Porcine Kidney Cells Research Articles

Analysis of differentially expressed genes related to cell death in porcine kidney-15 cells at 24 and 48 hours post porcine parvovirus infection.

This study aims to identify and characterize differentially expressed genes (DEGs) associated with porcine parvovirus (PPV)-induced cell death in porcine kidney-15 (PK-15) cells. By analyzing the biological processes enriched by these DEGs and exploring their interaction networks, we aim to gain a deeper understanding of the molecular mechanisms underlying PPV-mediated cell death. After infecting cultured PK-15 cells with PPV for 24 and 48 hours, cell viability and cysteine-requiring aspartate protease-3 (caspase-3) activity were assessed using an enzyme marker. Apoptosis was observed using fluorescence microscopy. The genome-wide gene expression levels were analyzed through RNA sequencing. The functional enrichment of DEGs was analyzed using the Kyoto Encyclopedia of Genes and Genomes database, and the protein-protein interaction network was generated using the Search Tool for the Retrieval of Interacting Genes/Proteins database. Porcine parvovirus inhibits cell viability, boosts caspase-3 activity, and enhances cell death at 24 and 48 hours postinfection (HPI). Porcine parvovirus-infected cells showed 547 DEGs at 24 HPI and 1,765 at 48 HPI. Different forms of cell death were enriched in 149 genes that were upregulated at both 24 and 48 HPI. More DEGs associated with cell death were involved at 48 than at 24 HPI. These DEGs are involved in multiple signaling pathways and interact within a complex protein network. Porcine parvovirus infection of PK-15 cells induces multiple cell death-related DEGs and signaling pathways. Our study presents a promising approach to investigating the mechanism of PPV infection, with a particular focus on the induction of cell death.

Palmitoylation regulates norepinephrine transporter uptake, surface localization, and total expression with pathogenic implications in postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is an adrenergic signaling disorder characterized by excessive plasma norepinephrine, postural tachycardia, and syncope. The norepinephrine transporter (NET) modulates adrenergic homeostasis via the reuptake of extracellular catecholamines and is implicated in the pathogenesis of adrenergic and neurological disorders. In this study, we reveal NET is palmitoylated in male Sprague-Dawley rats and Lilly Laboratory Cell Porcine Kidney (LLC-PK1) cells. S-palmitoylation, or the addition of a 16-carbon saturated fatty acid, is a reversible post-translational modification responsible for the regulation of numerous biological mechanisms. We found that LLC-PK1 NET is dynamically palmitoylated, and that inhibition with the palmitoyl acyltransferase (DHHC) inhibitor, 2-bromopalmitate (2BP) results in decreased NET palmitoylation within 90 min of treatment. This result was followed closely by a reduction in transport capacity, cell surface, and total cellular NET expression after 120 min of treatment. Increasing 2BP concentrations and treatment time revealed a nearly complete loss of total NET protein. Co-expression with individual DHHCs revealed a single DHHC enzyme, DHHC1, promoted wild-type (WT) hNET palmitoylation and elevated NET protein levels. The POTS-associated NET mutant, A457P, exhibits dramatically decreased transport capacity and cell surface levels which we have confirmed in the current study. In an attempt to recover A457P NET expression, we co-expressed the A457P variant with DHHC1 to drive expression as seen with the WT protein but instead saw an increase in NET N-terminal immuno-detectable forms and fragments. Elimination of a potential palmitoylation site at cysteine 44 in the N-terminal tail of hNET resulted in a low expression phenotype mimicking the A457P hNET variant. Further investigation of A457P NET palmitoylation and surface expression is necessary, but our preliminary novel findings reveal palmitoylation as a mechanism of NET regulation and suggest that dysregulation of this process may contribute to the pathogenesis of adrenergic disorders like POTS.

