Localization and expression of EBNA1-SIIN-GFP frameshift constructs.

<p>Panel A, GFP fluorescence of EGFP-N1, E1-GA(wild-type)-SIIN-GFP, E1ΔGA-SIIN-GFP, E1-GQE(frameshift 1)-SIIN-GFP or E1-GRS(frameshift 2)-SIIN-GFP expression constructs in 293KbC2 cells. The cells were examined using a laser-scanning Bio-Rad (Hercules, CA) MRC600 confocal microscope with original magnification ×63. Panel B, DAPI staining, and; Panel C, phase contrast images of the EBNA1-SIIN-GFP transfected cells. Panel D, Flow cytometric analysis of GFP expression in 293KbC2 cells following transfection with EGFP-N1 or EBNA1-SIIN-GFP frameshift variants. The Mean Fluorescence Intensity (MFI) of the EBNA1-GFP positive cells is indicated in the top right hand corner of each plot.</p

Schematic description of EBNA1 expression constructs containing identical mRNA sequences whilst encoding alternative repeat reading frames.

<p>A, The EBNA1 frameshift constructs were generated in either pcDNA3 for <i>in vitro</i> translation studies or in pEGFP-N1 for EBNA1-GFP expression and immunological studies. The overlapping DNA binding and dimerization domain, nuclear localization signal (NLS) and Glycine/Arginine (GR) repeat flanking sequences essential for genome maintenance functions are shown. The localizations of a model SIINFEKL epitope and the endogenous EBNA1 HPVGEADYFEY epitope used in the presentation assays are highlighted. B, Alignment of amino acid sequences of the EBNA1 (E1) repeat frameshift variants E1-GA(wild-type), E1-GQE(frameshift 1) and E1-GRS(frameshift 2). An asterisk indicates identical residues in all three proteins. Arrows denote nucleotide deletion positions at the start of the internal repeat and nucleotide insertion positions at the ends of the repeat in the E1-GA(wild-type) sequence to generate alternative repeat reading frames whilst maintaining wild-type EBNA1 sequence at both the N- and C-terminal domains flanking the internal repeat sequence.</p

Localization, expression and T-cell recognition of 293KbC2 cells transfected with an EBNA1-Ateline-SIIN-GFP expression construct.

<p>A, GFP fluorescence, DAPI staining and Flow cytometric analysis of GFP expression in 293KbC2 cells following transfection with an EBNA1-Ateline-SIIN-GFP expression construct. The cells were examined using a laser-scanning Bio-Rad (Hercules, CA) MRC600 confocal microscope with original magnification ×63. The Mean Fluorescence Intensity (MFI) of the EBNA1-GFP positive cells is indicated in the top right hand corner of the FACS plot. B, T-cell recognition of 293KbC2 cells transfected with EBNA1-SIIN-GFP variants. 293kbC2 cells expressing EBNA1-SIIN-GFP encoding either wildtype or Ateline repeat peptide sequences or no repeat were exposed to SIIN-specific OT-1 T-cells and incubated for 3 hours in the presence of brefeldin A at responder to stimulator ratios of 2.5∶1–20∶1. Following incubation, IFN-γ production by OT-1 T-cells was determined by intracellular cytokine staining. These data are representative of three separate experiments.</p

Dot-plot analyses illustrating pair wise local alignments between the EBNA1 mRNA sequence and the mRNAs for several gammaherpesvirus maintenance protein sequences.

<p>The over-all homology between sequences is shown as a straight line on the diagonal, while regions of repeats are shown as lots of lines in the same region, allowing visualization of where the repeated regions are between sequences. Panel A, Nucleotide sequence homology between <i>human herpesvirus-4</i>, <i>(HHV-4)</i> EBNA1 and <i>human herpesvirus-8 (HHV-8)</i> LANA1; Panel B, <i>(Papiine herpesvirus-1, HV-1)</i> baboon EBNA1; Panel C, <i>(Macacine HV-4)</i> rhesus EBNA1; Panel D, <i>(Alcelaphine HV-1)</i> ORF73; Panel E, <i>(Ovine HV-2)</i> ORF73, and; Panel F, <i>(Samirine HV-2)</i> ORF73. In each panel the intensity of the dot plots indicate the level of homology between the sequences being compared. Transcript sizes are shown in kb on both axes. The Genbank accessions numbers for these sequences are listed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#s4" target="_blank">Materials and Methods</a>.</p

Detection of H-2K^b–SIIN complexes on the surface of 293KbC2 cells expressing EBNA1-SIIN-GFP frameshift variants.

