong\u003e(D)\u003c/strong\u003e KEGG pathway analysis of the interacting proteins from Nsp1β bait screening, outlining the key signaling pathways and processes these proteins may participate in.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/271729604a3fd2991b39a4c2.png"},{"id":77869686,"identity":"969bbb90-35a8-4692-a520-09a3b6dfa6dd","added_by":"auto","created_at":"2025-03-06 10:04:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1853904,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal fluorescence microscopy confirms colocalization of Nsp1β with host proteins PCBP1, TXNIP, and CTSD. \u003cstrong\u003e(A)\u003c/strong\u003e HEK293T cells were co-transfected with mPCBP1-Myc, mTXNIP-Myc, or mCTSD-Myc along with Nsp1β-mCherry plasmids. Myc empty vector and Nsp1β-mCherry were used as controls. At 24 hpt, cells were subjected to indirect immunofluorescence analysis and visualized using confocal microscopy. Scale bars, indicated by white lines, represent the magnification. The \"m\" notation indicates that the species is monkey.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/96d318115ea487d97907f38f.png"},{"id":77869687,"identity":"52ffc699-0e98-4be8-b6a4-868cab49c24b","added_by":"auto","created_at":"2025-03-06 10:04:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1658326,"visible":true,"origin":"","legend":"\u003cp\u003eNsp1β interacts with THAP11. \u003cstrong\u003e(A)\u003c/strong\u003e Y2H assays confirmed the interaction between Nsp1β and THAP11. The pGBKT7-Nsp1β and pGADT7-THAP11 plasmids, or an empty vector, were co-transformed into Y2H Gold cells. pGBKT7-53 + pGADT7-T served as a positive control, and pGBKT7-Lam + pGADT7-T was used as a negative control. The transformed cells were grown on DDO and QDO/X/A media, with serial dilutions applied to assess the interaction. \u003cstrong\u003e(B)\u003c/strong\u003e Co-IP assays confirmed that Nsp1β interacts with THAP11. Anti-mCherry or anti-IgG antibodies were conjugated to magnetic A beads, and Myc-tagged proteins were detected in the immunoprecipitate. \u003cstrong\u003e(C)\u003c/strong\u003e Co-IP using anti-Myc antibodies conjugated to magnetic A beads detected Nsp1β-mCherry protein in the immunoprecipitate, further supporting the interaction between Nsp1β and THAP11. \u003cstrong\u003e(D)\u003c/strong\u003e Co-IP assays confirmed the interaction between endogenous THAP11 and Nsp1β in PRRSV-infected Marc-145 cells. Anti-THAP11 antibodies linked to magnetic A beads were used to detect Nsp1β in the immunoprecipitate. \u003cstrong\u003e(E)\u003c/strong\u003eCo-transfection of Nsp1β-mCherry and THAP11-Myc into HEK293T cells for 24 hours showed colocalization of the two proteins, as observed by confocal fluorescence microscopy. \u003cstrong\u003e(F)\u003c/strong\u003e The predicted spatial structure of monkey THAP11 protein, as determined by the AlphaFold3 server, reveals its folding pattern and key structural features. \u003cstrong\u003e(G)\u003c/strong\u003e The predicted spatial structure of Nsp1β, also obtained through AlphaFold3, shows the structural arrangement of this viral protein involved in immune modulation. \u003cstrong\u003e(H)\u003c/strong\u003e The predicted binding interface between Nsp1β and THAP11, as modeled by AlphaFold3, illustrates the potential interaction site, highlighting how these two proteins might interact at the molecular level.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/2dadf8978d35ef35719f407d.png"},{"id":77869691,"identity":"9bb51115-2e7b-4c36-bcc9-59e5eb51b7cc","added_by":"auto","created_at":"2025-03-06 10:04:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":412435,"visible":true,"origin":"","legend":"\u003cp\u003eTHAP11 negatively regulates PRRSV infection. \u003cstrong\u003e(A)\u003c/strong\u003eWestern blot analysis detected endogenous THAP11 protein levels at different time points (0, 6, 12, 24, and 36 h) following PRRSV infection, revealing that THAP11 protein accumulation remained relatively stable throughout the infection.\u003cstrong\u003e (B)\u003c/strong\u003e Marc-145 cells were transfected with either 2 μg of THAP11-Myc or the pcDNA3.1 plasmid as a control. At 12 hpt, cells were infected with PRRSV at a MOI of 0.5 for 12, 24, and 36 h. The accumulation of PRRSV N protein was assessed by western blot, showing reduced PRRSV replication in THAP11-Myc expressing cells compared to controls. \u003cstrong\u003e(C \u0026amp; D)\u003c/strong\u003e Knockdown of THAP11 enhanced PRRSV replication. Two siRNAs targeting THAP11 or a negative control siRNA were transfected into Marc-145 cells cultured in 12-well plates. At 12 hpt, cells were infected with PRRSV for 36 hours. The mRNA expression levels of THAP11 and the PRRSV ORF7 gene were measured by qPCR, and protein accumulation was analyzed by western blot.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/80db67d9e5673836afce5022.png"},{"id":77869692,"identity":"0a8dde3e-f851-40a4-a241-f780f7edaa1f","added_by":"auto","created_at":"2025-03-06 10:04:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2366956,"visible":true,"origin":"","legend":"\u003cp\u003eIFA assays confirm THAP11 suppresses PRRSV replication. \u003cstrong\u003e(A \u0026amp; B)\u003c/strong\u003e Marc-145 cells were transfected with either THAP11-Myc or the pcDNA3.1 empty vector for 12 h, followed by PRRSV infection for 24 or 36 h. After the infection period, cells were incubated overnight with primary antibodies: anti-N and anti-Myc. The anti-mouse 488 fluorescent secondary antibody was used to label the N protein, while the anti-Rabbit 555 fluorescent secondary antibody detected THAP11. Fluorescence images were captured using a Leica fluorescence microscope (Nikon, Germany), demonstrating that THAP11 expression reduced PRRSV replication as indicated by decreased N protein fluorescence.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/072e6f786cdfc0cf38ff9f34.png"},{"id":77870762,"identity":"69aef252-b9cd-414b-a531-8d41ee4bbded","added_by":"auto","created_at":"2025-03-06 10:12:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":913327,"visible":true,"origin":"","legend":"\u003cp\u003eTHAP11 promotes Nsp1β ubiquitination and degradation via the ubiquitin-proteasome pathway. \u003cstrong\u003e(A)\u003c/strong\u003e HEK293T cells were co-transfected with Nsp1β-HA and varying doses (0, 1.0, 1.5 μg) of THAP11-Myc plasmids. At 24 hpt, the accumulation of Nsp1β protein was analyzed by western blot, showing dose-dependent regulation by THAP11. \u003cstrong\u003e(B)\u003c/strong\u003e A total of 2.0 μg of Nsp1β-HA and THAP11-Myc or empty vector were co-transfected into HEK293T cells for 24 h. At 10 hpt, cells were treated with the proteasome inhibitor MG132, the autophagy inhibitors 3-MA and BafA1, or DMSO as a control. Western blot analysis detected accumulations of Nsp1β for 36 h. \u003cstrong\u003e(C)\u003c/strong\u003eHEK293T cells were co-transfected with Nsp1β-mCherry and Ub-HA plasmids, or with Nsp1β-mCherry or Ub-HA plasmids alone as controls. At 24 hpt, ubiquitination levels of Nsp1β were detected by Co-IP assays using anti-HA antibody. mCherry antibody was used to link to magnetic A beads for immunoprecipitation, confirming the ubiquitination of Nsp1β. \u003cstrong\u003e(D)\u003c/strong\u003e HEK293T cells were co-transfected with Nsp1β-mCherry, Ub-HA, and THAP11-Myc or pcDNA3.1 plasmids. At 24 hpi, western blot analysis showed increased ubiquitination of Nsp1β in the presence of THAP11, confirming its role in promoting Nsp1β ubiquitination. \u003cstrong\u003e(E)\u003c/strong\u003e HEK293T cells were co-transfected with Nsp1β-mCherry, Ub-HA, and dose-dependent THAP11-Myc plasmids. At 24 hpi, western blot analysis detected the ubiquitination levels of Nsp1β.