Nanoplastics trigger the aging and inflammation of porcine kidney cells

Nanoplastics have now become a pervasive contaminant, being detected in various environmental media. However, our understanding of the specific toxicological effects of nanoplastics (NPs) on the kidneys remains unclear, which is a scientific problem that needs to be solved. To address this question, we employed two kidney cell lines as in vitro models to study the toxicological effects of NPs on porcine kidney cells. Firstly, we observed that NPs can be internalized into the cytoplasm in a time- and dose-dependent manner by using a laser confocal microscope. We further discovered that NPs can trigger inflammatory responses and lead to porcine kidney cell senescence by detection of senescence marker molecules. Furthermore, the potential molecular mechanism(s) by which NPs induce porcine kidney cell senescence were explored, we found that NPs induce oxidative stress in the porcine kidney cells, leading to the accumulation of reactive oxygen species (ROS) within mitochondria, ultimately triggering inflammatory responses and senescence in the kidney cells. In summary, our experimental results not only provide new evidence for the toxicity of NPs but also offer new ideas and directions for future research. This discovery will aid in our deeper understanding of the potential health impacts of NPs on domestic pigs.

CircRNA_8521 promotes Senecavirus A infection by sponging miRNA-324 to regulate LC3A

Senecavirus A (SVA) causes outbreaks of vesicular disease in pigs, which imposes a considerable economic burden on the pork industry. As current SVA prevention measures are ineffective, new strategies for controlling SVA are urgently needed. Circular (circ)RNA is a newly characterized class of widely expressed, endogenous regulatory RNAs, which have been implicated in viral infection; however, whether circRNAs regulate SVA infection remains unknown. To investigate the influence of circRNAs on SVA infection in porcine kidney 15 (PK-15) cells, RNA sequencing technology was used to analyze the circRNA expression profiles of SVA-infected and uninfected PK-15 cells, the interactions between circRNAs, miRNAs, and mRNAs potentially implicated in SVA infection were predicted using bioinformatics tools. The prediction accuracy was verified using quantitative real-time (qRT)-PCR, Western blotting, as well as dual-luciferase reporter and RNA pull-down assays. The results showed that 67 circRNAs were differentially expressed as a result of SVA infection. We found that circ_8521 was significantly upregulated in SVA-infected PK-15 cells and promoted SVA infection. circ_8521 interacted with miR-324. miR-324 bound to LC3A mRNA which inhibited the expression of LC3A. Knockdown of LC3A inhibited SVA infection. However, circ_8521 promoted the expression of LC3A by binding to miR-324, thereby promoting SVA infection. We demonstrated that circ_8521 functioned as an endogenous miR-324 sponge to sequester miR-324, which promoted LC3A expression and ultimately SVA infection.

Identification of the Effects of 5-Azacytidine on Porcine Circovirus Type 2 Replication in Porcine Kidney Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.

Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.

Molecular and functional characterization of porcine poly C binding protein 1 (PCBP1)

BackgroundPoly C Binding Protein 1 (PCBP1) belongs to the heterogeneous nuclear ribonucleoprotein family. It is a multifunctional protein that participates in several functional circuits and plays a variety of roles in cellular processes. Although PCBP1 has been identified in several mammals, its function in porcine was unclear.ResultsIn this study, we cloned the gene of porcine PCBP1 and analyzed its evolutionary relationships among different species. We found porcine PCBP1 protein sequence was similar to that of other animals. The subcellular localization of PCBP1 in porcine kidney cells 15 (PK-15) cells was analyzed by immunofluorescence assay (IFA) and revealed that PCBP1 was mainly localized to the nucleus. Reverse transcription-quantitative PCR (RT-qPCR) was used to compare PCBP1 mRNA levels in different tissues of 30-day-old pigs. Results indicated that PCBP1 was expressed in various tissues and was most abundant in the liver. Finally, the effects of PCBP1 on cell cycle and apoptosis were investigated following its overexpression or knockdown in PK-15 cells. The findings demonstrated that PCBP1 knockdown arrested cell cycle in G0/G1 phase, and enhanced cell apoptosis.ConclusionsPorcine PCBP1 is a highly conserved protein, plays an important role in determining cell fate, and its functions need further study.

