Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs). Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD) motif, which is conserved in the kinesin-1-binding sequence (KBS) of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage. Vaccinia virus (VACV), the vaccine used to eradicate smallpox, exploits the host cell motor kinesin-1 to export virus particles to the cell surface. We demonstrate that the VACV F12 protein has structural similarity with kinesin light chain (KLC) and facilitates viral transport using a kinesin binding sequence (KBS) that is conserved in several neuronal proteins. Dysfunction of some of these neuronal proteins can contribute to diseases, such as Alzheimer's. Mutation of the KBS in protein F12 showed it is essential for kinesin recruitment to virions and for virion transport to the cell surface. These findings enhance our understanding of how viruses hijack the host cell transport system, demonstrate conservation of a kinesin binding motif in cellular and viral proteins and identify targets for drug development. F12 and A36 are each important for virus transport within or between cells and have been called transport proteins F12L, vΔF12L, also produced a small plaque, did not form actin tails and was highly attenuated in vivoF12 is a 65-kDa protein that lacks transmembrane sequences and is conserved in chordopoxviruses Here we show that F12 has structural similarity to KLC and contains TPRs and a WD motif that is required for kinesin-1 recruitment and virion transport. In contrast, WD motifs in A36 were not required for either process. Furthermore, TPRs were identified in proteins A36 and E2. We propose that F12 functions as a bridge to bind IEVs to the kinesin-1 motor. F12 interaction with IEV may be mediated by TPR∶TPR contacts between F12, E2/A36, and kinesin-1 binding is mediated by the WD motif found in F12 and cellular kinesin-1 binding proteins.The F12 protein from VACV strain WR F12 (WRF12) is conserved in other VACV strains, orthopoxviruses and chordopoxviruses The TPR (NCBI CDD cd00189) is a common structural motif facilitating protein-protein interactions and the assembly of multi-protein complexes. The repeat comprises a pattern of small and large amino acids [WLF]-X(2)-[LIM]-[GAS]-X(2)-[YLF]-X(8)-[ASE]-X(3)-[FYL]-X(2)-[ASL]-X(4)-[PKE] with no position invariant in vitro with KLC TPRs via aa 81–111 F12 interacts with VACV proteins A36 Based on the x-ray crystallographic structure of the KLC2 6 TPRs (pdb ID∶3CEQ), and its alignment with the F12 protein , a strucF12L gene showed the protein is not required for IEV formation but is needed for IEV transport to the cell periphery and CEV formation F12L alleles were transfected into cells infected with vΔF12L and rescue of IEV transport to the cell surface was measured by first staining live cells with anti-B5 mAb for counting CEV, and second by staining permeabilised cells with phalloidin and counting virus-tipped actin tails In previous studies, deletion of the In cells infected with vΔF12L, IEV particles were located in the centre of the cell, and CEV and actin tails were not present The important role of F12 in IEV movement suggests the protein may function in kinesin recruitment and/or activation The requirement of F12 for IEV motility is consistent with the suggested role in kinesin-1 recruitment and/or function. This was investigated further by confocal microscopy using viruses that have EGFP fused to the A5 core protein, and do or do not express F12, vEGFPA5L In the first paper describing E2 and its role in the VACV life cycle Domi et al n = 100) for each virus in our study, it was unlikely that any defect in IEV wrapping would have been missed in the thin sections examined. Similarly, Domi et al By counting many virus particles (Data presented show that F12 is required for kinesin-1 recruitment and IEV transport. To investigate further how F12 might recruit kinesin-1 we searched for known kinesin-1 binding sequences (KBS) within the F12 protein. A recent study identified two copies of a KBS in the neuronal transmembrane protein calsyntenin/alcadein conforming to the consensus sequence L/M–E/D–W–D–D–S, termed