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/4603db392a66b38c723c6d66.png"},{"id":77869689,"identity":"6e93c5b3-25ef-4978-8895-d0f038e6ae36","added_by":"auto","created_at":"2025-03-06 10:04:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":617176,"visible":true,"origin":"","legend":"\u003cp\u003eTHAP11 mediates Nsp1β degradation dependent on K48 and K63 ubiquitination. \u003cstrong\u003e(A)\u003c/strong\u003e HEK293T cells were co-transfected with Nsp1β-mCherry, THAP11-Myc, and mutant Ub (K48, K48R, K63, K63R) plasmids. At 24 hpt, western blot analysis detected Nsp1β ubiquitination levels, anti-mCherry was used in Co-IP assays. \u003cstrong\u003e(B)\u003c/strong\u003e HEK293T cells were co-transfected with Nsp1β-mCherry, Ub(K48)-HA and THAP11-Myc or pcDNA3.1 plasmids, or co-transfected with Nsp1β-mCherry, Ub(K63)-HA and THAP11-Myc or pcDNA3.1 plasmids. At 24 hpt, ubiquitination levels of Nsp1β were detected by Co-IP assays using anti-HA antibody. mCherry antibody was used to couple to magnetic A beads for immunoprecipitation.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/fd8f4919648c1f5e8a2248bd.png"},{"id":85686181,"identity":"39b1eabe-cc5e-4c7b-b2d6-2ecc61bad786","added_by":"auto","created_at":"2025-06-30 16:04:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10504288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/27e12ff6-5ba8-4e66-b64f-548113d59d69.pdf"},{"id":77870766,"identity":"e9b3d9ed-7e1b-4100-ba32-c0b7040e2dd7","added_by":"auto","created_at":"2025-03-06 10:12:32","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":17812,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6114224/v1/28faf5809b95c5f7ed3b8d0c.docx"}],"financialInterests":"","formattedTitle":"THAP11-mediated K48- and K63-linked ubiquitination is essential for the degradation of porcine reproductive and respiratory syndrome virus nonstructural protein 1β","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePorcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases in the global pig industry, leading to significant economic losses worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), which severely affects the reproductive health of sows and the respiratory health of piglets [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease results in reduced productivity and substantial economic burdens on pig farming. PRRSV exhibits rapid mutation and high genetic variability, which makes it particularly difficult to control with existing vaccines and antiviral therapies [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, new research aimed at identifying critical host-virus interactions and discovering novel antiviral targets is essential for improving PRRS management and treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePRRSV is a single-stranded, positive-sense RNA virus, approximately 15 kilobases (kb) in length. It belongs to the family \u003cem\u003eArteriviridae\u003c/em\u003e, containing 10\u0026ndash;12 open reading frames (ORFs), encoding several nonstructural (Nsp1-Nsp12) and structural proteins (GP2a, E, GP3, GP4, GP5, M and N) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. PRRSV is known to inhibit type I interferon responses through its nonstructural proteins (Nsp1α, Nsp1β, Nsp2, Nsp4 and Nsp11) [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nonstructural protein 1β (Nsp1β) is one of the key nonstructural proteins encoded by the PRRSV. As an essential viral protein, Nsp1β plays a critical role in the pathogenesis of PRRSV by modulating the host's immune responses and assisting the virus in evading immune detection [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The protein is encoded by the PRRSV genome as part of the viral replicase complex, which is responsible for viral RNA replication and transcription [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe functional analysis of Nsp1β has primarily focused on its immune evasion mechanisms, as this is a critical factor in PRRSV pathogenesis. Early studies demonstrated that Nsp1β interferes with interferon signaling by preventing the activation of the interferon regulatory factor 3 (IRF3) pathway [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This inhibits the production of type I interferons, which are typically induced upon viral infection as part of the host\u0026rsquo;s early antiviral responses. Further studies have shown that Nsp1β may also directly interact with other components of the innate immune system. For example, Nsp1β has been reported to interact with the host\u0026rsquo;s cellular RNA sensing pathways, preventing the host from detecting viral RNA effectively [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It inhibits the JAK-STAT pathway by preventing the nuclear translocation of STAT1 and STAT2, which are essential for initiating the interferon (IFN) responses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This blockade significantly hinders the host\u0026rsquo;s ability to mount an effective immune defense, allowing the virus to evade immune recognition. Furthermore, Nsp1β binds to nucleoporin 62 (Nup62), a component of the nuclear pore complex, to block the nuclear export of antiviral mRNA and proteins. This action impairs the host\u0026rsquo;s capacity to produce antiviral proteins, further contributing to the virus\u0026rsquo;s ability to replicate unchecked [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, Nsp1β stabilizes its own protein form by interacting with host deubiquitinating enzymes, such as USP1, preventing its degradation through the ubiquitin-proteasome pathway [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This stabilization not only preserves Nsp1β\u0026rsquo;s ability to suppress immune responses but also promotes viral replication. Nsp1β also stabilizes hypoxia-inducible factor 1 alpha (HIF-1α), a key regulator of inflammation, through deubiquitination. This process enhances viral replication by promoting inflammation and modulating immune responses to favor the virus\u0026rsquo;s survival [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, Nsp1β inhibits NLRP3 inflammasome activation, a crucial player in triggering inflammatory responses. This inhibition further suppresses the host\u0026rsquo;s immune responses, facilitating viral persistence [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Nsp1β also interacts with host proteins like DEAD-box RNA helicase DDX21 and GTPase-activating protein SH3 domain\u0026ndash;binding protein 1 (G3BP1), which regulate viral replication and stress granule dynamics [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These interactions help modulate the host cellular environment to favor viral replication while avoiding immune detection. Lastly, Nsp1β induces the degradation of karyopherin α1 (KPNA1), a key molecule in the nuclear import of immune signaling complexes like Interferon-stimulated gene factor 3 (ISGF3) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. By disrupting the nuclear translocation of these complexes, Nsp1β effectively inhibits the host immune responses, further enhancing viral replication. In summary, Nsp1β uses multiple mechanisms, including the disruption of immune signaling pathways, the stabilization of key host factors, and interference with antiviral mRNA export, to create a favorable environment for PRRSV replication and immune evasion.