Identification of signaling pathways induced by porcine parvovirus in porcine kidney-15 cells based on weighted correlation network analysis.

Porcine parvovirus (PPV) is an important trigger of reproductive issues in pigs. Infection of the porcine kidney-15 (PK-15) cells with PPV induces cell death and inflammation. To explore the impact of PPV infection on gene expression in PK-15 cells and to identify the associated signaling pathways, we performed weighted correlation network analysis (WGCNA) on both PPV-infected and uninfected cells. We identified the blue and brown modules, with the blue module demonstrating decreased gene expression and the brown module showing increased gene expression at 48 hr post-PPV induction. Gene Ontology (GO) analysis revealed that genes in the blue module were predominantly associated with cellular components, while those in the brown module were enriched in biological processes, including the immunological response to PPV infection. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the modules indicated that the "Cytokine-cytokine receptor interaction" was linked to PPV. Furthermore, we screened 124 target genes involved in more than 100 pathways that interact with C-X-C motif chemokine ligand 8 (CXCL8), which plays an essential role in regulating numerous biological processes. The signaling pathways we identified facilitate a comprehensive examination of the mechanisms of interactions initiated by PPV infection.

Transcriptome Analysis of LLC-PK Cells Single or Coinfected with Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural coinfections, which worsen as the disease progresses and cause increased mortality in piglets. To better understand the transcriptomic changes after PEDV and PDCoV coinfection, we compared LLC porcine kidney (LLC-PK) cells infected with PEDV and/or PDCoV and evaluated the differential expression of genes by transcriptomic analysis and real-time qPCR. The antiviral efficacy of interferon-stimulated gene 20 (ISG20) against PDCoV and PEDV infections was also assessed. Differentially expressed genes (DEGs) were detected in PEDV-, PDCoV-, and PEDV + PDCoV-infected cells at 6, 12, and 24 h post-infection (hpi), and at 24 hpi, the number of DEGs was the highest. Furthermore, changes in the expression of interferons, which are mainly related to apoptosis and activation of the host innate immune pathway, were found in the PEDV and PDCoV infection and coinfection groups. Additionally, 43 ISGs, including GBP2, IRF1, ISG20, and IFIT2, were upregulated during PEDV or PDCoV infection. Furthermore, we found that ISG20 significantly inhibited PEDV and PDCoV infection in LLC-PK cells. The transcriptomic profiles of cells coinfected with PEDV and PDCoV were reported, providing reference data for understanding the host response to PEDV and PDCoV coinfection.

CD46 inhibits the replication of swine influenza viruses by promoting the production of type I IFNs in PK-15 cells.

Swine flu caused by swine influenza A virus (swIAV) is an acute respiratory viral disease that is spreading in swine herds worldwide. Although the effect of some host factors on replication of swIAV has been identified, the role of CD46 in this process is unclear. Here, we report that CD46 inhibits the replication of swIAV by promoting the production of type I interferons (IFNs) in porcine kidney (PK-15) cells. CD46 knockout (CD46-KO) and stably expressing (CD46-overexpression) PK-15 cells were prepared using lentivirus-mediated CRISPR/Cas9 gene editing and seamless cloning technology. The results of virus infection in CD46-overexpression PK-15 cells showed that the replication of H1N1 and H3N2 swIAVs were inhibited, and the production of type I IFNs (IFN-α, IFN-β), interferon regulatory factor (IRF) 3, and mitochondrial antiviral-signaling protein (MAVS) was enhanced. Virus infection in CD46-KO PK-15 cells showed the opposite results. Further results showed that CD46-KO PK-15 cells have a favorable ability to proliferate influenza viruses compared to Madin-Darby canine kidney (MDCK) and PK-15 cells. These findings indicate that CD46 acts as promising target regulating the replication of swIAV, and help to develop new agents against infection and replication of the virus.

