Deletion mutants show that the 23-bp deletion in the upstream region of <i>PRNP</i> and/or the 12-bp deletion in intron1, coupled with the absence of the Sp1 SNP and the presence of exon1, are required for the negative feedback response to PrP overexpression during regulation of prion protein expression.

<p>(A) Deletion mutants of the DelDel constructs as shown on the left were used. Dotted box = Luciferase gene; black boxes = exon 1 and exon 2, which include numbers denoting the position of the reported transcription start site (+1) of the <i>PRNP</i> promoter region. The 23-bp indel, 12-bp indel, and SNP regions are also indicated above the reporter gene constructs. The absence (−) and presence (+) of each region in the reporter gene constructs are shown in the Table on the right. (B) Graph representing relative luciferase activities obtained with the above reporter plasmids in the presence of either an empty vector or pEF-boPrP. Relative luciferase activities (Mean ± S.D.) for 3 replicate experiments were compared with the pGL3-control plasmid (1%). A significant difference of luciferase activity in pEF-boPrP-transfected cells as compared with corresponding empty vector-transfected cells is shown by two asterisks (**, <0.01). NS indicates no significant difference.</p

SNP constructs show that the 23-bp deletion in the upstream region of <i>PRNP</i> and/or a 12-bp deletion in intron1, coupled with the absence of the Sp1 SNP and the presence of exon1, are required for the negative feedback response to PrP overexpression.

<p>(A) Map of the portion of bovine <i>PRNP</i> containing the 5′-flanking region and exons 1 and 2 is shown on the top line. Dotted box = Luciferase gene; black boxes = exon 1 and exon 2, which include numbers denoting the position of the reported transcription start site (+1) of the <i>PRNP</i> promoter region. The 23-bp indel, 12-bp indel, and SNP regions are also indicated above the reporter gene constructs. The absence (−) and presence (+) of each region in the reporter gene constructs are shown in the Table on the right. (B) Graph representing the relative luciferase activities obtained with the above reporter plasmids in the presence of either an empty vector, pEF-BOS (EM, open bars), or pEF-boPrP (PrP, solid bars). The pGL3-Control vector (with the standard SV40 promoter) was used for normalization between different experiments (relative light units (<i>RLU</i>) = (firefly luciferase_construct/<i>total protein</i>_construct)/(firefly luciferase_control/<i>total protein</i>_control)). Relative luciferase activities (Mean ± S.D.) for 3 replicate experiments were compared with that of the pGL3-control plasmid (1%). A significant difference of luciferase activity in pEF-boPrP-transfected cells as compared with corresponding empty vector-transfected cells is shown by one asterisk (*, <0.05) or two asterisks (**, <0.01). NS indicates no significant difference.</p

Expression of PrP in pEF-boPrP-transfected N2a cells.

<p>The bovine PrP expression vector pEF-boPrP was transfected into N2a cells. Forty eight hours after transfection, cells were lysed, and then the proteins were separated by 12% SDS-PAGE and blotted. The resulting blots were analyzed using anti-PrP mAb 6H4 as a specific antibody for bovine PrP protein, SAF 32 for PrP of all species, or B-5-1-2 for α-tubulin as an internal control.</p

Expression analyses of HIV-1 Vpr protein in human monocyte-derived macrophages (MDMs).

<p>(A) Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors through leukophoresis, cultured <i>in vitro</i>, and differentiated into MDMs as described in Materials and Methods. At day 7, the MDMs were infected with either Ad-Vpr or Ad-Zs, or were left untreated as mock-infected controls (left). At 48 h post-infection, the cells from Donor 1 were visualized by fluorescence (FL) and bright field phase contrast (BF) microscopy. (B) The cells from the two donors (upper panel, Donor 1; lower panel, Donor 2) were lysed and subjected to Western blot analyses using Vpr, ZsGreen1, and β-actin antibodies.</p

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p

Validation of differentially expressed genes at the protein level.

<p>Human monocyte-derived macrophages (MDMs) were infected with Ad-Vpr or Ad-Zs, or mock-infected as a control. At 48 h post-infection, the cells were washed, lysed, and subjected to Western blot analyses with the indicated antibodies. A β-actin antibody was used as a loading control.</p

Differentially expressed genes (fold change >2.0) associated with immune response (GO: 0006955) upon Ad-Vpr infection in Donor 1 and Donor 2.

<p>Differentially expressed genes (fold change >2.0) associated with immune response (GO: 0006955) upon Ad-Vpr infection in Donor 1 and Donor 2.</p

Gene ontology of differentially expressed genes after infection of human monocyte-derived macrophages (MDMs) with Ad-Vpr.

<p>(A) Venn diagram representing the number of differentially expressed cellular genes (>2-fold change in both donors) after infection of human MDMs with Ad-Vpr. (B) The top ten genes ontology classified by corrected p-value, and (C) heat map of hierarchical gene clustering of the 66 differentially regulated in both donors. Gene up-regulation is denoted in red and gene down-regulation is denoted in blue.</p

Schematic diagram of the Ad-Vpr and Ad-Zs vectors and analysis of their functional expression.

<p>(A) Recombinant adenovirus vectors expressing either FLAG-Vpr and ZsGreen1 or ZsGreen1 were generated using the Adeno-X™ expression system, as described in Materials and Methods. The transgene cassettes that replace the deleted E1 region contain a cytomegalovirus (CMV) promoter driving the expression of FLAG-Vpr and ZsGreen1 or ZsGreen1 protein, followed by an SV40 polyadenylation signal. The solid triangles indicate the regions deleted in the recombinant adenovirus (rAd) backbone. ITR: Inverted terminal repeats. (B) HeLa cells were infected with Ad-Vpr or Ad-Zs at MOI 50. At 48 h post-infection, cells were fixed and stained with propidium iodide for the analysis of DNA content. ZsGreen1-positive cells were analyzed by flow cytometry using Cell Quest for acquisition and ModFit LT. Arrowheads indicate peaks representing cells in the G1 and G2+M phases. The G2+M: G1 ratio is indicated in the upper right of each graph.</p

Differential expression profiling of cellular genes after infection with Ad-Vpr in human monocyte-derived macrophages (MDMs).

<p>Heat map of hierarchical gene clustering showing all genes that were either up- or down-regulated (>2-fold change) upon Ad-Vpr infection in MDMs from both donors. The color represents the normalized expression of genes in MDMs infected with Ad-Vpr or Ad-Zs (see color key). Gene up-regulation is denoted in red and gene down-regulation is denoted in blue.</p