Phagosomal TAP transports peptides into the lumen.

<p>A) Isolated GFP-Mtb-phagosomes were incubated with TMR-conjugated CFP10_2–12 peptide (blue), then fixed and stained with anti-TAP1 (red) and analyzed by fluorescence microscopy. B) TMR-conjugated CFP10_2–12 peptide (500 nM) and UL49.5-NP (10 uM) or UL49.5-SCR (10 uM) were added simultaneously to isolated Mtb phagosomes and incubated at 37° C for 30 min. Washed and fixed phagosomes were analyzed by FACS. FACS plot is representative of three independent experiments. C) Prior to infection, Mtb was coupled to UL49.5-NP or UL49.5-SCR to target the inhibitor specifically to the Mtb phagosome. Isolated phagosomes were then incubated with TMR-conjugated CFP10_2–12 peptide as indicated above. FACS plot is representative of two independent experiments.</p

UL49.5 peptide inhibits TAP-dependent presentation of antigen to CD8⁺ T cell clones.

<p>A-B) Murine DCs were incubated with 0.5 mg/ml OVA and the indicated concentrations of the UL49.5-NP. DCs were then co-cultured with OT-I T (A) or OT-II (B) cells and supernatants were analyzed for IL-2 by ELISA. Shown is the mean and standard error of 2 independent experiments. * p = 0.07; ** p = 0.01; *** p = 0.007 (Student's two-tailed t test). C) Human monocyte derived DC were incubated for 1 hr with UL49.5-NP (5 uM) or UL49.5-SCR (5 uM) peptides and then infected with H37Rv-eGFP (MOI = 10) overnight. IFNγ production by the TAP-dependent HLA-E-restricted T cell clone D160 1-23 or the TAP-independent Class II clone D454 E12 was assessed by ELISPOT. IFNγ response by each T cell clone following UL49.5-NP treatment was normalized to the response in the presence of UL49.5-SCR. Shown is the mean response and standard error from at least 4 independent experiments. *** denotes significantly reduced IFNγ production by D160 1–23 in the presence of UL49.5-NP compared to D454 E12 (Student's two-tailed t test, p<0.001).</p

Interactions between herpesvirus-encoded TAP-inhibitors and their target.

<p>Upper illustration: model of the TAP transporter, comprising the two subunits TAP1 and TAP2. Each subunit contains a transmembrane domain (TMD), encompassing 10 and 9 transmembrane (TM) helices for TAP1 and TAP2, respectively. The outer N-terminal helices of TAP1 and TAP2 (TMD0) form an autonomous binding platform for tapasin, whereas the core 6 TM helices are necessary for peptide transport. A peptide-binding domain is located within the cytosolic extensions of the TM helices. In addition, TAP1 and TAP2 contain a nucleotide-binding domain (NBD) in the cytosol, which harbors two ATP-binding sites. Lower illustrations: schematic representations of the interaction between the viral proteins and TAP. The sites where TAP is affected are indicated. HSV-1 ICP47 prevents peptide transport by physically obstructing the peptide-binding site. PRV, BoHV-1 and EHV (EHV-1 and EHV-4) UL49.5 leave the transporter in a transformation-incompetent conformation, thereby preventing the structural changes that are needed to translocate peptides over the ER membrane. BoHV-1 UL49.5 is known to interact with a region within the core domain of TAP, comprising the C-terminal 6 TM domains of both TAP1 and TAP2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref132" target="_blank">132</a>]. BoHV-1 UL49.5 induces the degradation of both TAP subunits, and EHV UL49.5 prevents ATP binding to TAP. HCMV US6 blocks TAP by inducing conformational changes that result in diminished ATP binding to TAP1. The protein interacts with TM domains 7–10 of TAP 1 and TM 1–4 of TAP2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref095" target="_blank">95</a>]. EBV BNLF2a inhibits peptide transport by interfering with both peptide and ATP binding to TAP.</p

Phylogenetic tree for selected members of the family Herpesviridae.