Biological properties of foot-and-mouth disease virus А 2205/G-IV strain

According to the World Organisation for Animal Health, foot-and-mouth disease (FMD) is regularly reported in domestic and wild cloven-hoofed animals in Africa. G-I, G-IV, G-VI, G-VII, ASIA/Iran-05 genetic lineages of serotype А FMD virus are considered to be the most widespread on the African continent. Given the close economic and trade relations maintained by the Russian Federation with the countries of North Africa, of particular interest for us is studying the FMD virus of serotype A G-IV genetic lineage, which has been responsible for the infection outbreaks in the naturally susceptible animal population of the said region every year since 2012, and there is a risk of introduction of this virus genotype in to the Russian Federation. Therefore, the issues of FMD introduction risk assessment and timely diagnosis are relevant for the Veterinary Service of Russia. FMD virus A 2205/G-IV strain tested for its biological and antigenic properties in cell cultures and naturally susceptible animals (cattle and pigs) was adapted for its reproduction in initially trypsinized porcine kidney (PK) cell culture, continuous monolayer cell cultures (IB-RS-2, PSGK-30, YaDK-04, ВНК-21) by five serial passages. The virus was considered to be adapted when 90–95% cytopathic effect developed within 14–19 hours after the cell culture infection. The virus adapted to the cell cultures was tested for its infectivity with microtitration in IB-RS-2 cell culture. The virus strain tested for vaccine matching with microneutralization test (MNT) demonstrated significant difference from production A/Turkey/06, А22 No. 550/Azerbaijan/64, A22/Iraq/64, A/Iran/97, A No. 2155/Zabaikalsky/2013, A No. 2166/Krasnodarsky/2013, A No. 2269/ARRIAH/2015 strains of FMD virus.

Identification of a Novel Porcine Teschovirus Subtype 19 within the Species Teschovirus A

Porcine teschovirus (PTV) is a member of the genus Teschovirus in the family Picornaviridae. Several novel PTVs in Teschovirus were identified in our previous study. To elucidate the taxonomic status of the species and subtypes, PTV-positive samples were inoculated into porcine kidney (PK-15) and swine testicular (ST) cells. A total of 58 PTV strains were successfully isolated and 21 different VP1 gene sequences were obtained. A phylogenetic analysis of the VP1 gene revealed that these PTV isolates comprised 11 genotypes, including 10 known genotypes (PTV 1–6, PTV 8–9, PTV 11, and Teschovirus B3), and a newly identified genotype. A nearly complete genome sequence of the novel PTV genotype isolate, PTV-SG2, was obtained. The homologies of the nucleotides and amino acids between PTV-SG2 and other PTVs were 69.7%–85.0% and 76.1%–90.4%, respectively. A phylogenetic analysis of the P1, polyprotein, and 3 CD genes revealed that PTV-SG2 belonged to a novel genotype within Teschovirus A, and a genetic divergence analysis of the novel PTV isolate further verified its taxonomic status. Based on the PTV naming convention, the novel PTV genotype identified in the present study was provisionally named PTV 19. Serum neutralization tests indicated that PTV 19 is a serotype-specific and completely different from previously reported PTV serotypes. Therefore, PTV 19 is a novel PTV serogroup. In conclusion, this study identified a PTV genotype and serotype within Teschovirus A.

Phenethyl isothiocyanate induces cytotoxicity and apoptosis of porcine kidney cells through Mitochondrial ROS-associated ERS pathway

Glucosinolates (GLS) in cruciferous vegetables are anti-nutritional factors. Excessive or long-term intake of GLS-containing feed is harmful to animal health and may cause kidney damage. Phenethyl isothiocyanate (PEITC) is a GLS. In this study, we investigated the inhibitory effect of PEITC on a porcine kidney (PK-15) cell line and explored the mechanism of PEITC-induced apoptosis. We found that PEITC could affect cell viability and induce cell apoptosis after incubating cells for 24 h. High concentrations of PEITC can induce intracellular ROS accumulation, resulting in impaired mitochondrial function (decreased MMP, decreased ATP) and DNA damage (increased 8-OHdG), cytochrome c in mitochondria is released into the cytoplasm and activates mitochondrial pathway apoptosis-related proteins (Bcl-2 family and caspase-9, -3). Meanwhile, PEITC could induce intracellular Ca2+ accumulation, disrupt ER homeostasis, and activate the expression levels of three ER-resident transmembrane proteins orchestrating the UPR (PERK, IRE-1α and ATF6) and ER-related proteins (GRP78 and CHOP), thereby activating ERS-pathway apoptosis-related proteins (caspase-12, -7). Our results showed that low concentration (2.5 μM) of PEITC had no damaging effect on cells. In comparison, a high concentration (10 μM) of PEITC could induce cell damage in porcine kidney cells and induce apoptosis in PK-15 cells via the Mitochondrial ROS-associated ERS pathway.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.

