Influence of the HERV-K TM protein on the cytokine release by donor PBMCs.

<p><b>a,</b> Dose-dependent induction of Il-10 release in PBMCs from two donors by the TM protein of HERV-K (0– medium control) compared to control protein BSA as measured by ELISAs (mean±SD; n = 3). <b>b,</b> Cytokine array measuring simultaneous release of cytokines from human donor PBMCs incubated with the TM protein of HERV-K, or control protein BSA, or medium alone after 24 hrs incubation. The up-regulated cytokines are circled. A list of all analysed cytokines and their full names are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070399#pone.0070399.s002" target="_blank">Table S1</a>.</p

Induction of IL-10 release in human PBMCs by HERV-K and PERV particles.

<p><b>a,</b> Characterisation and estimation of the amount of added HERV-K and PERV by SDS-PAGE and Coomassie blue staining. HERV-K containing 60 ng Gag (capsid, CA) and PERV containing 100 ng Gag (capsid, CA) were loaded per lane and the amount of the Gag (CA) proteins was used for comparison. <b>b,</b> Detection of the TM protein gp36 of HERV-K in the pellet used for incubation with human PBMCs by a TM specific antiserum, M – marker, control – pelleted supernatant from uninfected 293 cells, HERV-K – pelleted supernatant from GH cells. <b>c,</b> IL-10 release by human PBMCs induced by HERV-K particles, by the pellet from the supernatant of uninfected 293 cells corresponding to the same amount of supernatant (control), and by purified PERV particles produced on 293 cells. Virus containing approximately 100 ng Gag (CA) protein, which was used for comparison (see below) and 10 ng TM protein, was added to the first well (1), the dilutions 50/5 ng (2) and 25/2.5 ng (3) are also shown. IL-10 was measured after 24 hrs of incubation.</p

Influence of the recombinant TM protein of HERV-K on the proliferation of ConA-stimulated PBMCs from one healthy human blood donor (a, b) or murine splenocytes (c).

<p>Cell proliferation was measured using the Alamar blue assay (<b>a, c</b>) or by ³H-thymidine incorporation (<b>b</b>) (mean±SD; n = 3). ³H-thymidine was added on day three and cells were then harvested one the next day and the counts per minute were determined, dark gray – isu peptide-BSA conjugates, gray – BSA conjugates, added at day 0. Black (Pos, positive control) – medium alone.</p

Genes with the highest up-regulation (upper part) or down-regulation (lower part) of their expression in PBMC of one donor in response to the incubation with the TM protein of HERV-K.

<p>Fold changes indicate gene expression compared to control cells incubated in medium. The full names of the genes and 40 other up- or down-regulated genes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070399#pone.0070399.s003" target="_blank">Tables S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070399#pone.0070399.s004" target="_blank">S3</a>.</p

Influence of the homopolymer of the isu peptide of HERV-K on the IL-10 release by donor PBMCs.

<p><b>a,</b> Comparison of the isu peptide homopolymer (grey) with a randomised peptide (dark grey). <b>b,</b> Comparison of the IL-10 release from PBMCs of six donors treated with one batch and the same amount of the isu peptide homopolymer, the IL-10 release of their PBMCs incubated with medium alone was zero.</p

Localisation and sequence of the immunosuppressive (isu) domain of the TM protein of HERV-K (accession number Q69384).