<p>The Bayesian tree is based on amino acid sequence alignments for six large, well-conserved genes, namely the orthologs of HSV-1 genes UL15, UL19, UL27, UL28, UL29, and UL30, and is derived from McGeoch and Davison [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004743#ppat.1004743.ref081" target="_blank">81</a>]. Assignments to genera and subfamilies are shown on the right. Abbreviations not mentioned in the text are: MDV-1, Marek's disease virus type 1; MDV-2, Marek's disease virus type 2; HVT, herpesvirus of turkey; ILTV, infectious laryngotracheitis virus; PsHV-1, psitticid herpesvirus 1; GTHV, green turtle herpesvirus; THV, tupaia herpesvirus; GPCMV, guinea pig cytomegalovirus; CalHV-3, callitrichine herpesvirus 3; AHV-1, alcelaphine herpesvirus 1; OHV-2, ovine herpesvirus 2; PLHV-1, porcine lymphotropic herpesvirus 1; HVS, herpesvirus saimiri; HVA, herpesvirus ateles; and RRV, rhesus rhadinovirus. Red, blue, orange, and green shading indicate viruses that encode the ICP47, UL49.5, US6, or BNLF2a TAP inhibitor genes, respectively, and corresponding coloring of virus abbreviations indicate viruses in which these genes have been shown to be functional TAP inhibitors. Light orange shading identifies all members of the <i>Cytomegalovirus</i> genus that have a US6 gene. Light blue shading indicates all members of the herpesvirus family that code for a UL49.5 gene that might be involved in chaperoning maturation of glycoprotein M.</p

Adenoviral Vectors Stimulate Glucagon Transcription in Human Mesenchymal Stem Cells Expressing Pancreatic Transcription Factors

<div><p>Viral gene carriers are being widely used as gene transfer systems in (trans)differentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising <em>in vitro</em> and <em>in vivo</em>. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs) toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin) in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1)-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.</p> </div

E1-deleted adenoviral vector transduction triggers epigenetic changes in BM-MSC.

<p>(A–B) LV-CMV-GFP or LV-CMV-Pdx-1-modified BM-MSC were treated for 3 days with TSA (1 µM). Pdx-1 and glucagon gene expression was assessed by RT-PCR (A) or qPCR (B). GAPDH was used as reference. (C) ChIP analysis of acetylated histone H4 in the human glucagon gene in BM-MSC modified by adenoviral vector (Ad) or in control non-transduced cells (NT). Position of the primer sets over the glucagon gene are indicated: GCG-207 = glucagon promoter; GCG+297 = Intron 1; GCG+3142 Exon 2. (D) Micrococcal accessibility assay of the glucagon promoter upon adenoviral transduction. DNA from BM-MSC (GFP) or adenovirus-modified BM-MSC (Pdx-1/Ngn-3/Maf-A) was extracted and digested with 1000 mU of micrococcal nuclease. Digested DNA was compared to undigested DNA by qPCR (set to 100%).</p

Pdx-1, Ngn-3, and Maf-A expression transactivate the human insulin promoter.

<p>(A) 293T cells transduced with the LV-HIP-Luc lentivirus (left panel) and with the LV-GCG-Luc lentivirus (right panel) were infected with with LV-CMV-Pdx-1 (MOI = 1); LV-CMV-Ngn-3 (MOI = 1); LV-CMV-Maf-A (MOI = 1) or with all tree lentiviruses combined at a total MOI of 1. Luciferase activity in cell lysates was analysed 48 h post-infection. Luciferase activity of the 293T-LV-HIP-Luc and 293T_LV-GCG-Luc exposed to the lentivirus LV-CMV-GFP was set to 1. (B) Exposure of BM-MSC cells overexpressing Pdx-1, Ngn-3, and Maf-A to an adenovirus carrying an irrelevant transgene specifically induces glucagon gene expression. Human BM-MSC were transduced with LV-CMV-GFP (MOI = 2) or LV-CMV-Pdx-1 (MOI = 2); LV-CMV-Ngn-3 (MOI = 2) and LV-CMV-Maf-A (MOI = 2). Four days post- transduction, the modified cells were maintained for 3 additional days before analysis or infected with HAdV-5/fib50-EF1α-DsRed (MOI = 30). Cells were analyzed for insulin, glucagon and somatostatin gene expression by qPCR 3 days post adenovirus infection (i.e. 1 week post lentivirus infection). Data are presented as mRNA level relative to GAPDH mRNA as a reference.</p

Genes retained in E1- deleted adenoviral vector affect glucagon gene expression.