Porcine deltacoronavirus enters ST cells by clathrin-mediated endocytosis and does not require Rab5, Rab7, or Rab11.

Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.

Quercetin alleviates zearalenone-induced apoptosis and necroptosis of porcine renal epithelial cells by inhibiting CaSR/CaMKII signaling pathway

Zearalenone (ZEA) is a mycotoxin that is highly contaminated in feed and can cause severe toxic effects on the kidneys and other organs of animals. Quercetin (QUE) is a plant-derived flavonoid with a variety of detoxification properties, but the mechanism by which QUE detoxifies the toxic effects induced by ZEA has not yet been fully elucidated. We treated porcine kidney cells (PK15) with 80 μM ZEA and/or 30 μM QUE. The results showed that ROS and MDA levels were increased, antioxidant system levels were down-regulated, anti-apoptotic factor expression levels were decreased, and apoptotic and necroptosis-related factors were up-regulated after ZAE exposure. In addition, the results of Ca2+ staining, mitochondrial membrane potential, and mitochondrial dynamics-related indicators showed that ZEA induced Ca2+ overload in PK15 cells and increased mitochondrial Ca2+ uptake (MCU expression increased). The accumulated ROS and free Ca2+ further aggravate mitochondrial damage and eventually lead to mitochondrial pathway apoptosis and necroptosis. Nevertheless, QUE targets CaSR to inhibit the CaSR/CaMKII pathway and regulate calcium homeostasis, thereby alleviating apoptosis and necroptosis mediated by mitochondrial dynamic disorder and dysfunction. The present study demonstrated the mechanism by which ZEA induces apoptosis and necroptosis in PK15 and the protective role of QUE in this process.

Experimental infection of a porcine kidney cell line with hepatitis A virus

The hepatitis A virus (HAV) induces severe acute liver injury and is adapted to human and monkey cell lines but not other cells. In this study, the HAV was inoculated into porcine kidney (PK-15) cells to determine its infectivity in porcine cells. The growth pattern of the HAV in PK-15 cells was compared with its growth pattern in fetal rhesus kidney (FRhK-4) cells. The growth of HAV was less efficient in PK-15 cells. In conclusion, HAV replication was verified in PK-15 cells for the first time. Further investigations will be needed to identify the HAV-restrictive mechanisms in PK-15 cells.

The porcine piRNA transcriptome response to Senecavirus a infection.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. PIWI-interacting RNAs (piRNAs) are a class of small Ribonucleic Acids (RNAs) that have been found in mammalian cells in recent years. However, the expression profile of piRNAs in the host during SVA infection and their roles are poorly understood. Here, we found the significant differential expression of 173 piRNAs in SVA-infected porcine kidney (PK-15) cells using RNA-seq and 10 significant differentially expressed (DE) piRNAs were further verified by qRT-PCR. GO annotation analysis showed that metabolism, proliferation, and differentiation were significantly activated after SVA infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that significant DE piRNAs were mainly enriched in AMPK pathway, Rap1 pathway, circadian rhythm and VEGF pathway. It was suggested that piRNAs may regulated antiviral immunity, intracellular homeostasis, and tumor activities during SVA infection. In addition, we found that the expression levels of the major piRNA-generating genes BMAL1 and CRY1 were significantly downregulated after SVA infection. This suggests that SVA may affect circadian rhythm and promote apoptosis by inhibiting the major piRNA-generating genes BMAL1 and CRY1. The piRNA transcriptome in PK-15 cells has never been reported before, and this study will further the understanding of the piRNA regulatory mechanisms underlying SVA infections.