\u003c/p\u003e \u003cp\u003eThanatos-associated protein 11 (THAP11) is a transcriptional regulator containing a highly conserved THAP domain [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. THAP11 has been implicated in regulating apoptosis, oxidative stress, and immune responses, making it a potential factor in viral infections. The THAP domain is found in a variety of proteins involved in cellular stress responses and DNA repair, and THAP11 itself is known to interact with key host proteins to control cellular processes such as cell survival and inflammation [\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. While THAP11's role in cellular processes is well-documented, its involvement in viral infections, particularly in PRRSV infection, has not been thoroughly studied. Given THAP11\u0026rsquo;s ability to modulate immune responses and its role in cellular stress pathways, it is possible that it plays a role in the host\u0026rsquo;s defense against viral infections, including PRRSV.\u003c/p\u003e \u003cp\u003eIn this study, we utilized a porcine alveolar macrophages (PAMs) cDNA library and a yeast two-hybrid system to screen for host proteins interacting with PRRSV Nsp1β. A total of 34 potential interacting host proteins were identified. Among these, THAP11 demonstrated a particularly strong interaction with Nsp1β, making it a prime candidate for further investigation. Co-localization experiments, as well as Co-IP assays, confirmed the interaction between Nsp1β and THAP11. Furthermore, overexpression of THAP11 resulted in a significant reduction in PRRSV N protein accumulation, suggesting that THAP11 exerts an antiviral effect by inhibiting PRRSV replication. On the other hand, silencing THAP11 enhanced PRRSV replication, indicating that THAP11 plays a critical role in controlling viral replication. Further mechanistic studies revealed that THAP11 promotes the degradation of Nsp1β through the ubiquitin-proteasome system, specifically by enhancing K48- and K63-linked ubiquitination. This finding suggests that THAP11 functions not only as a regulatory protein but also as an important factor in the degradation of Nsp1β, thereby limiting the virus's ability to evade immune detection and replicate efficiently. These results highlight THAP11 as a novel antiviral host factor in PRRSV infection, providing a potential target for developing new antiviral strategies.\u003c/p\u003e \u003cp\u003eThis study contributes to the growing body of knowledge on host-virus interactions in PRRSV infection and provides new insights into the role of THAP11 in modulating viral replication through the ubiquitin-proteasome system. Understanding how PRRSV manipulates host proteins, such as THAP11, to evade immune detection and promote replication could pave the way for the development of more effective antiviral therapies and vaccines.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and cell lines\u003c/h2\u003e \u003cp\u003eThe bait plasmid for the yeast library screen was constructed using a traditional method. The cDNA encoding Nsp1β from the classical CH-1a strain of PRRSV-2 subtype 2 was amplified by PCR with specific primers listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The amplified product was then cloned into the yeast pGBKT7 vector using \u003cem\u003eEcoRI\u003c/em\u003e and \u003cem\u003eBamHI\u003c/em\u003e restriction sites to express the Gal4 DNA-binding domain fusion protein.\u003c/p\u003e \u003cp\u003eThe cDNA library for PAMs was constructed using SMART technology and cloned into the pGADT7-Rec vector through gene recombination techniques. The full-length cDNA sequence of the gene of interest was synthesized via reverse transcription PCR (RT-PCR) and then cloned into the pGADT7 yeast vector with \u003cem\u003eEcoRI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e restriction sites added to the primers listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eMarc-145 and HEK293T cells were obtained from previous laboratory stocks and cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum. All cells were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. All experiments were performed in the level 2 Biosafety Laboratory at the College of Veterinary Medicine, South China Agricultural University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eYeast two hybrid (Y2H) assays\u003c/h3\u003e\n\u003cp\u003eIn Y2H assays, a PAMs cDNA library for \u003cem\u003eSus scrofa\u003c/em\u003e was constructed to screen for proteins interacting with PRRSV Nsp1β using the GAL4 system as described in the BD Matchmaker Library Construction and Screening Kits User Manual (Clontech, Palo Alto, CA). Nsp1β was cloned into the yeast pGBKT7 vector and used as the bait by co-transformation into Y2H Gold yeast cells with PAMs cDNA library. Transformants were grown on SD/-Leu/-Trp/-His medium (TDO), positive clones were selected on the Ade/Leu/Trp/His-deficient medium and then confirmed by 5-Br-4-Cl-3-indoxyl α-D-galactoside (X-α-Gal) and Aureobasidin A (AbA)/(QDO/X/A) assays. To confirm the interaction between proteins, full length host proteins were fused into the pGADT7 vector (Clontech, Palo Alto, CA) as a prey. The corresponding proteins were in in Y2H assays. Transformants were plated on synthetic defined medium lacking Leu and Trp (SD/-Leu/-Trp, DDO), grown for three days, and then transferred to synthetic defined medium lacking Ade, Leu, Trp and His, supplemented with 70 \u0026micro;g/ml X-α-Gal and AbA (SD/-Ade/-Leu/-Trp/-His/X-α-Gal, QDO/X/A). Positive control experiments were performed by co-transforming pGBK-53 and pGAD-T, while negative controls were performed with pGBK-Lam and pGAD-T. Three independent experiments were conducted to confirm the results.\u003c/p\u003e\n\u003ch3\u003eDetection of auto-activating and toxicity\u003c/h3\u003e\n\u003cp\u003eBD-Nsp1β and the blank pGADT7 vector were co-transformed into Y2H cells. The transformants were then plated onto DDO/X, TDO/X, and QDO/X/AbA selective media for screening.\u003c/p\u003e\n\u003ch3\u003eConfocal assays\u003c/h3\u003e\n\u003cp\u003eHEK293T cells were cultured in 12-well plates containing 20 mm polylysine-coated coverslips. When the cells reached 70\u0026ndash;80% confluence, they were co-transfected with plasmids expressing Nsp1β and THAP11 or appropriate controls. At 24 hours post-transfection (hpt), the cells were processed for immunofluorescence (IF) staining according to standard procedures. Finally, the polylysine-coated coverslips were mounted onto glass slides using nail polish. Fluorescent images were captured using a confocal fluorescence microscope (Leica, Germany).