<p>(A) LV-CMV-Pdx-1 modified cells were transduced with HAdV-5/fib50-EF1α-DsRed (MOI = 30) or HD-HAdV-5/Fib50. Four days post transduction glucagon gene expression was evaluated by qPCR. GAPDH was used as reference. (B) BM-MSC(GFP) or BM-MSC (Pdx-1) were transduced with HAdV-5/fib50-EF1α-DsRed or LV-CMV-E4-ORF3 (MOI = 2). Four days post transduction glucagon gene expression was evaluated by PCR (C) or qPCR (D). GAPDH was used as reference.</p

BILF1 associates with MHC class I molecules at the cell surface and increases their rate of internalization.

<p>(A) BILF1 is predominantly localized at the cell surface. 293 cells stably transduced with BILF1 retrovirus were grown on glass slides, fixed and permeabilized, then stained with rat anti-HA (3F10) primary antibodies and Alexa Fluor® 594 goat anti-rat IgG. The nuclei were counterstained with DAPI. The stained slides were analyzed with a laser scanning confocal microscope, and the three photographs show different 1 micron-thick sections through representative cells. BILF1 stained red, and the nuclei stained blue. (B) BILF1 and MHC class I molecules co-precipitate at the cell surface. 293 cells (2×10⁶) stably transduced with control (c) or BILF1 (b) retrovirus were incubated with saturating concentrations of antibodies specific for MHC class I (W6/32), TfR (H68.4) or HA tagged BILF1 (3F10) on ice. After washing away excess antibody, the cells were lysed with NP40 detergent buffer, then precipitated with protein A/G beads and subjected to western-blotting as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000255#ppat-1000255-g006" target="_blank">Fig. 6B</a>, using antibodies specific for MHC class I (HC10) and HA tagged BILF1 (3F10). (C) BILF1 increases the rate of internalization of MHC class I, but not class II, from the cell surface. MJS cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32; top graph) or MHC class II (L234; bottom graph), then washed and incubated at 37°C for different periods of time. The cells were subsequently stained with PE-conjugated goat anti-mouse IgG antibody, and analyzed by flow cytometry. The mean fluorescence intensities of staining were averaged for triplicate samples, and normalized to the initial time 0 min samples. (D) BILF1 increases the rate of internalization, but not the rate of appearance, of MHC class I at the cell surface. Top graph: 293 cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32), then treated exactly as for the internalization assay performed with MJS cells in panel C. Bottom graph: replicate aliquots of the saturated W6/32-bound cells were harvested at the indicated time points, and the appearance of new MHC class I molecules was assayed by staining with PE-conjugated W6/32 antibody. The mean fluorescence intensities of staining were averaged for triplicate samples.</p

Characterization of cells stably transduced with a BILF1 retroviral vector.

<p>(A) 293 or MJS cells were stably transduced with control (pQCXIH) or BILF1 (pQCXIH-HABILF1) retrovirus. Surface MHC class I molecules were stained with PE-conjugated W6/32 antibodies and analyzed by flow cytometry. The solid line histograms depict the surface HLA class I staining of control cell lines, while the dashed line histogram depicts the surface HLA class I staining of cell lines expressing BILF1. The grey histogram illustrates background staining obtained with an isotype control PE-conjugated antibody. (B) Total cell lysates were generated from the retrovirus-transduced 293 and MJS cell lines, and 2×10⁵ cell equivalents were separated by SDS-PAGE and analyzed by Western Blotting with mAbs specific for BILF1 (3F10, anti-HA tag), MHC class I (HC10), MHC class II (DA6.147), TAP-1 (148.3), TAP-2 (435.3), TfR (H68.4) or with polyclonal antibodies to calregulin as a loading control.</p