the WD motif, after the conserved tryptophan and aspartic acid residues A search for the WD motif in other cellular proteins identified similar sequences in the collapsin response mediator protein-2, 14-3-3 protein and amyloid precursor protein with the consensus sequence (L-E/D-W-D-D-S) . These pThe roles of the F12 WD motif in kinesin-1 recruitment and IEV transport were investigated by mutation of WDD to AAA, a mutation that abrogates kinesin-1 binding by calsyntenin/alcadein A36R gene, where IEV still move on microtubules and reach the cell surface at 60% of wild type levels In parallel we also investigated the role of the WD motif (L–I–W–D–N–E) within protein A36 reported in the calsyntenin study We also identified another putative KBS in VACV A36  that only conforms partially to the WD consensus sequence. Although this motif is not conserved outside of the orthopoxvirus A36 orthologues, we undertook a similar mutagenic analysis. Mutation of the WED to AAA alone, or in addition to the WDN to AAA at residues 97–99 to form a double mutant, gave no phenotype (data not shown). This mutagenic analysis demonstrates the WD motif present within F12, but not those in A36, is critical for microtubule-mediated virion transport.How motor proteins select and bind intracellular cargo is an important biological question. The importance of intracellular transport is highlighted by the fact that mutations in motors lead to genetic diseases such as amyotrophic lateral sclerosis, paraplegia and Griscelli syndrome type 1 This paper concerns how VACV exploits kinesin-1 for transport of virions to the cell periphery. We have discovered that VACV transport protein F12 is structurally related to KLC and shares with KLC a similar size, pI, hydrophobicity profile and TPRs. Bioinformatic analysis and structural modelling of F12 TPRs 7–12 with the recently described crystal structure of KLC2 TPRs showed a striking structural similarity. A close relationship was also found for F12 TPRs 1–6, suggesting that F12 might have evolved by duplication of the TPR domain of KLC accompanied by deletion of the N-terminal heptad repeats. Mutagenesis of the F12 protein showed that deletion of these TPRs from either terminus abrogated F12 function, implying an important role for TPRs. Although these mutant proteins were reasonably stable in virus-infected cells, we cannot exclude the possibility that the loss of function was due to aberrant folding of truncated proteins. F12 also contains a WD kinesin-1 binding motif that is found in several cellular cargo proteins and mutagenesis demonstrated that the F12 WD motif is critical for recruitment of kinesin-1 and IEV transport. A model consonant with these observations is that F12 binds cargo (virions) via TPR∶TPR interactions with E2 , and that it uses the WD motif to attach to KLC and thereby the kinesin-1 motor. Thus F12 functions as a bridge between microtubule motor and virus particle. Recently, the Cayman ataxia protein caytaxin was proposed to have an analogous function and shown to bind to KLC TPRs via a WD motif and mediate transport of cargos, such as mitochondria, by specific binding to these structures This example of VACV mimicry of a host cell protein adds to a growing list. Other examples include K3, a mimic of eukaryotic initiation factor 2α in vitro (aa 81–111) In addition to finding TPRs in F12, we report that TPRs are also present in proteins E2 and A36 which, like F12, have a role in IEV formation or transport. TPRs mediate protein-protein interactions and therefore the presence of TPRs in several VACV proteins that associate with each other suggests that these interactions might be via TPR∶TPR contacts. Consistent with this, F12 and A36 interact through residues 351–458 of F12, and 91–111 of A36, which overlap the TPRs of both proteins Previously, immunoelectron microscopy demonstrated that F12 is associated transiently with VACV during morphogenesis and egress and is present only on IEV, but not IV, IMV or CEV F12L gene and then are transfected with wild type F12 tagged with HA (F12-HA), or with mutants thereof. F12 function was assessed by kinesin-1 