\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eCell samples cultured in 6-well or 12-well plates were lysed with RIPA buffer (high) (Solarbio) as per the manufacturer's protocol. The lysates underwent ultrasonic disruption and were then centrifuged at 12,000 rpm for 10 minutes (min). The resulting supernatant was collected into 500 \u0026micro;L sterile tubes, combined with 5\u0026times;SDS-PAGE loading buffer, heated at 100\u0026deg;C for 10 min, and cooled on ice for 2 min. Protein separation was performed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), utilizing the One-Step PAGE Gel Fast Preparation Kit (Vazyme Biotechnology, China). The proteins were subsequently transferred to a PVDF membrane via semi-dry transfer. After blocking the membrane with a solution containing 5% nonfat dry milk and 0.1% Tween 20 for 1 hour (h) at room temperature, it was incubated with specific primary or endogenous antibodies overnight at 4\u0026deg;C. Following this, the membrane was incubated with HRP-conjugated anti-mouse/rabbit IgG secondary antibody for 1 h at room temperature. Finally, protein bands were visualized using the Amersham ImageQuant 800 (Cytiva, Washington, DC, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP) assays\u003c/h2\u003e \u003cp\u003eMagnetic beads conjugated with anti-Myc or anti-mCherry antibodies were prepared following the instructions provided in the Protein A/G immunoprecipitation Kit (Selleck). In the Co-IP assay, cells cultured on 6-well plates were lysed for 30 min on ice with 250 \u0026micro;L of cell lysis buffer (Beyotime), supplemented with 1 mM PMSF protease inhibitor. The lysates were disrupted using ultrasonic treatment and centrifuged at 12,000 rpm for 15 min. A small aliquot of the supernatant was collected for input detection, while the remainder was incubated with protein A/G magnetic beads (Selleck) for antibody binding, according to the manufacturer\u0026rsquo;s protocol. The antigen-antibody complexes were incubated for 2 h at room temperature, and the beads were washed five times with a washing buffer (50 mM Tris, 150 mM NaCl, 0.5% detergent, pH 7.5). To elute the proteins, 40 \u0026micro;L of 1\u0026times;SDS-PAGE loading buffer was added to the beads, followed by denaturation at 100\u0026deg;C for 5 min. The proteins were then separated by SDS-PAGE for detection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence assays (IFA)\u003c/h3\u003e\n\u003cp\u003eCells were fixed in 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% Triton X-100 for 5 min. After blocking with 1% BSA for 1 h at room temperature, cells were incubated with primary antibodies overnight. Following washes, the cells were exposed to secondary antibodies conjugated with Alexa Fluor 488 or 555 fluorochromes (Cell Signaling Technology) for 1 h at room temperature. After further washing, the cells were stained with DAPI (Beyotime, China) for 5 min. Finally, fluorescence images were captured using an inverted microscope (Nikon ECLIPSE Ti2).\u003c/p\u003e\n\u003ch3\u003eQuantitative RT-PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from Marc-145 cells using TRizol reagent. The first strand of cDNA was synthesized via reverse transcription polymerase chain reaction (RT-PCR). Quantitative PCR (qPCR) was subsequently performed using ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Vazyme, Nanjing, China), following the manufacturer's instructions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid transfection and RNA interference (RNAi)\u003c/h2\u003e \u003cp\u003eHEK293T or Marc-145 cells were grown in 6/12-well plates with 80% confluence, the different doze recombinant plasmids were separately transfected or co-transfected into the cells via transfection reagents polyethylenimine (PEI) or Lipofectamine 2000. Two small interfering RNAs (siRNAs) targeting THAP11 gene coding sequence (CDS) region was designed and ordered by SYNBIO Technologies, Suzhou, China (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). siTHAP11-1 and siTHAP11-2 or negative control (siNC) was respectively transfected into Marc-145 cells using the Lipofectamine 2000 transfection reagent. At 24 hpt, cells were harvested for the analysis of THAP11-knockdown efficiency through real-time PCR and western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFunctional enrichment analysis of interacting protein\u003c/h2\u003e \u003cp\u003eGO enrichment and KEGG pathway enrichment analysis against screening interacted proteins were conducted using online the Metascape website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html\u003c/span\u003e\u003cspan address=\"https://metascape.org/gp/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and visualization of enriched data was analyzed via bioinformatics website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn this study, assays were conducted with at least three independent replications as per the instruction manual. The qPCR experiments were performed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher, USA), and data were analyzed using GraphPad Prism software (version 8.0). Statistical analyses were carried out using Student's t-test and one-way ANOVA. A difference with p-values less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePRRSV Nsp1β does not exhibit self-activation in Y2H screening assays\u003c/h2\u003e \u003cp\u003eNsp1β serves as a critical interferon inhibitor during PRRSV infection. To identify host factors interacting with Nsp1β, the full-length sequence of Nsp1β was inserted into the yeast library bait vector pGBKT7. The pGBKT7-Nsp1β plasmid was co-transformed with the pGADT7-T plasmid into Y2H Gold cells and cultured on selective media with varying nutritional deficiencies. The co-transformed cells produced only a few clones on the TDO selective medium supplemented with X-α-gal (TDO/X), but no clones were observed on the QDO/X medium supplemented with AbA (QDO/X/A) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Under these conditions, Y2H screening assays using pGBKT7-Nsp1β as bait were performed on the most stringent QDO/X/A medium, which presents the highest level of nutritional deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThirty-four potential host proteins interacting with Nsp1β were identified\u003c/h2\u003e \u003cp\u003eTo screen for host proteins that interact strongly with Nsp1β, bait plasmids and prey (cDNA library) were co-transformed into Y2H Gold cells, which were then cultured on the stringent TDO/X medium plates. A total of 74 clones grew on the QDO/X/A medium plates, which were further tested for interaction. Positive transformants were identified through sequencing, revealing 34 potential binding partners of Nsp1β, as listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, including THAP11, proteasome subunit beta type-10 (PSMB10), poly(rC)-binding protein 1 (PCBP1), thioredoxin-interacting protein (TXNIP), and cathepsin D (CSTD).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInformation for potential host factors interacting with PRRSV Nsp1β\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene symbol\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001038079.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ethioredoxin-interacting protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTXNIP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001038030.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eproteasome subunit beta type-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePSMB10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAY42145.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecathepsin D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020922421.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eprobable ATP-dependent RNA helicase DDX5 isoform X3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDDX5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013843801.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL-lactate dehydrogenase B chain isoform X1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLDHB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_005655974.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epersulfide dioxygenase ETHE1, mitochondrial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eETHE1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001229990.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ealpha-actinin-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACTN1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_003125105.