Further evidence for the immunosuppressive activity of the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV)

Further evidence for the immunosuppressive activity of the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV) | 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 Article Further evidence for the immunosuppressive activity of the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV) Joachim Denner, Reinhard Schwinzer, Claudia Pokoyski, Benedikt B Kaufer, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5967592/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Retroviruses are immunosuppressive and there is evidence that a highly conserved immunosuppressive domain (isu domain) in their transmembrane envelope protein contributes to this activity. Studies have shown that disrupted retroviruses, their purified transmembrane envelope proteins and synthetic peptides corresponding to the isu domain inhibit mitogen-triggered proliferation of peripheral blood mononuclear cells (PBMCs) and modulate their cytokine expression in vitro. In vivo, in a mouse tumour model, tumour cells that were unable to induce tumours in immunocompetent animals, gained the ability to do so when expressing the transmembrane envelope protein or the isu domain of various retroviruses on their surface. However, criticism arose that endotoxin contaminations in retroviral preparations might explain the observed cytokine modulation, as endotoxins are capable to induce similar effects. Here we demonstrate that in an endotoxin-free system, the transmembrane envelope protein p15E of PERV can modulate cytokine expression in human PBMCs. Human 293 cells were transfected with constructs expressing p15E. These transfected cells were co-cultured with human PBMCs resulting in the release of IL-10 protein and modulation of several cytokines and other markers, including IL-6, IL-10, IFN-, TNF-, MMP1, and SEPP1. Additionally, p15E expression reduced MHC class I expression and had a protective effect against cellular cytotoxicity. Notably, the expression of p15E was minimal, which explains why no effect was observed in certain experiments. This finding underscores the need for further research to elucidate the dynamics of p15E expression and its immunosuppressive activity. Biological sciences/Cell biology Biological sciences/Immunology Biological sciences/Microbiology Retroviruses porcine endogenous retroviruses (PERV) immunosuppression transmembrane envelope protein immunosuppressive peptide cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Retroviruses are immunosuppressive. This is not only true for the immunodeficiency viruses such as the human immunodeficiency virus 1 and 2 (HIV-1 and -2) [1], but also for most other retroviruses including gammaretroviruses [2]. The feline leukaemia viruses (FeLVs) [3], the murine leukaemia viruses (MuLVs) [4] and the KoRV [5] induce in the infected host not only leukaemia and lymphoma, but also a severe immunodeficiency which precedes usually the tumour development. Immunosuppression without tumour development was also observed and more cats died from immunosuppression than from leukaemia [3]. Whereas only 5 to 10% of FeLV-infected cats suffer from leukaemia and lymphoma, more than 65% of them died from opportunistic infections based on an underlying immunodeficiency [3, 6, 7]. In the case of the KoRV the infected animals also suffer from opportunistic infections, e.g., chlamydia infection [8]. The mechanism how retroviruses induce immunosuppression is not well studied. However, the fact that all retroviruses induce immunodeficiencies suggested that there may be a common mechanism [9]. Meanwhile it was shown that non-infectious retroviruses, their transmembrane envelope proteins or synthetic peptides corresponding to a highly conserved domain in their transmembrane envelope protein, called the immunosuppressive (isu) domain, are inhibiting different in vitro activities of immune cells (for review see [10, 11]). The assays used in these investigations were mitogen-triggered proliferation of peripheral blood mononuclear cells (PBMCs), mixed lymphocyte reaction, IL-2-stimulated proliferation of T cells, mitogen-triggered proliferation of B cells, neutrophilic and erythroid cell function, receptor motility on the cell surface of immune cells, measurement of cytokine release as well as measurement of cytokine and general gene expression. In some of the system it may be assumed in retrospect that the retroviral materials were contaminated with traces of endotoxin, which is also able to induce IL-10 and other cytokines [12, 13]. However, in most systems a contamination can be excluded, for example when the purified viral Gag protein used as control was inactive, whereas purified viral p15E was highly active [14]. The retroviral transmembrane envelope proteins are also immunosuppressive in vivo . Immunisation with p15E of FeLV increased tumour development: After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats [15]. The most convincing in vivo results came from a tumour rejection assay: Expression of different retroviral transmembrane envelope proteins on mouse tumour cells, which did not grow in immunocompetent mice, allowed them to produce tumours in immunocompetent animals by suppression of their immune system. This was shown for the p15E of MuLV [16], the transmembrane envelope proteins of the Mason-Pfizer monkey virus [17], of the human endogenous retrovirus - H (HERV-H) [18], of FeLV [19] and of one of two murine and one of two human syncytins. Syncytins are envelope proteins of endogenous retroviruses expressed in the placenta [20]. Experiments deleting parts of the isu sequence of syncytin 2 showed that this domain is the sequence responsible for the immunosuppressive activity [20]. Only one of the murine and human syncytins was immunosuppressive, the human syncytin-2 (HERV-FRD) and the mouse syncytin-B, in contrast, human syncytin 1 (HERV-W) and murine syncytin-A were not immunosuppressive. Mutations of relevant amino acids allowed to switch from an immunosuppressive syncytin into a non-immunosuppressive and vice versa [20]. Furthermore, immunization with the non-immunosuppressive form (wild-type syncytin-1 and mutated syncytin-2) induced immunoglobulin G titres 10- to 30-fold higher than the corresponding immunosuppressive form (mutant syncytin-1 and wild-type syncytin-2) [20]. This indicates that the immunosuppressive activity acts not only local, on the surface of the tumour cells, but is generalized, influencing also antibody production. Retrovirus infections modulate the cytokine release in the infected individuals, for example in AIDS patients [21-23] and the transmembrane envelope proteins and synthetic peptides corresponding to the immunosuppressive domain have also been shown to modulate cytokine mRNA expression and release in human PBMCs. Using cytokine arrays, it was shown that the transmembrane envelope proteins of HIV-1, KoRV, PERV, HERV-K and the corresponding isu peptides, increased the release of the following cytokines: IL1-b, IL-10, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, MCP-2, tumour necrosis factor (TNF)-a, macrophage inflammatory protein (MIP)-1a and MIP-3 [24-27]. In contrast, the expression of IL-2 and chemokine (C-X-C motif) ligand (CXCL-9, also called monokine induced by gamma interferon, MIG) decreased. Microarray analysis of the expression of more than 25 000 genes in human PBMCs treated with the homopolymer of the HIV-1 isu peptide or with the recombinant transmembrane envelope protein of HERV-K confirmed the cytokine data and showed up-regulation and down-regulation of more than 300 genes [25, 26]. Among the genes with the highest up-regulation were IL-6, matrix metalloproteinase 1 (MMP-1), triggering receptor expressed on myeloid cells 1 (TREM-1). Among the down-regulated genes were ficolin-1 (FCN1), selenoprotein P, plasma, 1 (SEPP1), TREM-2 and CXCL-10 (also called interferon gamma-induced protein 10, IP-10), all these proteins are involved in innate immunity [25, 26]. The activity of the retroviral transmembrane envelope proteins and synthetic peptides corresponding to the immunosuppressive domain is interspecies-reactive, for example, p15E of FeLV inhibits feline and human PBMCs [15], indicating a conserved receptor and a conserved way of action. Binding proteins on the surface of immune cells have been identified for both the isu peptide of HIV-1 [28–34] and p15E of FeLV [35], suggesting the presence of specific receptors. The knowledge of the mechanisms of the immunosuppressive activity of retroviruses may have importance for the vaccine development against retroviruses: The mutation of the isu domain increased significantly the efficacy of a vaccine against FeLV [19], and against the simian-human immunodeficiency virus (SHIV) [36]. Cynomolgus macaques were vaccinated with measles virus replicative vectors expressing antigens of SHIV. Antigens were either the wild type or the mutated in the isu domain the envelope protein. The inactivation of the isu domain led to the induction of significantly enhanced cellular immune responses and in reduced proviral loads after the challenge of the vaccinees [36]. A mutation in the isu domain of gp41 of HIV-1 increased the antibody production when immunizing rats with the mutated protein in contrast to the unmutated protein [25]. Furthermore, immunization with the non-immunosuppressive form (wild-type syncytin-1 and mutated syncytin-2) induced immunoglobulin G titres 10- to 30-fold higher than the corresponding immunosuppressive form (mutant syncytin-1 and wild-type syncytin-2) [20]. Here, we describe a novel and guaranteed endotoxin-free system for testing the immunosuppressive properties of a retroviral transmembrane envelope protein. For this, the transmembrane envelope protein p15E of the porcine endogenous retrovirus-A/C (PERV-A/C), a gammaretrovirus closely related to MuLV, FeLV and KoRV (all three viruses induce severe immunodeficiencies in infected hosts), was expressed on human cells. These cells were incubated with human PBMCs and the changes in their cytokine release and the cytokine expression in this endotoxin-free system were analysed in comparison to cells not expressing p15E and with human 293 cells infected with and producing moderate amounts of PERV-A/C [37,38]. Furthermore, these cells were used to study the impact of the expression of p15E on human cytotoxic cells and the expression of MHC class 1 molecules. Results Cloning and transfection of p15E of PERV. To establish a cellular and endotoxin-free system for studying the immunosuppressive properties of p15E of PERV, two distinct expression constructs were designed according to the PERV-A/C sequence AY570980 and inserted in a vector. Both constructs contained the ectodomain of p15E including the immunosuppressive domain, the membrane spanning domain (MSD), the ENV signal peptide (SP), the furin peptidase cleavage site and a short sequence derived from gp70 (Figure 1A, B). Both constructs did not contain the fusion peptide (FP) of p15E. One construct carried a mutation at position 1652, resulting in a cysteine to serin substitution, which removed one of the cysteines in the immunodominant region of p15E (Figure 1C). One construct, abbreviated p15E-link, contained a longer portion of the N-terminal part of p15E, referred as the linker region. Analysis of p15E of PERV expression in transfected human cells. The expression of p15E on the surface of transfected and virus-producing cells was analyzed using two methods: immunofluorescence and flow cytometry analysis. In both cases a specific antiserum against p15E of PERV was employed. This antiserum (#355) had previously been shown to react with recombinant and viral p15E in Western blot assays and the epitopes of the antibody binding had been defined: GPQQLEK/T in the fusion peptide proximal region (FPPR) of the N-terminal helix and FEGWFN in the membrane proximal external region (MPER) of p15E [39, 40]. Low intracellular expression of p15E was observed in the transfected cells when the immunofluorescence was performed with cell membrane permeabilization (Figure 2). The intracellular expression of p15E was much stronger in virus-producing 293 cells compared with the transfected cells. While the expression of p15E on the cell membrane of virus-producing cells was moderate, the expression of p15E on the cell surface was very low, as shown by immunofluorescence without permeabilization of the cell membrane (Figure 2). Flow cytometry studies confirmed significant differences in p15E expression between the cell surface and the intracellular compartment. (Figure 3). Cell surface staining of 293T wt cells with the anti-p15E antiserum revealed a small shift in fluorescence intensity (9 arbitrary units, solid histograms) as compared to incubation of cells with the secondary reagents alone (broken histograms). This shift could be due to some unspecific binding of the antiserum to 293T cells. Fluorescence