<p><b>a,</b> Functional domains of the TM protein: FP, fusion peptide; FPPR, fusion peptide proximal region; NHR, N-terminal helical region; ISU, isu domain; C-C, cystein-cystein loop; CHR, C-terminal helical region; MPER, membrane proximal external region; MSD, membrane spanning domain. In the amino acid sequence of the isu domain stars (*) indicate NH₂ groups, points (.) mark COOH groups relevant for polymerisation. <b>b,</b> Sequence comparison of the core (1 corresponds to the amino acid 552, 14 to 535, acc. Nr. Q69384) of the immunosuppressive domain of different retroviruses (MuLV, murine leukaemia virus; CKS-17 consensus, consensus sequence of the gammaretroviruses PERV, porcine endogenous retrovirus; HERV-K, -W, -FRD; human endogenous retroviruses-K, -W, -FRD; HIV-1, -2, human immunodeficiency viruses - 1, -2¸ MMTV, mouse mammary tumour virus; HERV-K, human endogenous retrovirus-K). Amino acids identical to that in the first sequence of each group are indicated gray. In addition, amino acids present in all retroviruses are marked green, in all gammaretroviruses and HERV-K pink, in HIV-1 and HERV-K orange and in MMTV and HERV-K blue. In the sequence of HIV-1 the amino acids with high importance are shown in bold, mutation of these amino acids totally abrogated the activity to induce IL-10 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070399#pone.0070399-Morozov1" target="_blank">[27]</a>.</p

Summary table.

<p>*- indeterminate</p><p>**—maternal anti-HEV IgG; n.a.-not available; n.d.-not done.</p><p>HEV in minipigs.</p

Western blot analysis of sera from Göttingen Minipigs.

<p>(A) Western blot analysis of sera from retired breeders (Group 1). The numbers on strips corresponded to the order of animals in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.t002" target="_blank">Table 2</a>. “+”–serum from a HEV infected pig diluted 1:300; “+ (bold)” serum from infected pig diluted 1:150. The antigen load was 300 ng/strip. Animal sera were tested twice in dilution 1:150. (B) Analysis of sera from sow-piglet pairs (Group 4). Odd numbers 1, 3, 5, 7, 9, 11 –strips incubated with sera from sows; even numbers 2, 4, 6, 8, 10, 12 –strips incubated with sera from piglets. Strip 4 was incubated with serum of piglet #319428. Strips 7, 8 (underlined) were treated with serum of the sow #314253 and serum of piglet #320282, respectively. “+” -serum from HEV infected pig; “-” serum from non-infected pig. The antigen load was 300 ng/strip. The sera specimens were diluted 1:150.</p

Microorganisms tested negative in three “retired breeders” minipigs, age 24–48 months.

<p>Microorganisms tested negative in three “retired breeders” minipigs, age 24–48 months.</p

HEV genome, primers and probes, recombinant proteins and real-time RT-PCR parameters.

<p>(A) Schematic presentation of the HEV genome with three open reading frames (ORF 1–3). UTR—untranslated region. The numbers of nucleotides are given from the first nucleotide of ORF 2. Cap- cap structure, Poly (A)—poly A sequence. PCR primers (black arrows) and probes (red arrows) used in the real-time RT-PCR methods “A”, “J”, “M1”,and “M2” for the detection of HEV ORF2. Primers for methods M1 and M2 are given in brackets as 1 or 2, respectively. Numbers are given as in A. ^*Method “A” was established by Adlhoch et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.ref041" target="_blank">41</a>]; **Method “J” was established by Jothikumar et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.ref059" target="_blank">59</a>]. (B) Parameters of the used real-time RT-PCR methods. Standard curves and PCR efficiencies are shown. One reference plasmid was used for the real-time RT-PCR method “A” and another, for the other three methods. (C) HEV ORF 2 is coding for the 660 aa long capsid glycoprotein. Putative glycosylation sites are marked in green, a low probability glycosylation site is marked in grey. Positions of the asparagine are numbered. The immunodominant region (IDR) is given in yellow. The signal peptide is shown as a blue arrow. (D) Recombinant proteins (Prospec and GT3) used as antigens in Western blot analysis are shown. GT3 contains the entire immunodominant region (IDR). The Prospec antigen contains glutathion-S-transferase (GST) on the N-terminus. GT3 contains a 6His-tag on the N-terminus. The fragments corresponding to the ORF2 are given in green. The numbers started from the first amino acids of the capsid protein. (E) Comparative analysis of the recombinant antigens by SDS-PAGE is shown on the right. 500 ng of proteins were loaded on the gel, the gel was stained with Coomassie brilliant blue. Lane 1 –Prospec, lane 2 –GT3, M–Size markers.</p