<p>H-2K^b–SIIN expression was assessed by flow cytometry of 293KbC2 cells transfected with E1-GA(wild-type)-SIIN-GFP, E1ΔGA-SIIN-GFP, E1-GQE(frameshift 1)-SIIN-GFP or E1-GRS(frameshift 2)-SIIN-GFP following staining with the monoclonal antibody 25D1.16 conjugated to Allophycocyanin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#ppat.1003112-Porgador1" target="_blank">[38]</a>. Values shown in each FACS plot are the percentage of GFP⁺H-2K^b–SIIN⁺ cells as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#s4" target="_blank">Materials and Methods</a>. These data are representative of three separate experiments.</p

T-cell recognition of 293KbC2 cells transfected with EBNA1-SIIN-GFP frameshift variants.

<p>Panels, A and B: 293kbC2 cells expressing EBNA1-SIIN-GFP encoding either alternative repeat peptide sequences or no repeat were exposed to SIIN-specific OT-1 T-cells and then incubated for 3 hours in the presence of brefeldin A at a responder to stimulator ratio of 5∶1 (panel A) and ratios of 2.5∶1–40∶1 (panel B). Following incubation, IFN-γ production by OT-1 T-cells was determined by intracellular cytokine staining. The top right hand corner of the FACS plot in panel A indicates the percentage of OT-1-specific CD8⁺ lymphocytes producing IFN-γ. Panels, C and D: EBV-negative DG75 cells were co-transfected with EBNA1-GFP expression constructs encoding alternative repeat peptide sequences or no repeat and a HLA B*3508-GFP expression vector. The transfected cells were exposed to HPV-specific T-cells and incubated in the presence of brefeldin A overnight at a responder to stimulator ratio of 5∶1 (panel C) and ratios of 2.5∶1–20∶1 (panel D). Following incubation, IFN-γ production by HPV-specific T-cells was determined by intracellular cytokine staining and shown in the top right hand corner of the FACS plot in panel C as the percentage of HPV-specific CD8⁺ lymphocytes producing IFN-γ. These data are representative of three separate experiments.</p

Intracellular degradation kinetics and expression of alternative EBNA1 repeat peptide sequences.

<p>A, 293KbC2 cells were transfected with the following expression constructs E1ΔGA-SIIN-GFP; E1-GA(wild-type)-SIIN-GFP; E1-GQE(frameshift 1)-SIIN-GFP or E1-GRS(frameshift 2)-SIIN-GFP in the presence or absence of the proteasome inhibitor MG132 (10 µM). At 24 h post-transfection, the cells were incubated with cycloheximide (50 µg/ml) and then monitored over a 30-h time course as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#s4" target="_blank">Materials and Methods</a>. EBNA1-SIIN-GFP expression of the cells at each time point was monitored by flow cytometry and plotted as the relative change in the levels of EBNA1-GFP expression following the addition of cycloheximide at time point 0. B, <i>In vitro</i> translation (IVT) assay of pcDNA3 expression constructs encoding E1ΔGA (lane 1), E1-GA(wild-type) (lane 2), E1-GQE(frameshift 1) (lane 3) or E1-GRS(frameshift 2) (lane 4).The constructs were transcribed and translated <i>in vitro</i> with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. ³⁵S-methionine-labeled proteins were visualized by autoradiography (upper panel). An asterisk indicates the full-length translation product of each EBNA1 frameshift variant. Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities (lower panel). These data are representative of three separate experiments.</p

Homologies among gammaherpesvirus maintenance proteins.

1<p>CR 1–3 designates sub-regions of internal central repeats displaying varying peptide sequences.</p>2<p>Homology data for protein sequences was acquired using the Strider sequence alignment program that uses the BLOSUM62 score matrix to score pairs of aligned residues <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003112#ppat.1003112-Henikoff1" target="_blank">[42]</a>.</p

A network of miRNAs (arrow shapes) that were identified as being differentially expressed in BMEs at 4 hours post-infection with <i>S. uberis</i> and their predicted target genes (circles).

<p>Red arrow shapes represent up-regulated miRNAs at 4 hpi; green down-regulated. Red circles represent target genes that are annotated by <a href="http://www.innatedb.com" target="_blank">www.innatedb.com</a> as having a role in innate immunity. The network was constructed and visualised in Cytoscape v2.8.2. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057543#pone.0057543-Shannon1" target="_blank">[69]</a>.</p

miRNA target predictions by miRanda and TargetScan and their intersect.

<p>miRNA target predictions by miRanda and TargetScan and their intersect.</p