intensity was not enhanced after staining of 293T-p15E-NHR-His (8 units) or 293T-p15E-link-His cells (9 units), suggesting that p15E is not expressed on the cell surface or with very low density which is below detection level. However, clear-cut binding of the anti-p15E antiserum could be demonstrated in permeabilized transfectants. Thus, mean fluorescence intensity of 16 units in 293T wt cells significantly increased to 283 and 502 units after staining of 293T-p15E-NHR-His and 293T-p15E-link-His cells, respectively. Thus, the p15E transgene is expressed in this cell model and the protein can readily be detected intracellularly but barely on the cell surface. Effect of p15E of PERV on cytokine expression in human PBMCs. To investigate whether 293T cells expressing p15E can induce IL-10 secretion in human PBMCs, similar to the synthetic isu peptides, recombinant transmembrane envelope proteins and virus preparations of HIV-1 and HERV-K [25,26], p15E-expressing cells were co-incubated with purified human PBMCs. After 24 hours, IL-10 levels in the supernatant were quantified using an ELISA. An increase in IL-10 release was observed when PBMCs were incubated with 293T cells expressing both p15E constructs compared to wild-type 293T cells (Figure 4A-C). However, in some experiments no increased expression was observed (not shown). Human PBMCs incubated with porcine embryonic kidney PK15 cells producing PERV also showed increased IL-10 release (Figure 4B). Notably, a much higher release of IL-10 was observed when PBMCs were incubated with 293T cells producing PERV-A/C (Figure 4C). These cells produced virus, as demonstrated by measuring viral RNA using a real-time PCR in the supernatant (data not shown), and exhibited a higher p15E surface expression (Figure 2C). Differences in induced IL-10 levels between the p15E-link-His and p15E-NHR-His constructs observed in some experiments (e.g., Figure 4A) but not in others (e.g., Figures 4B and 4C), along with the absence of IL-10 induction in certain cases, suggest variability in the expression of active p15E on the surface of transfected cells. Moreover, it remains unclear to what extent p15E released from disrupted cells contributes to the induction of IL-10. After clearly showing the increased release of IL-10 by human PBMCs after incubation with p15E-expressing cells in some experiments, the impact on the expression of other cytokines and markers was assessed. Real-time RT-PCRs specific for the mRNA of IL-6, IL-10, INF-g, TNF-a, and SEPP1 were established and the expression was measured after 4, 6, 8 and 10 hours of incubation with 293T cells expressing p15E-link-His and 293 cells producing PERV-A/C (Figure 5). In some experiments expression of IL-6, IL-10, INF-g, TNF-a, and SEPP1 mRNA increased, either steadily increasing as in the case of IL-10, or peaking at 8 hours as in the case of IL-6, TNF-a and INF-g. However, when MMP-1, TNF-a, IL-8 and IL-6 were analyzed in another experiment, an increase in expression of the mRNA of these molecules was only observed for PK15 cells, but not for the transfected cells with exception of a slight increase of MMP-1, and IL-6 by cells expressing p15E-link (Figure 6). Effect of p15E of PERV on human cytotoxic effector cells. Another in vitro assay was applied to evaluate the immunosuppressive effect of p15E of PERV on cytotoxicity of effector cells. Thus, PBMC were cultivated for 5 days with IL-2 to induce cytotoxic activity and then co-cultivated for two hours with wild-type 293 cells, and 293 cells expressing p15E as p15E-link-His (Figure 7). In a series of experiments using effector populations from different blood donors, 3 to 7% of gated CD56 + CD45 + cells (effector population) expressed CD107a. An increased proportion of CD107 + cells (11 to 34%) was observed in co-cultures with wild-type 293T cells, indicating degranulation of the effector cells by contact with 293T cells. In two experiments (Exp. 1 and 2), we observed a slight reduction of CD107a expression when p15E expressing transfectants were used as targets. However, no reduction was seen in the other two experiments. This data indicates that expression of p15E on target cells may have a mild protective effect against cellular cytotoxicity. This correlates obviously with the level of expression of p15E, but may also depend on the donor of the PBMCs used Effect of p15E of PERV on MHC class-I expression. Retroviruses are known to downregulate MHC molecules at the cell surface. For example, HIV-1 reduces MHC class-I A and B molecules, thereby shielding infected cells from cytotoxic T lymphocyte (CTL)-mediated killing [41]. A similar effect has been reported for gammaretroviruses closely related to PERV [42]. To examine whether p15E expression affects MHC class-I (HLA-ABC) levels, 293T wild-type cells, and cells expressing p15E either as p15E-NHR-His or p15E-link-His were stained with a monoclonal antibody against human MHC class-I molecules. A significant reduction of 16-20% in MHC class-I expression was observed (Figure 8). Discussion To gain further evidence for the immunosuppressive properties of the transmembrane envelope protein p15E of PERV, part of this molecule including the isu domain was expressed in human 293T cells and its effect on human PBMCs was investigated. Unfortunately, the protein expression on the cell surface was very low, leading to variability in its effects across experiments. Since the expression of p15E was the only parameter fluctuating in the experiments, the modulation of the cytokine release found in some experiments must be associated with this molecule. It remains unclear why the expression of p15E, especially on the cell surface, is so low. Surprisingly an arginine repeat was found in the protein sequence of p15E of PERV, which was absent in the sequence of p15E of MuLV [43]. This short arginine repeat suggests that the PERV protein could be, in contrast to the MuLV protein p15E, retained in the cell [43]. Arginine/serine rich proteins are mainly localised in the cytoplasm and are targeted to the nucleus [44,45]. Nevertheless, few p15E molecules can be found at the surface of the transfected cells (Figure 2). Two methods, immunofluorescence and flow cytometry showed independently the low expression in the cytoplasma of human 293 cells and the lower expression at the cell surface. However, it remains unclear whether in addition to p15E on the cell surface, released p15E molecules from the cytoplasma of disrupted cells have been also involved in generation of the observed immunosuppressive effects. Despite the low expression of p15E, cytokine expression and release from PBMCs of healthy humans were modulated in a manner consistent with previous observations for synthetic peptides corresponding to the ISU domain of PERV p15E and purified PERV particles [46, 47]. The sequence of the isu domain of PERV is identical to the isu domains of related gammaretroviruses such as murine leukaemia virus (MuLV), feline leukaemia virus (FeLV) and koala retrovirus (KoRV) [10]. Therefore, theoretically, evidence of immunosuppressive properties in synthetic peptides, viral or recombinant p15E, or virus particles from MuLV, FeLV, and KoRV inherently extends to the ISU domain of PERV, and vice versa [27, 48]. The immunosuppressive properties of MuLV, FeLV and KoRV as well as human endogenous retroviruses such as HERV-K are well studied in vitro and in vivo (for review see [10, 11, 50]. The envelope proteins of endogenous retroviruses called syncytins play not only a role in the placentogenesis, but may also immunoprotect the embryo [50]. However, an involvement of PERV in pig placentogenesis was not yet demonstrated. Immunosuppression is a general property of all retroviruses, and immunodeficiency viruses such as the human immunodeficiency viruses HIV-1 and HIV-2 are well studied examples (for review see [10]). The changes in cytokine expression observed here are in agreement with changes in cytokine expression observed when human PBMCs were incubated with polymers of synthetic peptides corresponding to the isu domain of HIV [26] or with HERV-K particles released from a human teratocarcinoma cell line, with a recombinant transmembrane envelope protein of HERV-K or with peptides corresponding to the isu domain of HERV-K [25]. Modulation of cytokine expression was also observed when FeLV was analyzed (for review see [11]). One major advantage of the established system is the absence of endotoxin. Endotoxin is able to induce cytokine modulation resembling the modulation observed here [13] and the probability of an endotoxin contamination below the detection limit of the used detection assay (EndoLISA System from Hyglos, Germany) was given when working with synthetic peptides or recombinant proteins produced in bacteria. Endotoxin is a lipopolysaccharide (LPS) of the outer membrane of most gram-negative bacteria, it binds first to the LPS-binding protein (LBP) and is transferred to cluster of differentiation 14 (CD14), where myeloid differentiation-2 protein (MD-2) and the Toll-like receptor 4 (TLR4) re-associate. The receptor binding leads to a signal transduction involving activation of the transcription factor nuclear factor-kappa B (NF-κB), resulting in the release of cytokines [51]. This was the reason why in our later experiments gp41 produced in human 293 cells, was used [52]. The secreted and purified to homogeneity recombinant gp41 produced in 293 cells was soluble, glycosylated and assembled into trimers. The protein bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal PBMCs [52]. The immunosuppressive properties of the transmembrane envelope protein gp41 of HIV-1 was also studied in a cellular system which was endotoxin-free. For this, murine cTRAMP prostate cancer cells were transfected with a gp41-expressing vector, and gp41 expression on the cell surface was demonstrated by FACS analysis, and the cells released gp41 into the cell supernatant [53]. These cells were pulsed with the ovalbumin-derived MHC-I peptide SIINFEKL and co-cultured with naïve CD8 + T cells from OT-1 mice, which carry the corresponding SIINFEKL T-cell receptor. The gp41-expressing cells, but not the vector control cells, strongly inhibited IFNγ production and reduced CD25 (IL-2 receptor) expression. These findings indicated that gp41 impairs the antigen-specific response of murine CD8 + T cells by drastically suppressing IFNγ production. Furthermore, this result corroborates previous findings that retroviral transmembrane proteins or peptides corresponding to their isu-domain exhibit interspecies reactivity by modulating immune cells across species (for review, see [10]). To summarize, using a novel endotoxin-free cellular system to express the transmembrane envelope protein p15E of PERV, we gained new insights into the immunosuppressive properties of this molecule. Further experiments are required to enhance p15E expression levels to achieve more pronounced and conclusive results. Methods Cell culture and viruses Human kidney epithelial 293T cells, 293T cells infected with and producing PERV-A/C and porcine embryonic kidney pig PK15 cells were grown in Dulbecco Eagle Medium (DMEM) with 10% foetal bovine serum (FCS, PAN Biotech, Aidenbach, Germany, Lot P160616), and 1% penicillin-streptomycin (DMEM culture medium). Cells were maintained at 37°C in a humidified chamber with 5% CO 2 . PK15 cells harbour PERV-A and PERV-B, but not PERV-C proviruses in their genome, they release infectious virus particles. These cells were provided by Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). The PERV-A/C produced by 293T cells is the result of passaging cell-free virus on human 293T cells and is characterized by multimerized transcription factor binding sites in the long-terminal repeat (LTR) [37,38]. Since the number of repeats in the LTR changes during cultivation, a PCR was performed using LTR-specific primers in order to characterize the virus used in the present experiments. The length of the amplicon indicated that the virus had four and a half repeats as described previously [37] (Supplementary Figure 1). 