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epoly(rC)-binding protein 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePCBP1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001090927.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecathepsin B precursor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTSB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001233111.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHAP domain-containing protein 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTHAP11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_003124995.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emacrophage-capping protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAPG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_003127414.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eER membrane protein complex subunit 10 isoform X1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEMC10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_021090073.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esortilin 1, transcript variant X4, mRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSORT1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013854598.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efolate receptor 1 isoform X2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFOLR1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020936382.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efilamin-A isoform X1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFLNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001072147.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCD81 molecule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCD81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020922821.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eacyl-CoA synthetase family member 2, mitochondrial isoform X2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACSF2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABC17921.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMHC class I antigen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSLA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_003123039.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATP synthase subunit delta, mitochondrial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eATP5D\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001278705.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emacrosialin precursor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCD68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020936879.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eapical endosomal glycoprotein isoform X7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMAMDC7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_005660860.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eferritin heavy chain isoform X1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFTH1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_005669190.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emyb-binding protein 1A isoform X2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMYBBP1A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_001927370.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ebeta-1,4-galactosyltransferase 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB4GALT3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001027548.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehypoxanthine-guanine phosphoribosyltransferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHPRT1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020936398.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efilamin-A isoform X11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFLNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_999066.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epropionyl-CoA carboxylase beta chain, mitochondrial precursor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePCCB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_001929153.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTOX high mobility group box family member 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTOX4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020940051.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edocking protein 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDOK3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020942955.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSH2 domain-containing protein 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSH2D6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_005660642.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elysine acetyltransferase 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKAT5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_020939148.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emicrotubule-associated serine/threonine-protein kinase 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMAST3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_021080945.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eputative GTP-binding protein 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGTPBP6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP_001230636.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecytosolic non-specific dipeptidase 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCNDP2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGO enrichment and KEGG pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eTo investigate the role of Nsp1β in regulating host cell metabolism, immune responses, and self-replication processes through interactions with host factors, Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the 34 identified interacting proteins. GO cellular component analysis revealed enrichment in the lytic vacuole, mitochondrial matrix, and melanosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). GO molecular function analysis identified that proteins interacting with Nsp1β were associated with cell adhesion molecule binding, ligase activity, and integrin binding, while some were involved in regulating DNA binding, receptor activity, and enzyme activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). GO biological process analysis indicated enrichment in the enzyme-linked receptor protein signaling pathway, cellular metabolism, regulation of biosynthetic processes, and antiviral immune responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). KEGG pathway analysis highlighted that the interacting host proteins were primarily involved in cell growth and death, transport and catabolism, the host immune system, and signal transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In conclusion, PRRSV Nsp1β may evade host immune responses by interacting with various cellular signaling molecules and pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCo-localization of Nsp1β with multiple host proteins\u003c/h2\u003e \u003cp\u003eTo better visualize the interaction between Nsp1β and PCBP1, TXNIP, and CSTD in vivo, the full-length sequence of Nsp1β was inserted into the pCAGGS expression vector, with a C-terminal fusion to the mCherry tag. The open reading frames of PCBP1, TXNIP, and CSTD were separately cloned into the pcDNA3.1 vector, with a C-terminal fusion to the Myc tag. At 24 hpt of Nsp1β-mCherry with host proteins, and using pcDNA3.1-Myc as a negative control, an indirect IFA was performed to assess the co-localization between Nsp1β and the selected host proteins. Confocal fluorescence microscopy was used