A Single Amino Acid Substitution in Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 2 Significantly Impairs Its Infectivity in Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism for macrophages and CD163 is a key receptor for infection. In this study, the PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, and two plaque-clones (C1 and C2) were randomly selected for further analysis. The C1 virus nearly lost the ability to infect porcine alveolar macrophages (PAMs), as well as porcine kidney cells expressing porcine CD163 (PK15-pCD163), while the C2 virus replicates well in these two cell types. Pretreatment of MARC-145 cells with an anti-CD163 antibody nearly blocked C1 virus infection, indicating that the virus still required CD163 to infect cells. The C1 virus carried four unique amino acid substitutions: three in the nonstructural proteins and a K160I in GP2. The introduction of an I160K substitution in GP2 of the C1 virus restored its infectivity in PAMs and PK15-pCD163 cells, while the introduction of a K160I substitution in GP2 of the low-passaged, virulent PRRSV strain NCV13 significantly impaired its infectivity. Importantly, pigs inoculated with the rNCV13-K160I mutant exhibited lower viremia levels and lung lesions than those infected with the parental rNCV13. These results demonstrated that the K160 residue in GP2 is one of the key determinants of PRRSV tropism.

African Swine Fever Virus MGF110-7L Induces Host Cell Translation Suppression and Stress Granule Formation by Activating the PERK/PKR-eIF2α Pathway.

African swine fever (ASF) is a highly contagious and often lethal disease of pigs caused by ASF virus (ASFV) and recognized as the biggest killer in global swine industry. Despite exhibiting incredible self-sufficiency, ASFV remains unconditionally dependent on the host translation machinery for its mRNA translation. However, less is yet known regarding how ASFV-encoded proteins regulate host translation machinery in infected cells. Here, we examined how ASFV interacts with the eukaryotic initiation factor 2α (eIF2α) signaling axis, which directs host translation control and adaptation to cellular stress. We found that ASFV MGF110-7L, a previously uncharacterized member of the multigene family 110, remarkably enhanced the phosphorylation level of eIF2α. In porcine alveolar macrophage 3D4/21 and porcine kidney-15 cells, MGF110-7L triggered eIF2α signaling and the integrated stress response, resulting in the suppression of host translation and the formation of stress granules (SGs). Mechanistically, MGF110-7L-induced phosphorylation of eIF2α was mediated via protein kinase R (PKR) and PKR-like endoplasmic reticulum (ER) kinase (PERK), and this process was essential for host translation repression and SG formation. Notably, our subsequent analyses confirmed that MGF110-7L was overwhelmingly retained in the ER and caused a specific reorganization of the secretory pathway. Further proteomic analyses and biochemical experiments revealed that MGF110-7L could trigger ER stress and activate the unfolded protein response, thus contributing to eIF2α phosphorylation and translation reprogramming. Overall, our study both identifies a novel mechanism by which ASFV MGF110-7L subverts the host protein synthesis machinery and provides further insights into the translation regulation that occurs during ASFV infection. IMPORTANCE African swine fever (ASF) has become a socioeconomic burden and a threat to food security and biodiversity, but no commercial vaccines or antivirals are available currently. Understanding the viral strategies to subvert the host translation machinery during ASF virus (ASFV) infection could potentially lead to new vaccines and antiviral therapies. In this study, we dissected how ASFV MGF110-7L interacts with the eIF2α signaling axis controlling translational reprogramming, and we addressed the role of MGF110-7L in induction of cellular stress responses, eIF2α phosphorylation, translation suppression, and stress granule formation. These results define several molecular interfaces by which ASFV MGF110-7L subverts host cell translation, which may guide research on antiviral strategies and dissection of ASFV pathogenesis.