293T cells were split twice in a week in a 1:3 ratio, PK15 were split 1:2 ratio every 3 days after washing with phosphate buffered solution (PBS) and trypsinization 0.25% trypsin/0.02% EDTA (PAN Biotech). Cloning of p15E . Synthetic p15E constructs were produced based on the env gene of PERV-A/C (AY570980) as gene blocks (gBlock) (Integrated DNA Technologies IDT, Coralville, Iowa, USA) (Figure 1). All constructs contained the signal peptide of the env gene and a linker part of the gp70 gene coding for its first 23 amino acids, followed by the sequence for the furin cleavage site and modified sequence of the p15E gene. All constructs did not contain the fusion peptide (position 1390-1431). The constructs designated p15E-link contained the following modifications: a nucleotide change at position 1652 from g to c (leading to a cysteine to serin conversion). The constructs designated p15E-NHR does not contain the sequence coding for the unstructured N-terminal part including the fusion peptide but starts with the N-terminal helix (NHR) (nucleotide position 1456) of p15E; the nucleotide at position 1652 was not changed. For cloning purposes an Eco-R1 restriction site was added to the 5‘end including a Kozak sequence for optimal translation initiation. The 3‘end contained a sequence coding for a 6x histidine tag (p15E-link-His, p15E-NHR-His), and a stop codon followed by a Nhe-1 cutting site. The p15E gBlock with Eco-R1 and Nhe-1 site were cloned into pVitro2-EGFP [53] using the same two enzymes replacing the EGFP gene in the plasmid. All plasmids were sequenced before transfection into 293T cells. Transfection of p15E. For plasmid transfection 10 5 293T cells were seeded in a 12 well plate the day before transfection. Next day medium was changed to DMEM with 5% FCS. For each transfection 3 µL of polyethylenimine (PEI) solution (1mg/mL) were added to 50 µL PBS; in parallel 1 µg plasmid DNA was added to 50 µL PBS [54]. Both solutions were vortexed at high speed for 1 min. After 10 min rest at room temperature PEI and DNA solution were gently mixed and incubated for 3 min at room temperature. The transfection solution was added dropwise to the cells. After 3 h medium was changed to DMEM culture medium. Selection was started 2 days after transfection with 500 µg/mL hygromycin B. Peripheral blood mononuclear cells (PBMCs) . At the Institute of Virology in Berlin, PBMCs were isolated from buffy coats from human blood from an anonymous donor using Ficoll-Hypaque density centrifugation with the use of Leucosep Tubes 50 mL (Greiner Bio-One, Kremsmünster, Austria) according to the instructions of the manufacturer (Greiner Bio-One). Buffy coat was diluted in a 1:2 ratio with PBS beforehand. Leucosep tubes were filled with 15 mL of Ficoll-Hypaque and centrifuged for 30 seconds at 1000x g at room temperature to move Ficoll--Hypaque below the porous barrier. 30 mL of diluted buffy coat was layered on top of the porous barrier and centrifuged at 1000xg for 10 minutes at room temperature without brakes. After centrifugation, the following layers were observed: plasma, enriched cell fraction (PBMCs), granulocytes and erythrocytes. The fraction containing PBMCs was harvested using a Pasteur pipette. The porous barrier effectively avoids recontamination with pelleted erythrocytes and granulocytes. Harvested PBMCs were washed twice with 10 mL of PBS and subsequently centrifuged for 10 minutes at 250x g. The PBMC pellet was resuspended in cell culture medium. Resuspended PBMCs were counted using Neubauer chamber and 1x10 8 PBMCs were frozen in cryopreserved tubes and stored in nitrogen tanks at -80° C in a freezing medium containing 70% DMEM, 20% FCS and 10% dimethyl sulfoxide (DMSO). Freshly isolated PBMCs were used for co-culture experiments. The use of human blood has been approved by the ethical commission at the Medical Faculty of the Humboldt University Berlin. At the Transplant Laboratory in Hannover, PBMC were isolated from discarded material of normal routine apheresis samples from anonymized donors. Ethics declarations. At the Institute of Virology in Berlin, buffy coats from human blood from an anonymous donor were provided by Deutsches Rotes Kreuz, Blutspendedienst Nord-Ost, Berlin. The use of human blood has been approved by the ethical commission at the Medical Faculty of the Humboldt University Berlin. At the Transplant Laboratory in Hannover, PBMC were isolated from discarded material of normal routine apheresis samples obtained from the Department of Transfusion Medicine of the Hannover Medical School. Samples were anonymized and could not be assigned to an individual donor. The local ethics committee of Hanover Medical School approved this procedure. All methods were carried out in accordance with DFG guidelines of Good Scientific Practice. Informed consent to participate was waived by an Institutional Review Board (IRB). Co-cultivation. 293T and PK-15 are adherent cells and were used at 80 to 90% confluence, the cells were washed with PBS, incubated with 0.25 % trypsin/0.02 %EDTA at 37 o C for 2 to 15 minutes. 10 mL of culture medium containing FCS was added cells were collected in a 15 mL falcon tube, centrifuged at 300x g for 5 minutes. Pellets were resuspended in 10 ml culture medium. Cells were counted twice using a Neubauer Chamber, centrifuged at 300 x g for 5 minutes and culture medium was added to obtain 3 x 10 5 cells/100 μL. 100 μL of cells were added to each well of 96 well plate and 100 μL of culture medium without hygromycin was added into the wells respectively. Cells were incubated overnight at 37°C in a humidified chamber with 5% CO 2 . The next day, 100 μL of media was removed as 293T and PK-15 adhere to the surface of the plate and 3 x 10 5 PBMCs in a volume of 100 μL was added for co-incubation and left overnight in 37°C in a humidified chamber with 5% CO 2 . For the PCR expression studies 2.5 x 10 4 /100µl 293T cells and 7.5 x 10 4 /100µl PBMCs were used. Fluorescence analysis. 293T cells were gently dislodged from cell culture flasks with ice-cold PBS and counted with a Neubauer chamber after trypan blue staining to detect dead cells. Cell suspension was adjusted to 250 x 10 3 cells/mL. For each sample 50 x 10 3 cells in 200µL PBS were transferred to a glass slide using a Cellspin 1 device (Tharmac, Limburg/Lahn, Germany) at 8,000 rpm for 10 min according to the manufacturer’s instruction. Slides were kept at room temperature until completely dry. Cells were fixed in 4% formaldehyde in PBS for 15min followed by washing in PBS, 3´ for 10 min. Perforation of the cell membrane was achieved by 15 min incubation in PBS with 0.5 % Triton X-100 (Carl Roth, Karlsruhe, Germany) followed by PBS washes as described above. Slides were incubated with 3 % BSA in PBS for 1 h to reduce unspecific binding followed by incubation with a 1:100 dilution of goat anti-p15E (goat #355, [39]) in 3 % BSA/PBS for 1 h. After PBS wash (3´, 10 min) FITC conjugated anti-goat antibody from donkey (Merck/Sigma Aldrich, Darmstadt, Germany) was incubated for 1 h at room temperature. Cover slips were added after all liquid was removed and a mounting dye containing 4′,6-diamidino-2-phenylindole (DAPI) was added (Carl Roth, Karlsruhe, Germany). Samples were analysed with a Zeiss Axio fluorescence microscope equipped with an Axiocom 503 mono camera and a Colibri 7 LED light source using ZEN 2.3 software (Zeiss, Oberkochen, Germany). SMART set up provided by the software was used to adjust the fluorescence signals. Exposure time for the FITC channel was set to 5 sec (DAPI 50 msec). Unspecific background signals in the FITC channel were subtracted by setting the threshold in the negative control (untransfected 293T cells) to zero/black. These setting were used for all samples. DAPI staining was adjusted to highest contrast. Antibodies and flow cytometry. A goat anti-p15E serum was used to monitor p15E expression after transfection of human 293T. Generation and characterization of the goat anti-p15E serum #355 had been described previously [39]. The cells were incubated with the serum (30 min, 1:40 dilution), followed by two additional incubation steps using biotinylated bovine anti-goat Ig (Dianova, Hamburg, Germany) and allophycocyanin (APC)-conjugated streptavidin (BD Biosciences, San Jose, CA). To monitor intracellular levels of p15E, transfected and control cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with saponin (0.2%) for 10 min at room temperature and then incubated with the anti-p15E antiserum. Expression of MHC class-I molecules (HLA-ABC) on 293T cells was detected by indirect staining using monoclonal antibody (mAb) W6/32 (American Type Culture Collection, ATCC, Manassas, Virginia, USA) and phycoerythrin (PE)-conjugated rat anti-mouse kappa light chain (BD, Biosciences). Directly labelled mAb CD56-APC (B159; BD Biosciences) in combination with CD107a-PE (H4A3; BD Biosciences) were used to study degranulation of human NK cells in response to 293T or L23 cells. Analyses were performed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and data were processed by using FCS Express 7 (De Novo software, Pasadena, CA, USA). CD107a assay. Cytotoxic effector cells were generated by culturing PBMC for 5 to 7 days in the presence of 50 ng/ml IL-2. The cells (2x10 5 ) were then co-cultured for two hours with 2x10 5 293T wt cells or 293T cells transfected to express p15E (293T-p15E-NHR-His or 293T-p15E-link-His). Cytotoxic activity against 293T target cells was monitored by assessing CD107a expression (degranulation) on gated CD56 + CD45 + NK cells. Statistical Analysis. Statistical analysis was performed by using the Student’s t test. Levels of significance are given as P-values. DNA and RNA extraction. In order to demonstrate the presence of the sequence encoding p15E with its isu domain, the cell lines 293T-wt, 293T-p15E-NHR-His and 293T-p15E-Link-His were tested using conventional PCR. Genomic DNA was isolated from transfected and non-transfected cells using DNA Easy Blood and Tissue Kit (Qiagen). 5 x 10 6 cultured cells of each cell line were centrifuged for 5 min at 300 x g and resuspended in 200 μL of PBS. Further steps of DNA extraction were performed using the Instruction manual provided by Qiagen to purify total DNA from animal blood or cells (DNeasy Blood and Tissue Handbook, Spin-Column protocol). All centrifugation steps were performed at room temperature. DNA concentration was quantified twice using nanodrop 1000 spectrophotometer (PeqLab, Erlangen, Germany). Purified plasmids of p15E-NHR-His and p15E-Link-GFP (1:100 and 1:1000) were used as positive controls. 293T cells were used as a negative control. DNA concentrations were standardized using nuclease free water and 5 μL of DNA sample was added to each reaction mix. DNA from virus producing 293 cells was isolated using the DNeasy Blood and Tissue kit (Qiagen). In order to analyze the expression of cytokines, RNA was isolated using the RNeasy Kit (Qiagen) and DNAse treatment to remove cellular DNA. Polymerase chain reaction (PCR). In order to characterize PERV proviruses in the virus-producing 293T cells, PCRs using specific primers for the pol and LTR region of PERV (Table 1) were performed: 5 min denaturation at 95°C, and 45 cycles (15s 95°C, 30s 62°C, 30s 72°C). PCR was performed using a Biometra Thermocycler (Analytik Jena, Germany), electrophoresis in a 1.3% agarose gel and the GeneRuler 1 kbp DNA ladder (Thermo Scientific) were used for gel electrophoresis. Reverse transcriptase real-time polymerase chain reaction (RT-real-time PCR). In order to analyze the expression of different cytokines, RT-real time PCRs were established using specific primers for IL-6, IL-10, INF-g, TNF- a, MMP1 and SEPP1 (Table 1). Reaction mixes containing PCR buffer, forward and reverse primers and probes, dNTPs, AmpliTaq Polymerase (Thermo Scientific, Schwerte, Germany), nuclease free water and DNA sample were incubated 12 min at 95°C and 40 PCR cycles (20s denaturation at 95°C, 30 s annealing at 55°C, 1 min extension and 20s at 72°C followed by an extension step for 15 min at 72°C). Detection of cytokine release by ELISA. To detect IL-10, an ELISA for human IL-10 (R & D Biosciences) was used. Supernatants obtained from co-culture of human PBMCs with transfected and non-transfected cells (3 x 10 5 each sample group) were used for the assay. Supernatant containing proteins was collected after 24 hours of incubation by centrifuging at 2000 x g for 10 min. ELISAs were performed in duplicates according to protocols of the supplier: 200 μL of standard, control and sample supernatant were added per well of ELISA microplate and covered with an adhesive strip for incubation for 2 hrs at room temperature. After incubation, wells were washed with 400 μL of wash buffer using a squirt bottle for a total of 4 washes. 200 μL of human IL-10 conjugate was added to each well and incubated for an hour at room temperature. Washing was repeated and subsequently 200 μL of substrate solution was added to each well and incubated for 30 minutes at room temperature. Subsequently, 50 μl of stop solution was added to each well and colour change from blue to yellow was observed. Optical density was determined within 30 minutes using a microplate reader set to 450 nm. To detect IL-6, an ELISA for human IL-6 (Sigma Aldrich, St. Louis, MI, USA) was used. Supernatants containing proteins were collected after 24 hours of incubation by centrifuging at 2000g for 10 min. ELISAs were performed in duplicates according to protocols of the supplier. Standards, buffer solutions and detection antibodies were prepared as mentioned in manufacturers manual. 