to observe the subcellular localization patterns. The results showed that PRRSV Nsp1β was predominantly expressed in the nucleus. Co-transfection of Nsp1β-mCherry with each of the four host proteins resulted in merged yellow fluorescence, indicating co-localization, while co-transfection of the empty vector with Nsp1β-mCherry exhibited only single fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These findings suggest that Nsp1β co-localizes with the selected host proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTHAP domain-containing protein 11 interacts with Nsp1β in vivo and in vitro\u003c/h2\u003e \u003cp\u003eAmong the screened proteins, we selected genes with a nucleotide length of less than 2000 bp as potential study targets. In the yeast assay, strong interaction between Nsp1β and THAP11 was observed on QDO selective medium supplemented with X-α-gal and AbA (QDO/X/A) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Next, the full-length THAP11 sequence from \u003cem\u003eChlorocebus sabaeus\u003c/em\u003e was cloned into the pcDNA3.1 expression vector, fused with a C-terminal Myc tag. The Nsp1β sequence from the classical PRRSV CH-1a strain was constructed into the pCAGGS-Myc expression vector, with a C-terminal fusion to the mCherry tag. Co-transfection of THAP11-Myc and Nsp1β-mCherry into HEK293T cells for 24 h was followed by incubation with either an mCherry antibody or a control IgG antibody. Co-IP assay results showed that THAP11 could be detected in the immunoprecipitate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating an interaction between THAP11 and Nsp1β. Conversely, anti-Myc antibody also detected strong Nsp1β-mCherry protein in the immunoprecipitate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, endogenous THAP11 could be immunoprecipitated with Nsp1β in PRRSV-infected Marc-145 cells using anti-THAP11 antibody in a Co-IP assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Confocal fluorescence microscopy revealed overlapping yellow fluorescence in the cell nucleus when Nsp1β and THAP11 were co-expressed in HEK293T cells, demonstrating co-localization of Nsp1β with THAP11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Collectively, these results confirm that Nsp1β interacts with THAP11 both in vivo and in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTHAP11 is a transcriptional regulator containing a THAP (THAP-domain) domain. According to the AlphaFold3 prediction, the structure of THAP11 displays the characteristic THAP domain, which exhibits highly conserved secondary structural features, including β-sheets and α-helices, consistent with known structures of THAP family proteins. The protein features a tightly folded core, which is likely crucial for its roles in DNA and RNA binding, as well as transcriptional regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Nsp1β is a key protein of PRRSV, known for its function in disrupting host immune responses. The AlphaFold3 prediction indicates that Nsp1β adopts a highly folded structure, containing multiple conserved α-helices and β-sheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Through AlphaFold3 simulation of their interaction, the results suggest that THAP11 and Nsp1β likely bind through a network of hydrogen bonds and hydrophobic interactions, as indicated by a negative change in Gibbs free energy (∆iG\u0026thinsp;\u0026lt;\u0026thinsp;0). The THAP domain of THAP11 interacts with specific regions of Nsp1β, forming a stable binding interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), which could potentially influence the immune evasion mechanisms of PRRSV.\u003c/p\u003e \u003cp\u003eThe predicted interaction structure provides molecular-level insights into the THAP11-Nsp1β binding, contributing to a better understanding of the role of THAP11 in PRRSV infection and its impact on viral immune evasion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTHAP11 negatively regulates PRRSV replication\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between THAP11 and PRRSV and to understand the role of THAP11 during PRRSV infection, we first analyzed endogenous THAP11 expression in PRRSV-infected Marc-145 cells at 0, 6, 12, 24, 36 hours post-infection (hpi) with a multiplicity of infection (MOI) of 0.5. The results showed that the accumulation of THAP11 protein did not significantly change at different time points following PRRSV infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Next, Marc-145 cells were transfected with either THAP11-Myc or an empty pcDNA3.1 vector for 12 h, after which PRRSV at MOI of 0.5 was inoculated into the cells for 12, 24, and 36 h. The accumulation of the PRRSV N protein in the THAP11-Myc-expressing group was significantly lower than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that exogenous expression of THAP11 inhibits PRRSV replication.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further confirm the function of THAP11 during PRRSV infection, we performed siRNA-mediated RNA interference to knock down endogenous THAP11 expression. Two siRNAs targeting the THAP11 coding region (siTHAP11-1 and siTHAP11-2) were transfected into Marc-145 cells, with siNC (non-targeting control) as a negative control. At 12 hpt, PRRSV was added to the cells and cultured for an additional 36 h. The expression of THAP11 was significantly reduced in the siTHAP11 groups compared to the siNC control, and the accumulation of the PRRSV ORF7 gene was upregulated more than twofold (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Additionally, THAP11 protein levels were lower in the siRNA-silenced cells compared to the siNC control, while the accumulation of the PRRSV N protein was significantly higher in the siTHAP11 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), demonstrating that knockdown of THAP11 facilitates PRRSV replication.\u003c/p\u003e \u003cp\u003eFurthermore, IFA revealed that overexpression of THAP11 markedly reduced the fluorescence accumulation of the PRRSV N protein compared to cells expressing the empty vector, at both 12 and 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA \u0026amp; B). In conclusion, these results suggest that THAP11 negatively regulates PRRSV replication.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTHAP11 degrades Nsp1β via the ubiquitin-proteasome system\u003c/h2\u003e \u003cp\u003eTHAP11 has been reported to interact with PCBP1 to regulate transcription, suggesting that THAP11 may be a multifunctional protein. To explore the biological significance of its interactions, we constructed a plasmid pCAGGS-Nsp1β-HA, in which Nsp1β was fused with a C-terminal HA tag. Co-transfection of Nsp1β-HA and dose-independent THAP11-Myc plasmids into HEK293T cells was performed. Protein analysis revealed that the accumulation of Nsp1β gradually decreased as THAP11 expression increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo clarify the degradation pathway, HEK293T cells pre-transfected with Nsp1β-HA and THAP11-Myc plasmids for 12 h were treated with the proteasome inhibitor MG132, the autophagy inhibitor 3-MA, or BafA1, with DMSO as the control. Western blot analysis showed that only the MG132-treated group retained Nsp1β protein accumulation, indicating that MG132 inhibited Nsp1β degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This suggests that THAP11 promotes Nsp1β degradation through the proteasome pathway.