100 μL of supernatant from co-culture of different samples were added in the wells along with standard. Samples and standard were incubated overnight at 4°C with gentle shaking. The next day, solutions were discarded, and wells were washed with 300 μL wash buffer 4 times with a multichannel pipette. Following the washing step, 100 μL of biotinylated antibody was added to each well and incubated at room temperature for 60 minutes with gentle shaking. Biotinylated antibody was removed, and wells were washed and 100 μL of streptavidin solution was added to each well afterwards. Followed by an incubation for 45 minutes with gentle shaking. Streptavidin was removed from wells and washed, subsequently 100 μL of TMB one step substrate reagent was added to each well and incubated for 30 minutes in the dark with gentle shaking. After 30 minutes, 50 μL of stop solution was added to stop reaction and optical density of solution in wells were measured immediately at 450 nm on a microplate reader. Declarations Availability of data and materials Data is provided within the manuscript or supplementary information files. Authors contribution Conceptualization: JD; data curation, formal analysis, investigation, methodology, RS, CP, BD, LL; funding acquisition: JD, BK, RS; supervision: JD, BK, RS; writing – original draft: JD, RS; writing – review and editing: JD, RS, BK. All authors have read and approved the final manuscript. Funding This research was partially supported by the Deutsche Forschungsgemeinschaft, TRR127. Acknowledgments We would like to thank Ludwig Krabben, Institute of Virology, for the constructs, and transfected cells, Axel Teigeler, Institute of Virology, and Antje Brinkmann, Transplant Laboratory, for excellent technical support. Declarations Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at Correspondence and requests for materials should be addressed to J.D. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Moir S, Chun TW, Fauci AS. Pathogenic mechanisms of HIV disease. Annu Rev Pathol. 6, 223-48. doi: 10.1146/annurev-pathol-011110-130254 (2011). Schlecht-Louf G, Renard M, Mangeney M, Letzelter C, Richaud A, Ducos B, Bouallaga I, Heidmann T. Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses. Proc Natl Acad Sci U S A. 107(8), 3782-3787. doi: 10.1073/pnas.0913122107 (2010). Hardy WD Jr. Immunopathology induced by the feline leukemia virus. Semin Immunopathol 5, 75-106 (1982). 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Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells J Virol. 74(9), 4028-4038. doi: 10.1128/jvi.74.9.4028-4038.2000 (2000). Yang L, Güell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church G. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 350(6264), 1101–4 (2015). Tables Table 1 Primers used for the analysis of cytokine expression and provirus detection Primer Sequenz Reference Accession number Localisation PK 34 5´-AAAGGATGAAAATGCAACCTAACC-3´ Czauderna et al. [55] Y17012 3 - 26 PK 26 5´-ACGCACAAGACAAAGACACACGAA-3´ 1134 - 1111 PERV pol fw 5´-CGACTGCCCCAAGGGTTCAA-3´ Yang et al. [56] HM159246 3568–3587 PERV pol rev 5´-TCTCTCCTGCAAATCTFFGCC-3´ 3803–3783 GAPDH fw 5´-GGCCATGCTGGCGCTGAGTAC-3´ Denner et al. [26] NM 002046.3 364-386 GAPDH rev 5´-TGGTCCACACCCATGACGA-3´ 494-512 GAPDH probe 5´-HEX-CTTCACCACCATGGAGAAGGCTGGG-BHQ-1-3´ 405-429 IFN-γ fw 5´-TGCAGAGCCAAATTGTCTCC-3´ this manuscript NM 000619.3 328-347 IFN-γ rev 5´-TGCTTTGCGTTGGACATTCA-3´ 502-521 IFN-γ probe 5’-6-FAM-ACCATCAAGGAAGACATGAATGTCAAG-BHQ-1-3’ 408-434 IL-6 fw 5´-GGTACATCCTCGACGGCATCT-3´ Denner et al. [26] NM 000600.3 289-309 IL-6 rev 5´-GTGCCTCTTTGCTGCTTTCAC-3´ 349-369 IL-6 probe 5’-6-Fam-TGTTACTCTTGTTACATGTCTCCTTTCTCAGGGCT-BHQ-1-3’ 311-345 IL-10 fw 5´-CCACGCTTTCTAGCTGTT-3´ Denner et al. [26] NM 000572.2 966-983 IL-10 rev 5´-CTCCCTGGTTTCTCTTCCTAA-3´ 1058-1078 I-10 probe 5’-6-FAM-TCTTGTCTCTGGGCTT-BHQ-1-3’ 1015-1030 MMP1 fw 5´-CATCCAAGCCATATATGGACG-3´ Denner et al. [26] NM 002421.3 908-928 MMP1 rev 5´-TCTCTTAAAACTGAGAGGTCT-3´ 1498-1518 MMP1 probe 5’-6-FAM-CTGGGCTGTTCAGGGACAGAA-BHQ-1-3’ 1187-1207 SEPP1 fw 5’-CATGGACATCAGCACCTT-3’ Denner et al. [26] NM 005410.2 774-459 SEPP1 rev 5’-TCGACAGAGCTTCTTTTG-3´ 954-972 SEPP1 probe 5´-6-FAM-AGAATCAGCAACCAGGAGCA-BHQ-1-3´ 721-740 TNF-α fw 5´-GAGAAGCAACTACAGACCCC-3´ this manuscript NM 000594.4 48-67 TNF-α rev 5´-CATGCTTTCAGTGCTCATGG 176-195 TNF-α probe 5’-6-FAM-ACAACCCTCAGACGCCACATCC-BHQ-1-3’ 76-97 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.pdf Supplementary Figure 1. Agarose gel electrophoresis of the PCR amplicons of PERVs integrated in 293 cells. A, amplification of the pol sequence, indicating the presence of the provirus in the cell genome, M, marker, 1, PERV-A/C used in the experiment; 2, positive control; 3, negative control. B, amplification of the LTR sequence. M, marker,1-4 amplicons of PERV-A/C used in the experiment, 5, amplicon of PERV/3° plasmid; 6, amplicon of PERV/5° plasmid; 7, negative control. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5967592","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":421080468,"identity":"3411ca26-9419-4822-a484-9848e64d9b80","order_by":0,"name":"Joachim Denner","email":"data:image/png;base64,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","orcid":"","institution":"Free University Berlin","correspondingAuthor":true,"prefix":"","firstName":"Joachim","middleName":"","lastName":"Denner","suffix":""},{"id":421080469,"identity":"d02873ce-e229-4968-9e14-cd62a9798d68","order_by":1,"name":"Reinhard Schwinzer","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Reinhard","middleName":"","lastName":"Schwinzer","suffix":""},{"id":421080470,"identity":"8e1fa843-58d8-4e68-9bfd-e51ddd131b1b","order_by":2,"name":"Claudia Pokoyski","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Pokoyski","suffix":""},{"id":421080471,"identity":"ebe37590-fe12-4bef-8d63-42c48090d593","order_by":3,"name":"Benedikt B Kaufer","email":"","orcid":"","institution":"Free University Berlin","correspondingAuthor":false,"prefix":"","firstName":"Benedikt","middleName":"B","lastName":"Kaufer","suffix":""},{"id":421080472,"identity":"185fbd13-e31b-41d0-a01b-101c1ccb3f87","order_by":4,"name":"Björn Dierkes","email":"","orcid":"","institution":"Free University Berlin","correspondingAuthor":false,"prefix":"","firstName":"Björn","middleName":"","lastName":"Dierkes","suffix":""},{"id":421080473,"identity":"eddfddcd-772e-434e-a09f-e9728203ad97","order_by":5,"name":"Lovlesh Lovlesh","email":"","orcid":"","institution":"Free University Berlin","correspondingAuthor":false,"prefix":"","firstName":"Lovlesh","middleName":"","lastName":"Lovlesh","suffix":""}],"badges":[],"createdAt":"2025-02-05 16:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5967592/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5967592/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77334478,"identity":"151f56ee-7401-4f3c-b954-b093369ed7dc","added_by":"auto","created_at":"2025-02-27 14:00:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":133530,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of the expression constructs of PERV p15E: \u003cstrong\u003eA\u003c/strong\u003e, sequence of the envelope gene (env) used for the construction based on the PERV A/C NCBI database entry AY570980. SP, signal peptide; gp70, 23 amino acid residues of the surface envelope protein; FP, fusion peptide of the transmembrane envelope protein p15E sequence; isu, immunosuppressive domain; MSD, membrane spanning domain. The arrow indicates the furin peptidase cleavage site, which is positioned after the carboxy-terminal arginine (Arg) residue in the sequence –Arg–X–Lys/Arg–Arg↓– (where Lys is lysine, X is any amino acid and ↓ identifies the cleavage site). Numbers above entry indicate the nucleotide according to the PERV sequence, numbers below refer to the nucleotide position according to the env gene. \u003cstrong\u003eB\u003c/strong\u003e, Schematic presentation of p15E-link and p15E-NHR expression constructs, DFP, deletion of the fusion peptide coding region; C S, single nucleotide exchange at position 1652, that leads to a cysteine to serine substitution. \u003cstrong\u003eC\u003c/strong\u003e, partial sequence of the transmembrane envelope protein of PERV containing the immunosuppressive and immunodominant domains. * indicates the mutated cysteine in p15E-link.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/9cfbfcdd06a4e88e5a6ba76e.png"},{"id":77335700,"identity":"ad990650-b4cf-4daa-9d83-5849be91b525","added_by":"auto","created_at":"2025-02-27 14:08:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1273873,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of p15E in transfected and PERV-producing 293T cells analyzed by immunofluorescence using a p15E-specific goat antiserum (#355 [39]) and FITC conjugated donkey anti-goat antibody, the cells were mounted using antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). \u003cstrong\u003eA\u003c/strong\u003e, with permeabilization of the cell membrane by Triton X-100. \u003cstrong\u003eB\u003c/strong\u003e, without permeabilization. Magnification 63 times.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/c5ac3edce5affd91763dff33.png"},{"id":77334479,"identity":"301e2f2f-5610-4200-8dcf-bae0864d16bb","added_by":"auto","created_at":"2025-02-27 14:00:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110851,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of cell surface and intracellular expression of p15E in human 293T cells.\u003cstrong\u003e \u003c/strong\u003e293T wt, 293T-p15E-NHR-His, and 293T-p15E-link-His cells were incubated with anti-p15E goat serum #355 followed by incubations with biotinylated bovine anti-goat Ig and APC-conjugated streptavidin. Solid histograms were obtained by anti-p15E binding; the numbers represent mean fluorescence intensity. Histograms of broken lines represent reactivity of secondary reagents alone.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/7fc34663b96ffd0e64b9b2fb.png"},{"id":77334482,"identity":"4b2d1943-7616-452c-ac8c-5265cc9a174d","added_by":"auto","created_at":"2025-02-27 14:00:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109758,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of p15E on the IL-10 release by human PBMCs. In three independent experiments 293T wild-type cells, 293T cells transfected with p15E-link-His and p15E-NHR-His, PK15 cells and 293T producing PERV were co-incubated with human PBMCs and the IL-10 release was measured by ELISA. Stars indicate statistical significance.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/08802336027a6eca170ecd05.png"},{"id":77334489,"identity":"c147573f-fc4c-4ede-8f09-90718b705d4f","added_by":"auto","created_at":"2025-02-27 14:00:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":175185,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of the influence of PERV-producing 293T cells and 293T cells expressing p15E-link on the expression of different cytokines as well as SEPP1-10 release by human PBMCs.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/e5945438935f8412f05a00c9.png"},{"id":77336430,"identity":"71e2d1ca-f3cf-448e-8e94-b8f944e0b48e","added_by":"auto","created_at":"2025-02-27 14:16:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93554,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of p15E on cytokine and MMP1 expression. 293T wild-type cells, 293T cells transfected with p15E-link-His and p15E-NHR-His, and PK15 cells were co-incubated with human PBMCs and the expression of RNA of IL-6, TNFa, and MMP1 was analyzed by real-time PCR.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/44b117e7b40e38faaee9ab35.png"},{"id":77335721,"identity":"75ecfd63-ecbb-4f77-b160-c9bd7a4699c3","added_by":"auto","created_at":"2025-02-27 14:08:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":86837,"visible":true,"origin":"","legend":"\u003cp\u003eDegranulation of effector cells by co-culturing with 293T cells in four independent experiments (Exp.). IL-2 activated PBMC were cultured alone or with 293T wt or 293T cells expressing p15E-link-His. Expression of CD107a was monitored after 2 hours on gated CD56\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/95e901c5b28b50304f2c50b8.png"},{"id":77337605,"identity":"0e22b304-cf04-4d2d-8619-e7db3a04170d","added_by":"auto","created_at":"2025-02-27 14:24:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":120833,"visible":true,"origin":"","legend":"\u003cp\u003eReduced expression of MHC class-I (HLA-ABC) on p15E-transfected 293T cells\u003cstrong\u003e. \u003c/strong\u003e293T wt, 293T-p15E-NHR-His, and 293T-p15E-link-His cells were stained with mAb to human MHC class-I. The numbers represent percentage of cells carrying reduced levels of MHC class-I molecules (dashed area). Broken-line-histograms were obtained after incubation of cells with secondary reagents alone. Shown is one representative experiment out of a series of three.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/065c0f0b9f20cebb88d492c3.png"},{"id":77338114,"identity":"7685934f-7128-4580-a4bd-cbc4acf085ef","added_by":"auto","created_at":"2025-02-27 14:32:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3827361,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/aa92bb77-26e3-42bd-8a3b-d1cf16751f5a.pdf"},{"id":77334480,"identity":"710a32ce-22ab-4548-9cf1-06baf1084ed1","added_by":"auto","created_at":"2025-02-27 14:00:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":133768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. \u003c/strong\u003eAgarose gel electrophoresis of the PCR amplicons of PERVs integrated in 293 cells. \u003cstrong\u003eA\u003c/strong\u003e, amplification of the pol sequence, indicating the presence of the provirus in the cell genome, M, marker, 1, PERV-A/C used in the experiment; 2, positive control; 3, negative control. \u003cstrong\u003eB\u003c/strong\u003e, amplification of the LTR sequence. M, marker,1-4 amplicons of PERV-A/C used in the experiment, 5, amplicon of PERV/3° plasmid; 6, amplicon of PERV/5° plasmid; 7, negative control.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5967592/v1/889a097fd6c47b4e7b71cb5d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Further evidence for the immunosuppressive activity of the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRetroviruses are immunosuppressive. This is not only true for the immunodeficiency viruses such as the human immunodeficiency virus 1 and 2 (HIV-1 and -2) [1], but also for most other retroviruses including gammaretroviruses [2]. The feline leukaemia viruses (FeLVs) [3], the murine leukaemia viruses (MuLVs) [4] and the KoRV [5] induce in the infected host not only leukaemia and lymphoma, but also a severe immunodeficiency which precedes usually the tumour development. Immunosuppression without tumour development was also observed and more cats died from immunosuppression than from leukaemia [3]. Whereas only 5 to 10% of FeLV-infected cats suffer from leukaemia and lymphoma, more than 65% of them died from opportunistic infections based on an underlying immunodeficiency [3, 6, 7]. In the case of the KoRV the infected animals also suffer from opportunistic infections, e.g., chlamydia infection [8].\u003c/p\u003e\n\u003cp\u003eThe mechanism how retroviruses induce immunosuppression is not well studied. However, the fact that all retroviruses induce immunodeficiencies suggested that there may be a common mechanism [9]. Meanwhile it was shown that non-infectious retroviruses, their transmembrane envelope proteins or synthetic peptides corresponding to a highly conserved domain in their transmembrane envelope protein, called the immunosuppressive (isu) domain, are inhibiting different \u003cem\u003ein vitro\u003c/em\u003e activities of immune cells (for review see [10, 11]). The assays used in these investigations were mitogen-triggered proliferation of peripheral blood mononuclear cells (PBMCs), mixed lymphocyte reaction, IL-2-stimulated proliferation of T cells, mitogen-triggered proliferation of B cells, neutrophilic and erythroid cell function, receptor motility on the cell surface of immune cells, measurement of cytokine release as well as measurement of cytokine and general gene expression. In some of the system it may be assumed in retrospect that the retroviral materials were contaminated with traces of endotoxin, which is also able to induce IL-10 and other cytokines [12, 13]. However, in most systems a contamination can be excluded, for example when the purified viral Gag protein used as control was inactive, whereas purified viral p15E was highly active [14].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe retroviral transmembrane envelope proteins are also immunosuppressive \u003cem\u003ein vivo\u003c/em\u003e. Immunisation with p15E of FeLV increased tumour development:\u0026nbsp;After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats [15].\u003c/p\u003e\n\u003cp\u003eThe most convincing \u003cem\u003ein vivo\u003c/em\u003e results came from a tumour rejection assay: Expression of different retroviral transmembrane envelope proteins on mouse tumour cells, which did not grow in immunocompetent mice, allowed them to produce tumours in immunocompetent animals by suppression of their immune system. This was shown for the p15E of MuLV [16], the transmembrane envelope proteins of the Mason-Pfizer monkey virus [17], of the human endogenous retrovirus - H (HERV-H) [18], of FeLV [19] and of one of two murine and one of two human syncytins. Syncytins are envelope proteins of endogenous retroviruses expressed in the placenta [20]. Experiments deleting parts of the isu sequence of syncytin 2 showed that this domain is the sequence responsible for the immunosuppressive activity [20]. Only one of the murine and human syncytins was immunosuppressive, the human syncytin-2 (HERV-FRD) and the mouse syncytin-B, in contrast, human syncytin 1 (HERV-W) and murine syncytin-A were not immunosuppressive. Mutations of relevant amino acids allowed to switch from an immunosuppressive syncytin into a non-immunosuppressive and vice versa [20]. Furthermore, immunization with the non-immunosuppressive form (wild-type syncytin-1 and mutated syncytin-2) induced immunoglobulin G titres 10- to 30-fold higher than the corresponding immunosuppressive form (mutant syncytin-1 and wild-type syncytin-2) [20]. This indicates that the immunosuppressive activity acts not only local, on the surface of the tumour cells, but is generalized, influencing also antibody production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRetrovirus infections modulate the cytokine release in the infected individuals, for example in AIDS patients [21-23] and the transmembrane envelope proteins and synthetic peptides corresponding to the immunosuppressive domain have also been shown to modulate cytokine mRNA expression and release in human PBMCs. Using cytokine arrays, it was shown that the transmembrane envelope proteins of HIV-1, KoRV, PERV, HERV-K and the corresponding isu peptides, increased the release of the following cytokines: IL1-b, IL-10, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, MCP-2, tumour necrosis factor (TNF)-a, macrophage inflammatory protein (MIP)-1a\u0026nbsp;and MIP-3 [24-27]. In contrast, the expression of IL-2 and chemokine (C-X-C motif) ligand (CXCL-9, also called monokine induced by gamma interferon, MIG) decreased. Microarray analysis of the expression of more than 25 000 genes in human PBMCs treated with the homopolymer of the HIV-1 isu peptide or with the recombinant transmembrane envelope protein of HERV-K confirmed the cytokine data and showed up-regulation and down-regulation of more than 300 genes [25, 26]. Among the genes with the highest up-regulation were IL-6, matrix metalloproteinase 1 (MMP-1), triggering receptor expressed on myeloid cells 1 (TREM-1). Among the down-regulated genes were ficolin-1 (FCN1), selenoprotein P, plasma, 1 (SEPP1), TREM-2 and CXCL-10 (also called interferon gamma-induced protein 10, IP-10), all these proteins are involved in innate immunity [25, 26].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe activity of the retroviral transmembrane envelope proteins and synthetic peptides corresponding to the immunosuppressive domain is interspecies-reactive, for example, p15E of FeLV inhibits feline and human PBMCs [15], indicating a conserved receptor and a conserved way of action. Binding proteins on the surface of immune cells have been identified for both the isu peptide of HIV-1 [28\u0026ndash;34] and p15E of FeLV [35], suggesting the presence of specific receptors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe knowledge of the mechanisms of the immunosuppressive activity of retroviruses may have importance for the vaccine development against retroviruses: The mutation of the isu domain increased significantly the efficacy of a vaccine against FeLV [19], and against the simian-human immunodeficiency virus (SHIV) [36]. Cynomolgus macaques were vaccinated with measles virus replicative vectors expressing antigens of SHIV. Antigens were either the wild type or the mutated in the isu domain the envelope protein. The inactivation of the isu domain led to the induction of significantly enhanced cellular immune responses and in reduced proviral loads after the challenge of the vaccinees [36]. A mutation in the isu domain of gp41 of HIV-1 increased the antibody production when immunizing rats with the mutated protein in contrast to the unmutated protein [25]. Furthermore, immunization with the non-immunosuppressive form (wild-type syncytin-1 and mutated syncytin-2) induced immunoglobulin G titres 10- to 30-fold higher than the corresponding immunosuppressive form (mutant syncytin-1 and wild-type syncytin-2) [20].\u003c/p\u003e\n\u003cp\u003eHere, we describe a novel and guaranteed endotoxin-free system for testing the immunosuppressive properties of a retroviral transmembrane envelope protein. For this, the transmembrane envelope protein p15E of the porcine endogenous retrovirus-A/C (PERV-A/C), a gammaretrovirus closely related to MuLV, FeLV and KoRV (all three viruses induce severe immunodeficiencies in infected hosts), was expressed on human cells. These cells were incubated with human PBMCs and the changes in their cytokine release and the cytokine expression in this endotoxin-free system were analysed in comparison to cells not expressing p15E and with human 293 cells infected with and producing moderate amounts of PERV-A/C [37,38]. Furthermore, these cells were used to study the impact of the expression of p15E on human cytotoxic cells and the expression of MHC class 1 molecules.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCloning and transfection of p15E of PERV.\u0026nbsp;\u003c/strong\u003eTo establish a cellular and endotoxin-free system for studying the immunosuppressive properties of p15E of PERV, two distinct expression constructs were designed according to the PERV-A/C sequence AY570980 and inserted in a vector. Both constructs contained the ectodomain of p15E including the immunosuppressive domain, the membrane spanning domain (MSD), the ENV signal peptide (SP), the furin peptidase cleavage site and a short sequence derived from gp70 (Figure 1A, B). Both constructs did not contain the fusion peptide (FP) of p15E. One construct carried a mutation at position 1652, resulting in a cysteine to serin substitution, which removed one of the cysteines in the immunodominant region of p15E (Figure 1C). One construct, abbreviated p15E-link, contained a longer portion of the N-terminal part of p15E, referred as the linker region.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of p15E of PERV expression in transfected human cells.\u0026nbsp;\u003c/strong\u003eThe expression of p15E on the surface of transfected and virus-producing cells was analyzed using two methods: immunofluorescence and flow cytometry analysis. In both cases a specific antiserum against p15E of PERV was employed. This antiserum (#355) had previously been shown to react with recombinant and viral p15E in Western blot assays and the epitopes of the antibody binding had been defined: GPQQLEK/T in the fusion peptide proximal region (FPPR) of the N-terminal helix and FEGWFN in the membrane proximal external region (MPER) of p15E [39, 40]. Low intracellular expression of p15E was observed in the transfected cells when the immunofluorescence was performed with cell membrane permeabilization (Figure 2). The intracellular expression of p15E was much stronger in virus-producing 293 cells compared with the transfected cells. While the expression of p15E on the cell membrane of virus-producing cells was moderate, the expression of p15E on the cell surface was very low, as shown by immunofluorescence without permeabilization of the cell membrane (Figure 2).\u003c/p\u003e\n\u003cp\u003eFlow cytometry studies confirmed significant differences in p15E expression between the cell surface and the intracellular compartment. (Figure 3). Cell surface staining of 293T wt cells with the anti-p15E antiserum revealed a small shift in fluorescence intensity (9 arbitrary units, solid histograms) as compared to incubation of cells with the secondary reagents alone (broken histograms). This shift could be due to some unspecific binding of the antiserum to 293T cells. Fluorescence intensity was not enhanced after staining of 293T-p15E-NHR-His (8 units) or 293T-p15E-link-His cells (9 units), suggesting that p15E is not expressed on the cell surface or with very low density which is below detection level. However, clear-cut binding of the anti-p15E antiserum could be demonstrated in permeabilized transfectants. Thus, mean fluorescence intensity of 16 units in 293T wt cells significantly increased to 283 and 502 units after staining of 293T-p15E-NHR-His and 293T-p15E-link-His cells, respectively. Thus, the p15E transgene is expressed in this cell model and the protein can readily be detected intracellularly but barely on the cell surface.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of p15E of PERV on cytokine expression in human PBMCs.