\u003c/p\u003e \u003cp\u003eSince proteasomal degradation requires ubiquitination, we next investigated whether Nsp1β could be modified by ubiquitination. HEK293T cells were transfected with either Nsp1β-mCherry alone or in combination with Ub-HA for 24 h. Co-immunoprecipitation (Co-IP) assays analyzed the ubiquitination levels of Nsp1β. Immunoprecipitation with a mCherry antibody revealed a distinct ubiquitinated band in the co-transfection group, while the single transfection did not show any modified bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), confirming that Nsp1β can be ubiquitinated.\u003c/p\u003e \u003cp\u003eTo determine if THAP11 promotes Nsp1β ubiquitination and degradation, HEK293T cells were co-transfected with Nsp1β-mCherry, Ub-HA, and either an empty pcDNA3.1 vector or THAP11-Myc plasmids. At 24 hpt, Co-IP results showed that the ubiquitination levels of Nsp1β were significantly increased in cells expressing THAP11-Myc compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Additionally, the ubiquitination levels increased in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Collectively, these results indicate that THAP11 enhances the ubiquitination of Nsp1β, leading to its degradation via the proteasome.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTHAP11 promotes K48 and K63 conjugated ubiquitination of Nsp1β for degradation\u003c/h2\u003e \u003cp\u003eTo further elucidate the type of ubiquitin conjugation involved in THAP11-mediated Nsp1β degradation, we focused on the ubiquitin-proteasome system, where K48 and K63-linked ubiquitin chains are generally associated with the degradation of misfolded proteins or pathogens. We constructed ubiquitin mutants retaining either the K48 or K63 lysine site (K48 and K63), as well as mutants where K48 or K63 were replaced with arginine (K48R or K63R), all in an HA-tagged vector. Co-transfection of Nsp1β-mCherry, Ub(K48)-HA, and THAP11-Myc (or empty vector), as well as Nsp1β-mCherry, Ub(K63)-HA, and THAP11-Myc (or empty vector), into HEK293T cells was performed. Co-immunoprecipitation (Co-IP) analysis revealed that Nsp1β underwent significant ubiquitination modification when either the K48 or K63 lysine sites were retained in the ubiquitin molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Moreover, the expression of THAP11 further enhanced the levels of K48 and K63 ubiquitination of Nsp1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). These findings suggest that THAP11 promotes both K48- and K63-conjugated ubiquitination of Nsp1β, leading to its degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study used a yeast two-hybrid system to screen for host proteins interacting with PRRSV Nsp1β, identifying 34 potential interacting proteins. Among these, THAP11 showed a strong interaction with Nsp1β and was selected for further investigation. Overexpression of THAP11 significantly reduced PRRSV N protein accumulation, demonstrating antiviral activity, while knockdown of THAP11 enhanced PRRSV replication. Importantly, THAP11 facilitated the degradation of Nsp1β via increasing K48- and K63-linked ubiquitination, thereby inhibiting PRRSV replication.\u003c/p\u003e \u003cp\u003eTHAP11, a transcriptional regulator, may modulate host immune responses through its interaction with PRRSV Nsp1β. By reducing Nsp1β accumulation, overexpression of THAP11 weakens the virus's immune evasion ability and limits its replication. Conversely, THAP11 knockdown promotes Nsp1β accumulation, facilitating viral replication. Additionally, THAP11 mediates Nsp1β degradation via the ubiquitin-proteasome pathway, elucidating THAP11's regulatory role during PRRSV infection.\u003c/p\u003e \u003cp\u003eThis study is the first to reveal the interaction between THAP11 and PRRSV Nsp1β, as well as its potential antiviral role. Previous studies have reported interactions between THAP11 and other host factors like PCBP1 and TXNIP, which play critical roles in transcription and cellular responses. However, the relationship between THAP11 and PRRSV has not been widely explored. This study fills this gap, suggesting that THAP11 may be involved in the host antiviral response through the ubiquitin-mediated degradation of Nsp1β.\u003c/p\u003e \u003cp\u003eWhile this study identifies the potential antiviral role of THAP11, several limitations exist. First, the detailed mechanism by which THAP11 regulates Nsp1β degradation through ubiquitination remains to be fully elucidated and requires further experimental validation. Second, although the results observed in HEK293T cells are promising, whether these findings translate to in vivo pig models remains uncertain. Additionally, the roles of PCBP1, TXNIP, PSMB10, and CTSD in PRRSV infection need further confirmation through additional experiments.\u003c/p\u003e \u003cp\u003eAmong the identified interacting proteins, PCBP1 has been shown to interact with PRRSV Nsp1β and plays a key role in the lifecycle of several other viruses [\u003cspan additionalcitationids=\"CR39 CR40 CR41\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], indicating its potential importance in viral replication and immune modulation. In the context of Hepatitis C virus (HCV) infection, PCBP1 is involved in stabilizing viral RNA and promoting its replication. Research has demonstrated that PCBP1 binds to the 5' untranslated region (UTR) of the HCV RNA, stabilizing the RNA and facilitating its replication [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Similarly, PCBP1 has been implicated in regulating the translation of the SV40 large T antigen [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], further supporting its role as a multifunctional factor in viral processes. The influence of PCBP1 on viral RNA stability and replication is likely critical to the replication of many viruses. In the case of PRRSV, PCBP1 may influence the stability and transcription of viral RNA, potentially enhancing viral replication. Given that PRRSV relies on host cell machinery for RNA synthesis and stability, PCBP1's interactions with the viral genome could play a pivotal role in facilitating these processes. Future studies should explore the specific interactions between PCBP1 and the PRRSV genome to fully understand its contribution to viral replication. Understanding how PCBP1 regulates viral RNA could open new avenues for therapeutic intervention, particularly in modulating host-virus interactions to limit viral replication.\u003c/p\u003e \u003cp\u003eTXNIP has been studied extensively for its involvement in oxidative stress, inflammation, and immune-related pathways [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. While direct evidence linking TXNIP to PRRSV is scarce, its broader role in viral infections and immune responses points to its potential relevance in PRRSV pathogenesis. TXNIP is known to regulate the NLRP3 inflammasome, which plays a pivotal role in triggering inflammatory pathways and pyroptosis under oxidative stress conditions [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This inflammasome activation could impact viral infections by modulating immune responses, including those seen in PRRSV infections. TXNIP has also been implicated in apoptosis regulation and redox balance, both of which influence viral replication and immune evasion [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. TXNIP has been shown to regulate the cellular response during COVID-19 infection, where it inhibits oxidative stress in T cells, facilitating viral spread. TXNIP's role in viral replication appears to be closely linked to its ability to modulate cellular redox states [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. By maintaining the redox balance, TXNIP could create a more favorable environment for viral replication and immune evasion [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These insights suggest that TXNIP may help PRRSV manipulate host immune responses, offering an interesting avenue for future research on host-virus interactions. Further investigation is warranted to determine whether PRRSV influences TXNIP expression or activity, potentially contributing to its pathogenicity.