\u003c/strong\u003e To investigate whether 293T cells expressing p15E can induce IL-10 secretion in human PBMCs, similar to the synthetic isu peptides, recombinant transmembrane envelope proteins and virus preparations of HIV-1 and HERV-K [25,26], p15E-expressing cells were co-incubated with purified human PBMCs. After 24 hours, IL-10 levels in the supernatant were quantified using an ELISA. An increase in IL-10 release was observed when PBMCs were incubated with 293T cells expressing both p15E constructs compared to wild-type 293T cells (Figure 4A-C). However, in some experiments no increased expression was observed (not shown). Human PBMCs incubated with porcine embryonic kidney PK15 cells producing PERV also showed increased IL-10 release (Figure 4B). Notably, a much higher release of IL-10 was observed when PBMCs were incubated with 293T cells producing PERV-A/C (Figure 4C). These cells produced virus, as demonstrated by measuring viral RNA using a real-time PCR in the supernatant (data not shown), and exhibited a higher p15E surface expression (Figure 2C). Differences in induced IL-10 levels between the p15E-link-His and p15E-NHR-His constructs observed in some experiments (e.g., Figure 4A) but not in others (e.g., Figures 4B and 4C), along with the absence of IL-10 induction in certain cases, suggest variability in the expression of active p15E on the surface of transfected cells. Moreover, it remains unclear to what extent p15E released from disrupted cells contributes to the induction of IL-10.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter clearly showing the increased release of IL-10 by human PBMCs after incubation with p15E-expressing cells in some experiments, the impact on the expression of other cytokines and markers was assessed. Real-time RT-PCRs specific for the mRNA of IL-6, IL-10, INF-g, TNF-a, and SEPP1 were established and the expression was measured after 4, 6, 8 and 10 hours of incubation with 293T cells expressing p15E-link-His and 293 cells producing PERV-A/C (Figure 5). In some experiments expression of IL-6, IL-10, INF-g, TNF-a, and SEPP1 mRNA increased, either steadily increasing as in the case of IL-10, or peaking at 8 hours as in the case of IL-6, TNF-a and INF-g. However, when MMP-1, TNF-a, IL-8 and IL-6 were analyzed in another experiment, an increase in expression of the mRNA of these molecules was only observed for PK15 cells, but not for the transfected cells with exception of a slight increase of MMP-1, and IL-6 by cells expressing p15E-link (Figure 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of p15E of PERV on human cytotoxic effector cells.\u003c/strong\u003e Another \u003cem\u003ein vitro\u003c/em\u003e assay was applied to evaluate the immunosuppressive effect of p15E of PERV on cytotoxicity of effector cells. Thus, PBMC were cultivated for 5 days with IL-2 to induce cytotoxic activity and then co-cultivated for two hours with wild-type 293 cells, and 293 cells expressing p15E as p15E-link-His (Figure 7). In a series of experiments using effector populations from different blood donors, 3 to 7% of gated CD56\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e cells (effector population) expressed CD107a. An increased proportion of CD107\u003csup\u003e+\u003c/sup\u003e cells (11 to 34%) was observed in co-cultures with wild-type 293T cells, indicating degranulation of the effector cells by contact with 293T cells. In two experiments (Exp. 1 and 2), we observed a slight reduction of CD107a expression when p15E expressing transfectants were used as targets. However, no reduction was seen in the other two experiments. This data indicates that expression of p15E on target cells may have a mild protective effect against cellular cytotoxicity. This correlates obviously with the level of expression of p15E, but may also depend on the donor of the PBMCs used\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of p15E of PERV on MHC class-I expression.\u003c/strong\u003e Retroviruses are known to downregulate MHC molecules at the cell surface. For example, HIV-1 reduces MHC class-I A and B molecules, thereby shielding infected cells from cytotoxic T lymphocyte (CTL)-mediated killing [41]. A similar effect has been reported for gammaretroviruses closely related to PERV [42]. To examine whether p15E expression affects MHC class-I (HLA-ABC) levels, 293T wild-type cells, and cells expressing p15E either as p15E-NHR-His or p15E-link-His were stained with a monoclonal antibody against human MHC class-I molecules. A significant reduction of 16-20% in MHC class-I expression was observed (Figure 8).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo gain further evidence for the immunosuppressive properties of the transmembrane envelope protein p15E of PERV, part of this molecule including the isu domain was expressed in human 293T cells and its effect on human PBMCs was investigated. Unfortunately, the protein expression on the cell surface was very low, leading to variability in its effects across experiments. Since the expression of p15E was the only parameter fluctuating in the experiments, the modulation of the cytokine release found in some experiments must be associated with this molecule.\u003c/p\u003e\n\u003cp\u003eIt remains unclear why the expression of p15E, especially on the cell surface, is so low. Surprisingly an arginine repeat was found in the protein sequence of p15E of PERV, which was absent in the sequence of p15E of MuLV [43]. This short arginine repeat suggests that the PERV protein could be, in contrast to the MuLV protein p15E, retained in the cell [43]. Arginine/serine rich proteins are mainly localised in the cytoplasm and are targeted to the nucleus [44,45]. Nevertheless, few p15E molecules can be found at the surface of the transfected cells (Figure 2). Two methods, immunofluorescence and flow cytometry showed independently the low expression in the cytoplasma of human 293 cells and the lower expression at the cell surface. However, it remains unclear whether in addition to p15E on the cell surface, released p15E molecules from the cytoplasma of disrupted cells have been also involved in generation of the observed immunosuppressive effects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite the low expression of p15E, cytokine expression and release from PBMCs of healthy humans were modulated in a manner consistent with previous observations for synthetic peptides corresponding to the ISU domain of PERV p15E and purified PERV particles [46, 47]. The sequence of the isu domain of PERV is identical to the isu domains of related gammaretroviruses such as murine leukaemia virus (MuLV), feline leukaemia virus (FeLV) and koala retrovirus (KoRV) [10]. Therefore, theoretically, evidence of immunosuppressive properties in synthetic peptides, viral or recombinant p15E, or virus particles from MuLV, FeLV, and KoRV inherently extends to the ISU domain of PERV, and vice versa [27, 48]. The immunosuppressive properties of MuLV, FeLV and KoRV as well as human endogenous retroviruses such as HERV-K are well studied \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003e(for review see [10, 11, 50]. The envelope proteins of endogenous retroviruses called syncytins play not only a role in the placentogenesis, but may also immunoprotect the embryo [50]. However, an involvement of PERV in pig placentogenesis was not yet demonstrated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunosuppression is a general property of all retroviruses, and immunodeficiency viruses such as the human immunodeficiency viruses HIV-1 and HIV-2 are well studied examples (for review see [10]). The changes in cytokine expression observed here are in agreement with changes in cytokine expression observed when human PBMCs were incubated with polymers of synthetic peptides corresponding to the isu domain of HIV [26] or with HERV-K particles released from a human teratocarcinoma cell line, with a recombinant transmembrane envelope protein of HERV-K or with peptides corresponding to the isu domain of HERV-K [25]. Modulation of cytokine expression was also observed when FeLV was analyzed (for review see [11]).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne major advantage of the established system is the absence of endotoxin. Endotoxin is able to induce cytokine modulation resembling the modulation observed here [13] and the probability of an endotoxin contamination below the detection limit of the used detection assay (EndoLISA System from Hyglos, Germany) was given when working with synthetic peptides or recombinant proteins produced in bacteria. Endotoxin is a lipopolysaccharide (LPS) of the outer membrane of most gram-negative bacteria, it binds first to the LPS-binding protein (LBP) and is transferred to cluster of differentiation 14 (CD14), where myeloid differentiation-2 protein (MD-2) and the Toll-like receptor 4 (TLR4) re-associate. The receptor binding leads to a signal transduction involving activation of the transcription factor nuclear factor-kappa B (NF-\u0026kappa;B), resulting in the release of cytokines [51]. This was the reason why in our later experiments gp41 produced in human 293 cells, was used [52]. The secreted and purified to homogeneity recombinant gp41 produced in 293 cells was soluble, glycosylated and assembled into trimers.\u0026nbsp;The protein bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal PBMCs [52].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe immunosuppressive properties of the transmembrane envelope protein gp41 of HIV-1 was also studied in a cellular system which was endotoxin-free. For this, murine cTRAMP prostate cancer cells were transfected with a gp41-expressing vector, and gp41 expression on the cell surface was demonstrated by FACS analysis, and the cells released gp41 into the cell supernatant [53]. These cells were pulsed with the ovalbumin-derived MHC-I peptide SIINFEKL and co-cultured with na\u0026iuml;ve CD8\u003csup\u003e+\u003c/sup\u003e T cells from OT-1 mice, which carry the corresponding SIINFEKL T-cell receptor. The gp41-expressing cells, but not the vector control cells, strongly inhibited IFN\u0026gamma; production and reduced CD25 (IL-2 receptor) expression. These findings indicated that gp41 impairs the antigen-specific response of murine CD8\u003csup\u003e+\u003c/sup\u003e T cells by drastically suppressing IFN\u0026gamma; production. Furthermore, this result corroborates previous findings that retroviral transmembrane proteins or peptides corresponding to their isu-domain exhibit interspecies reactivity by modulating immune cells across species (for review, see [10]).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To summarize, using a novel endotoxin-free cellular system to express the transmembrane envelope protein p15E of PERV, we gained new insights into the immunosuppressive properties of this molecule. Further experiments are required to enhance p15E expression levels to achieve more pronounced and conclusive results.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture and viruses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman kidney epithelial 293T cells, 293T cells infected with and producing PERV-A/C and porcine embryonic kidney pig PK15 cells were grown in Dulbecco Eagle Medium (DMEM) with 10% foetal bovine serum (FCS, PAN Biotech, Aidenbach, Germany, Lot P160616), and 1% penicillin-streptomycin (DMEM culture medium). Cells were maintained at 37°C in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e. PK15 cells harbour PERV-A and PERV-B, but not PERV-C proviruses in their genome, they release infectious virus particles. These cells were provided by Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). The PERV-A/C produced by 293T cells is the result of passaging cell-free virus on human 293T cells and is characterized by multimerized transcription factor binding sites in the long-terminal repeat (LTR) [37,38]. Since the number of repeats in the LTR changes during cultivation, a PCR was performed using LTR-specific primers in order to characterize the virus used in the present experiments. The length of the amplicon indicated that the virus had four and a half repeats as described previously [37] (Supplementary Figure 1). 293T cells were split twice in a week in a 1:3 ratio, PK15 were split 1:2 ratio every 3 days after washing with phosphate buffered solution (PBS) and trypsinization 0.25% trypsin/0.02% EDTA (PAN Biotech).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning of p15E\u003c/strong\u003e. Synthetic p15E constructs were produced based on the \u003cem\u003eenv\u003c/em\u003e gene of PERV-A/C (AY570980) as gene blocks (gBlock) (Integrated DNA Technologies IDT, Coralville, Iowa, USA) (Figure 1). All constructs contained the signal peptide of the \u003cem\u003eenv\u0026nbsp;\u003c/em\u003egene and a linker part of the gp70 gene coding for its first 23 amino acids, followed by the sequence for the furin cleavage site and modified sequence of the p15E gene. All constructs did not contain the fusion peptide (position 1390-1431). The constructs designated p15E-link contained the following modifications: a nucleotide change at position 1652 from g to c (leading to a cysteine to serin conversion). The constructs designated p15E-NHR does not contain the sequence coding for the unstructured N-terminal part including the fusion peptide but starts with the N-terminal helix (NHR) (nucleotide position 1456) of p15E; the nucleotide at position 1652 was not changed. For cloning purposes an Eco-R1 restriction site was added to the 5‘end including a Kozak sequence for optimal translation initiation. The 3‘end contained a sequence coding for a 6x histidine tag (p15E-link-His, p15E-NHR-His), and a stop codon followed by a Nhe-1 cutting site. The p15E gBlock with Eco-R1 and Nhe-1 site were cloned into pVitro2-EGFP [53] using the same two enzymes replacing the EGFP gene in the plasmid. All plasmids were sequenced before transfection into 293T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection of p15E.\u0026nbsp;\u003c/strong\u003eFor plasmid transfection 10\u003csup\u003e5\u003c/sup\u003e 293T cells were seeded in a 12 well plate the day before transfection. Next day medium was changed to DMEM with 5% FCS. For each transfection 3 µL of polyethylenimine (PEI) solution (1mg/mL) were added to 50 µL PBS; in parallel 1 µg plasmid DNA was added to 50 µL PBS [54]. Both solutions were vortexed at high speed for 1 min. After 10 min rest at room temperature PEI and DNA solution were gently mixed and incubated for 3 min at room temperature. The transfection solution was added dropwise to the cells. After 3 h medium was changed to DMEM culture medium. Selection was started 2 days after transfection with 500 µg/mL hygromycin\u0026nbsp;B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeripheral blood mononuclear cells (PBMCs)\u003c/strong\u003e.\u0026nbsp;At the Institute of Virology in Berlin, PBMCs were isolated from buffy coats from human blood from an anonymous donor using Ficoll-Hypaque density centrifugation with the use of Leucosep Tubes 50 mL (Greiner Bio-One, Kremsmünster, Austria) according to the instructions of the manufacturer (Greiner Bio-One). Buffy coat was diluted in a 1:2 ratio with PBS beforehand. Leucosep tubes were filled with 15 mL of Ficoll-Hypaque and centrifuged for 30 seconds at 1000x g at room temperature to move Ficoll--Hypaque below the porous barrier. 30 mL of diluted buffy coat was layered on top of the porous barrier and centrifuged at 1000xg for 10 minutes at room temperature without brakes. After centrifugation, the following layers were observed: plasma, enriched cell fraction (PBMCs), granulocytes and erythrocytes. The fraction containing PBMCs was harvested using a Pasteur pipette. The porous barrier effectively avoids recontamination with pelleted erythrocytes and granulocytes. Harvested PBMCs were washed twice with 10 mL of PBS and subsequently centrifuged for 10 minutes at 250x g. The PBMC pellet was resuspended in cell culture medium. Resuspended PBMCs were counted using Neubauer chamber and 1x10\u003csup\u003e8\u003c/sup\u003e PBMCs were frozen in cryopreserved tubes and stored in nitrogen tanks at -80° C in a freezing medium containing 70% DMEM, 20% FCS and 10% dimethyl sulfoxide (DMSO). Freshly isolated PBMCs were used for co-culture experiments.\u0026nbsp;The use of human blood has been approved by the ethical commission at the Medical Faculty of the Humboldt University Berlin.\u003c/p\u003e\n\u003cp\u003eAt the Transplant Laboratory in Hannover, PBMC were isolated from discarded material of normal routine apheresis samples from anonymized donors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations.\u0026nbsp;\u003c/strong\u003eAt the Institute of Virology in Berlin, buffy coats from human blood from an anonymous donor were provided by Deutsches Rotes Kreuz, Blutspendedienst Nord-Ost, Berlin. The use of human blood has been approved by the ethical commission at the Medical Faculty of the Humboldt University Berlin. At the Transplant Laboratory in Hannover, PBMC were isolated from discarded material of normal routine apheresis samples obtained from the Department of Transfusion Medicine of the Hannover Medical School. Samples were anonymized and could not be assigned to an individual donor. The local ethics committee of Hanover Medical School approved this procedure. All methods were carried out in accordance with DFG guidelines of Good Scientific Practice. Informed consent to participate was waived by an Institutional Review Board (IRB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-cultivation.\u0026nbsp;\u003c/strong\u003e293T and PK-15 are adherent cells and were used at 80 to 90% confluence, the cells were washed with PBS, incubated with 0.25 % trypsin/0.02 %EDTA at 37\u003csup\u003eo\u003c/sup\u003eC for 2 to 15 minutes. 10 mL of culture medium containing FCS was added cells were collected in a 15 mL falcon tube, centrifuged at 300x g for 5 minutes. Pellets were resuspended in 10 ml culture medium. Cells were counted twice using a Neubauer Chamber, centrifuged at 300 x g for 5 minutes and culture medium was added to obtain 3 x 10\u003csup\u003e5\u003c/sup\u003ecells/100 μL. 100 μL of cells were added to each well of 96 well plate and 100 μL of culture medium without hygromycin was added into the wells respectively. Cells were incubated overnight at 37°C in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e. The next day, 100 μL of media was removed as 293T and PK-15 adhere to the surface of the plate and 3 x 10\u003csup\u003e5\u003c/sup\u003e PBMCs in a volume of 100 μL was added for co-incubation and left overnight in 37°C in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e. For the PCR expression studies 2.5 x 10\u003csup\u003e4\u003c/sup\u003e/100µl 293T cells and 7.5 x 10\u003csup\u003e4\u003c/sup\u003e/100µl PBMCs were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence analysis.\u0026nbsp;\u003c/strong\u003e293T cells were gently dislodged from cell culture flasks with ice-cold PBS and counted with a Neubauer chamber after trypan blue staining to detect dead cells. Cell suspension was adjusted to 250 x 10\u003csup\u003e3\u003c/sup\u003e cells/mL. For each sample 50 x 10\u003csup\u003e3\u003c/sup\u003e cells in 200µL PBS were transferred to a glass slide using a Cellspin 1 device (Tharmac, Limburg/Lahn, Germany) at 8,000 rpm for 10 min according to the manufacturer’s instruction. Slides were kept at room temperature until completely dry. Cells were fixed in 4% formaldehyde in PBS for 15min followed by washing in PBS, 3´\u0026nbsp;for 10 min. Perforation of the cell membrane was achieved by 15 min incubation in PBS with 0.5 % Triton X-100 (Carl Roth, Karlsruhe, Germany) followed by PBS washes as described above. Slides were incubated with 3 % BSA in PBS for 1 h to reduce unspecific binding followed by incubation with a 1:100 dilution of goat anti-p15E (goat #355, [39]) in 3 % BSA/PBS for 1 h. After PBS wash (3´, 10 min) FITC conjugated anti-goat antibody from donkey (Merck/Sigma Aldrich, Darmstadt, Germany) was incubated for 1 h at room temperature. Cover slips were added after all liquid was removed and a mounting dye containing 4′,6-diamidino-2-phenylindole (DAPI) was added (Carl Roth, Karlsruhe, Germany).\u003c/p\u003e\n\u003cp\u003eSamples were analysed with a Zeiss Axio fluorescence microscope equipped with an Axiocom 503 mono camera and a Colibri 7 LED light source using ZEN 2.3 software (Zeiss, Oberkochen, Germany). SMART set up provided by the software was used to adjust the fluorescence signals. Exposure time for the FITC channel was set to 5 sec (DAPI 50 msec). Unspecific background signals in the FITC channel were subtracted by setting the threshold in the negative control (untransfected 293T cells) to zero/black. These setting were used for all samples. DAPI staining was adjusted to highest contrast.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies and flow cytometry.\u0026nbsp;\u003c/strong\u003eA goat anti-p15E serum was used to monitor p15E expression after transfection of human 293T. Generation and characterization of the goat anti-p15E serum #355 had been described previously [39]. The cells were incubated with the serum (30 min, 1:40 dilution), followed by two additional incubation steps using biotinylated bovine anti-goat Ig (Dianova, Hamburg, Germany) and allophycocyanin (APC)-conjugated streptavidin (BD Biosciences, San Jose, CA). To monitor intracellular levels of p15E, transfected and control cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with saponin (0.2%) for 10 min at room temperature and then incubated with the anti-p15E antiserum. Expression of MHC class-I molecules (HLA-ABC) on 293T cells was detected by indirect staining using monoclonal antibody (mAb) W6/32 (American Type Culture Collection, ATCC, Manassas, Virginia, USA) and phycoerythrin (PE)-conjugated rat anti-mouse kappa light chain (BD, Biosciences). Directly labelled mAb CD56-APC (B159; BD Biosciences) in combination with CD107a-PE (H4A3; BD Biosciences) were used to study degranulation of human NK cells in response to 293T or L23 cells. Analyses were performed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and data were processed by using FCS Express 7 (De Novo software, Pasadena, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD107a assay.\u0026nbsp;\u003c/strong\u003eCytotoxic effector cells were generated by culturing PBMC for 5 to 7 days in the presence of 50 ng/ml IL-2. The cells (2x10\u003csup\u003e5\u003c/sup\u003e) were then co-cultured for two hours with 2x10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003e293T wt cells or 293T cells transfected to express p15E (293T-p15E-NHR-His or 293T-p15E-link-His). Cytotoxic activity against 293T target cells was monitored by assessing CD107a expression (degranulation) on gated CD56\u003csup\u003e+\u003c/sup\u003e CD45\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eNK cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis.\u0026nbsp;\u003c/strong\u003eStatistical analysis was performed by using the Student’s t test. Levels of significance are given as P-values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA and RNA extraction.\u003c/strong\u003e In order to demonstrate the presence of the sequence encoding p15E with its isu domain, the cell lines 293T-wt, 293T-p15E-NHR-His and 293T-p15E-Link-His were tested using conventional PCR. Genomic DNA was isolated from transfected and non-transfected cells using DNA Easy Blood and Tissue Kit (Qiagen). 5 x 10\u003csup\u003e6\u003c/sup\u003e cultured cells of each cell line were centrifuged for 5 min at 300 x g and resuspended in 200 μL of PBS. Further steps of DNA extraction were performed using the Instruction manual provided by Qiagen to purify total DNA from animal blood or cells (DNeasy Blood and Tissue Handbook, Spin-Column protocol). All centrifugation steps were performed at room temperature. DNA concentration was quantified twice using nanodrop 1000 spectrophotometer (PeqLab, Erlangen, Germany). Purified plasmids of p15E-NHR-His and p15E-Link-GFP (1:100 and 1:1000) were used as positive controls. 293T cells were used as a negative control. DNA concentrations were standardized using nuclease free water and 5 μL of DNA sample was added to each reaction mix. DNA from virus producing 293 cells was isolated using the DNeasy Blood and Tissue kit (Qiagen). In order to analyze the expression of cytokines, RNA was isolated using the RNeasy Kit (Qiagen) and DNAse treatment to remove cellular DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolymerase chain reaction (PCR).\u003c/strong\u003eIn order to characterize PERV proviruses in the virus-producing 293T cells, PCRs using specific primers for the pol and LTR region of PERV (Table 1) were performed: 5 min denaturation at 95°C, and 45 cycles (15s 95°C, 30s 62°C, 30s 72°C). PCR was performed using a Biometra Thermocycler (Analytik Jena, Germany), electrophoresis in a 1.3% agarose gel and the GeneRuler 1 kbp DNA ladder (Thermo Scientific) were used for gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcriptase real-time polymerase chain reaction (RT-real-time PCR).\u0026nbsp;\u003c/strong\u003eIn order to analyze the expression of different cytokines, RT-real time PCRs were established using specific primers for IL-6, IL-10, INF-g, TNF- a, MMP1 and SEPP1 (Table 1). Reaction mixes containing PCR buffer, forward and reverse primers and probes, dNTPs, AmpliTaq Polymerase (Thermo Scientific, Schwerte, Germany), nuclease free water and DNA sample were incubated 12 min at 95°C and 40 PCR cycles (20s denaturation at 95°C, 30 s annealing at 55°C, 1 min extension and 20s at 72°C followed by an extension step for 15 min at 72°C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cytokine release by ELISA.