\u003c/p\u003e \u003cp\u003ePSMB10, also known as MECL-1, is an integral component of the immunoproteasome, which plays a crucial role in antigen processing for MHC class I presentation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Induced by interferon-gamma, PSMB10 replaces constitutive proteasome subunits in immune cells and assists in the degradation of viral proteins for immune recognition. This process is essential for the immune response during viral infections, and the immunoproteasome, including PSMB10, is involved in modulating pathways like NF-κB signaling [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In viral infections such as classical swine fever virus, the proteasome, and specifically PSMB10, processes viral proteins, promoting their recognition by the immune system [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This suggests that PSMB10 may play a role in PRRSV infection, potentially contributing to the degradation of viral proteins and enhancing immune system recognition.\u003c/p\u003e \u003cp\u003eCTSD, an aspartic protease located primarily in lysosomes, plays a significant role in various viral infections by aiding different stages of the viral lifecycle. In viral contexts, CTSD facilitates the processing of viral proteins, promotes viral entry, and triggers immune responses, such as antigen presentation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. For instance, CTSD has been shown to activate viral glycoproteins after endocytosis, facilitating viral fusion with the host cell membrane [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In infections like SARS-CoV-2, CTSD, along with other cathepsins such as B and L, is essential for cleaving the viral spike protein, which is crucial for viral entry into the host cell [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. This highlights the importance of CTSD in viral infections and suggests that it may play a similar role in PRRSV, potentially influencing viral entry and immune modulation. By expanding the understanding of how proteins like TXNIP, PSMB10, and CTSD interact with viral proteins like Nsp1β, this research opens up new possibilities for exploring host-virus interactions in PRRSV and other viral infections. Further investigation into these pathways could lead to the development of targeted therapeutic strategies to modulate these interactions and combat PRRSV infections more effectively.\u003c/p\u003e \u003cp\u003eThis study reveals, for the first time, that THAP11 inhibits PRRSV replication by interacting with and degrading Nsp1β via the ubiquitin-proteasome pathway. By revealing the interaction between THAP11 and Nsp1β, Future studies will focus on exploring THAP11\u0026rsquo;s role in other viral infections and whether it can be considered a broad-spectrum antiviral factor. Furthermore, given the interactions between PCBP1, TXNIP, and CTSD with PRRSV in this study, future work will further investigate these proteins' roles in PRRSV infection and evaluate their potential as antiviral targets.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2023YFD1801500), the Basic and Applied Basic Research Foundation of Guangdong Province (2024A1515012991), the Science and Technology Planning Project of Guangzhou (2023B03J0947 and 2025D04J0072), and the Laboratory of Lingnan Modern Agriculture Project (NG2022003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBinghua Chen: Writing-original draft, Data curation, Conceptualization, Formal analysis. Yongsheng Xie: Investigation, Methodology, Validation, Data curation. Zhan He: Software, Validation, Data curation. Yongjie Chen, Jiecong Yan, Fangfang Li, Yunyan Luo, Yanfei Pan and Min Liu: Validation, Data curation. Chunhe Guo: Conceptualization, Funding acquisition, Supervision, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ.K. Lunney, Y. Fang, A. Ladinig, N. Chen, Y. Li, B. Rowland, and G.J. Renukaradhya, Porcine reproductive and respiratory syndrome virus (PRRSV): pathogenesis and interaction with the immune system. 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Epigenomics 14 (2022) 153-162.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Yeast two-hybrid screening, Virus-host interactions, PRRSV, Nsp1β, THAP11","lastPublishedDoi":"10.21203/rs.3.rs-6114224/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6114224/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePorcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen in the global pig industry, causing significant economic losses. Due to its rapid mutation, effective antiviral treatments or vaccines are still lacking. Therefore, it is essential to identify potential host factors that interact with PRRSV-encoded proteins. In this study, a porcine alveolar macrophage cDNA library was used to identify host proteins interacting with PRRSV nonstructural protein 1β (Nsp1β) through a yeast two-hybrid system. A total of 34 potential host factors were identified, with thanatos-associated protein 11 (THAP11) showing a strong interaction with Nsp1β. These interactions were further analyzed using Gene Ontology and KEGG pathway analysis. Co-localization of Nsp1β with THAP11, poly(rC)-binding protein 1 (PCBP1), thioredoxin-interacting protein (TXNIP), and cathepsin D (CTSD) was observed, and Co-IP assays confirmed the Nsp1β-THAP11 interaction. Overexpression of THAP11 reduced PRRSV N protein accumulation, indicating an antiviral effect, while silencing THAP11 enhanced PRRSV replication. Furthermore, THAP11 promoted the degradation of Nsp1β by increasing K48- and K63-linked ubiquitination, thereby restricting PRRSV replication. These findings suggest that THAP11 exerts an antiviral effect by interacting with and degrading Nsp1β via the ubiquitin-proteasome system, providing insights for future PRRSV defense strategies.\u003c/p\u003e","manuscriptTitle":"THAP11-mediated K48- and K63-linked ubiquitination is essential for the degradation of porcine reproductive and respiratory syndrome virus nonstructural protein 1β","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-06 10:04:27","doi":"10.21203/rs.3.rs-6114224/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-04-03T04:08:40+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-04T08:22:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-04T06:57:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-27T13:16:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-02-26T09:46:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea56b008-32bc-4e03-8cff-1cd236b27f57","owner":[],"postedDate":"March 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:00:40+00:00","versionOfRecord":{"articleIdentity":"rs-6114224","link":"https://doi.org/10.1007/s00018-025-05760-3","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-06-23 15:57:04","publishedOnDateReadable":"June 23rd, 2025"},"versionCreatedAt":"2025-03-06 10:04:27","video":"","vorDoi":"10.1007/s00018-025-05760-3","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05760-3","workflowStages":[]},"version":"v1","identity":"rs-6114224","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6114224","identity":"rs-6114224","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}