\u003c/strong\u003e To detect IL-10, an ELISA for human IL-10 (R \u0026amp; D Biosciences) was used. Supernatants obtained from co-culture of human PBMCs with transfected and non-transfected cells (3 x 10\u003csup\u003e5\u003c/sup\u003e each sample group) were used for the assay. Supernatant containing proteins was collected after 24 hours of incubation by centrifuging at 2000 x g for 10 min. ELISAs were performed in duplicates according to protocols of the supplier: 200 μL of standard, control and sample supernatant were added per well of ELISA microplate and covered with an adhesive strip for incubation for 2 hrs at room temperature. After incubation, wells were washed with 400 μL of wash buffer using a squirt bottle for a total of 4 washes. 200 μL of human IL-10 conjugate was added to each well and incubated for an hour at room temperature. Washing was repeated and subsequently 200 μL of substrate solution was added to each well and incubated for 30 minutes at room temperature. Subsequently, 50 μl of stop solution was added to each well and colour change from blue to yellow was observed. Optical density was determined within 30 minutes using a microplate reader set to 450 nm. To detect IL-6, an ELISA for human IL-6 (Sigma Aldrich, St. Louis, MI, USA) was used. Supernatants containing proteins were collected after 24 hours of incubation by centrifuging at 2000g for 10 min. ELISAs were performed in duplicates according to protocols of the supplier. Standards, buffer solutions and detection antibodies were prepared as mentioned in manufacturers manual. 100 μL of supernatant from co-culture of different samples were added in the wells along with standard. Samples and standard were incubated overnight at 4°C with gentle shaking. The next day, solutions were discarded, and wells were washed with 300 μL wash buffer 4 times with a multichannel pipette. Following the washing step, 100 μL of biotinylated antibody was added to each well and incubated at room temperature for 60 minutes with gentle shaking. Biotinylated antibody was removed, and wells were washed and 100 μL of streptavidin solution was added to each well afterwards. Followed by an incubation for 45 minutes with gentle shaking. Streptavidin was removed from wells and washed, subsequently 100 μL of TMB one step substrate reagent was added to each well and incubated for 30 minutes in the dark with gentle shaking. After 30 minutes, 50 μL of stop solution was added to stop reaction and optical density of solution in wells were measured immediately at 450 nm on a microplate reader.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JD; data curation, formal analysis, investigation, methodology, RS, CP, BD, LL; funding acquisition: JD, BK, RS; supervision: JD, BK, RS; writing – original draft: JD, RS; writing – review and editing: JD, RS, BK. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by the Deutsche Forschungsgemeinschaft, TRR127.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Ludwig Krabben, Institute of Virology, for the constructs, and transfected cells, Axel Teigeler, Institute of Virology, and Antje Brinkmann, Transplant Laboratory, for excellent technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to J.D.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher’s note\u0026nbsp;\u003c/strong\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMoir S, Chun TW, Fauci AS. 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Genome-wide inactivation of porcine endogenous retroviruses (PERVs). \u003cem\u003eScience.\u003c/em\u003e\u003cstrong\u003e350(6264),\u003c/strong\u003e 1101\u0026ndash;4 (2015).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1 Primers used for the analysis of cytokine expression and provirus detection\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequenz\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enumber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocalisation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ePK 34\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-AAAGGATGAAAATGCAACCTAACC-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCzauderna et al. [55]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eY17012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3 - 26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ePK 26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-ACGCACAAGACAAAGACACACGAA-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1134 - 1111\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ePERV \u003cem\u003epol\u0026nbsp;\u003c/em\u003efw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-CGACTGCCCCAAGGGTTCAA-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eYang et al. [56]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eHM159246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3568\u0026ndash;3587\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003ePERV \u003cem\u003epol\u0026nbsp;\u003c/em\u003erev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-TCTCTCCTGCAAATCTFFGCC-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3803\u0026ndash;3783\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGAPDH fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-GGCCATGCTGGCGCTGAGTAC-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eDenner et al. [26]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 002046.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e364-386\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGAPDH rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-TGGTCCACACCCATGACGA-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e494-512\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGAPDH probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-HEX-CTTCACCACCATGGAGAAGGCTGGG-BHQ-1-3\u0026acute; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e405-429\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIFN-\u0026gamma; fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-TGCAGAGCCAAATTGTCTCC-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003ethis manuscript\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 000619.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e328-347\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIFN-\u0026gamma; rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-TGCTTTGCGTTGGACATTCA-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e502-521\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIFN-\u0026gamma; probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-6-FAM-ACCATCAAGGAAGACATGAATGTCAAG-BHQ-1-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e408-434\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIL-6 fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-GGTACATCCTCGACGGCATCT-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eDenner et al. [26]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 000600.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e289-309\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIL-6 rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-GTGCCTCTTTGCTGCTTTCAC-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e349-369\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIL-6 probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-6-Fam-TGTTACTCTTGTTACATGTCTCCTTTCTCAGGGCT-BHQ-1-3\u0026rsquo; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e311-345\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIL-10 fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-CCACGCTTTCTAGCTGTT-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eDenner et al. [26]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 000572.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e966-983\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIL-10 rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-CTCCCTGGTTTCTCTTCCTAA-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1058-1078\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eI-10 probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-6-FAM-TCTTGTCTCTGGGCTT-BHQ-1-3\u0026rsquo;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1015-1030\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMMP1 fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-CATCCAAGCCATATATGGACG-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eDenner et al. [26]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 002421.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e908-928\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMMP1 rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-TCTCTTAAAACTGAGAGGTCT-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1498-1518\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMMP1 probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-6-FAM-CTGGGCTGTTCAGGGACAGAA-BHQ-1-3\u0026rsquo; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1187-1207\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eSEPP1 fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-CATGGACATCAGCACCTT-3\u0026rsquo;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eDenner et al. [26]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 005410.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e774-459\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eSEPP1 rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-TCGACAGAGCTTCTTTTG-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e954-972\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eSEPP1 probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-6-FAM-AGAATCAGCAACCAGGAGCA-BHQ-1-3\u0026acute; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e721-740\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha; fw\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-GAGAAGCAACTACAGACCCC-3\u0026acute;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003ethis manuscript\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNM 000594.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e48-67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha; rev\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026acute;-CATGCTTTCAGTGCTCATGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e176-195\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha; probe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 425px;\"\u003e\n \u003cp\u003e5\u0026rsquo;-6-FAM-ACAACCCTCAGACGCCACATCC-BHQ-1-3\u0026rsquo;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e76-97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Retroviruses, porcine endogenous retroviruses (PERV), immunosuppression, transmembrane envelope protein, immunosuppressive peptide, cytokines","lastPublishedDoi":"10.21203/rs.3.rs-5967592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5967592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Retroviruses are immunosuppressive and there is evidence that a highly conserved immunosuppressive domain (isu domain) in their transmembrane envelope protein contributes to this activity. Studies have shown that disrupted retroviruses, their purified transmembrane envelope proteins and synthetic peptides corresponding to the isu domain inhibit mitogen-triggered proliferation of peripheral blood mononuclear cells (PBMCs) and modulate their cytokine expression in vitro. In vivo, in a mouse tumour model, tumour cells that were unable to induce tumours in immunocompetent animals, gained the ability to do so when expressing the transmembrane envelope protein or the isu domain of various retroviruses on their surface. However, criticism arose that endotoxin contaminations in retroviral preparations might explain the observed cytokine modulation, as endotoxins are capable to induce similar effects. Here we demonstrate that in an endotoxin-free system, the transmembrane envelope protein p15E of PERV can modulate cytokine expression in human PBMCs. Human 293 cells were transfected with constructs expressing p15E. These transfected cells were co-cultured with human PBMCs resulting in the release of IL-10 protein and modulation of several cytokines and other markers, including IL-6, IL-10, IFN-, TNF-, MMP1, and SEPP1. Additionally, p15E expression reduced MHC class I expression and had a protective effect against cellular cytotoxicity. Notably, the expression of p15E was minimal, which explains why no effect was observed in certain experiments. This finding underscores the need for further research to elucidate the dynamics of p15E expression and its immunosuppressive activity.","manuscriptTitle":"Further evidence for the immunosuppressive activity of the transmembrane envelope protein p15E of the porcine endogenous retrovirus (PERV)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-27 14:00:29","doi":"10.21203/rs.3.rs-5967592/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd433bfd-3101-4a1e-b5f6-137d86be566d","owner":[],"postedDate":"February 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44884200,"name":"Biological sciences/Cell biology"},{"id":44884201,"name":"Biological sciences/Immunology"},{"id":44884202,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-02-27T14:00:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-27 14:00:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5967592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5967592","identity":"rs-5967592","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}