recruitment, dispersal of virions to the cell periphery, CEV formation and the production of actin tails from the cell surface. This showed that all F12 mutants tested with deletions from either terminus were non-functional, and mutation of the WDD motif to AAA also inhibited both kinesin-1 recruitment and IEV transport. A direct F12∶kinesin-1 interaction was not detected in previous yeast two hybrid screens in vitro has still to be demonstrated within infected cells.To investigate which domains of F12 are needed for kinesin-1 recruitment and IEV transport, we utilised a rescue assay in which cells are infected with a mutant virus lacking the An earlier study describing the WD kinesin-1 binding sequence in the neuronal transmembrane protein calsyntenin/alcadein reported that two WD motifs were needed for function, and these authors reported a WD motif in A36 Previously, protein A36 was described as essential for kinesin-1 recruitment to IEV and for IEV transport In summary, VACV F12 is a KLC structural mimic containing TPRs and a KBS, and VACV proteins A36 and E2 also contain TPRs that may mediate protein∶protein interactions. In addition, we show that the calsyntenin/alcadein WD motif for binding kinesin-1 is conserved in several other cellular kinesin-binding proteins and in VACV proteins F12 and A36. The F12 WD motif is critical for kinesin-1 recruitment and virion transport to the cell surface, whereas the A36 WD motif plays a supporting role. This report advances our understanding of VACV transport to the cell surface and illustrates how poxviruses use molecular mimicry for subversion of host cell biology.HeLa cells were grown in minimum essential medium (MEM) supplemented with 10% heat-inactivated foetal bovine serum (Gibco). The viruses used in this study were vaccinia virus (VACV) strain Western Reserve (WR) and derivative strains vΔF12L, F12-HA AAGCTTGCCATCATGGGTATCGAGAATACAGATTCCAT-3′5′-GAAC (F12-ΔN123); AAGCTTGCCATCATGTCCTCGTTAGATCAAACACA-3′5′- GAAC (F12-ΔN214) and AAGCTTGCCATCATGGGAATGGGGATGTATTATCCTA-3′5′- GAAC (F12-ΔN416) containing a HindIII restriction site (underlined) and GGATCCTTAAGCGTAATCAGGCACG-3′5′-GAAC (F12-HA R1) containing a BamHI restriction site (underlined) were used. The PCR product was digested with HindIII and BamHI and ligated into HindIII and BamHI- restricted pSEL, a pBluescipt vector containing the VACV synthetic early/late (E/L) GGATCCTTAGGTAGTATTGTCATCATCGTGAT-3′5′-GAAC (F12-ΔC128); GGATCCTTAGATTTTCCATCCACAATTATTG-3′5′-GAAC (F12-ΔC330) and GGATCCTTAATACATCTGTTTCCTATAATCGTT-3′5′-GAAC (F12-ΔC476) containing a BamHI restriction site (underlined) and 5′-GAACAAGCTTATGTTAAACAGGGTACAAATCTTG-3′ (F12-F1) containing a HindIII restriction site (underlined) were used. To generate A36-HA primers A36-F HindIII BamHI, AAGCTTATGATGCTGGTACCTCTTATCACGG-3′5′-CCC and A36-R HA BamHI, GGATCCTTAAGCGTAATCTGGAACATCGTATGGGTACACCAATGATACGACCGATGATTC-3′5′- GGA were used. The PCR fragments were digested with HindIII and BamHI and ligated into HindIII- and BamHI-digested pSEL to form the pSEL-F12-ΔC truncated mutants. The WD motifs within A36 and F12 were mutated to alanines (F12-HA WDD534AAA and A36-HA WDN95AAA) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) using pSEL-F12-HA and pSEL-A36-HA as template.PCR was performed with Pfu polymerase (Stratagene). The fidelity of the cloned fragments was verified by DNA sequencing. Mutants of F12 with N- or C-terminal truncations were constructed by PCR using VACV WR or vF12L-HA genomic DNA as template for the C- or N- terminal mutants, respectively. For the N-terminal mutants, the oligonucleotides HeLa cells were seeded on sterile glass coverslips and infected with VACV. At 2 h p.i. cells were transfected with 1 µg of plasmid using Fugene 6 (Roche Diagnostics) following the manufacturer's instructions. At 12 h post-transfection the cells were harvested or fixed and processed for immunofluorescence as detailed below.Antibodies used were mouse mAb 63–90 α-KLC (IgG1) Cells were partitioned using Triton X-114 as described previously t-test to test the significance of the results.In each experiment the number of actin tails per cell were counted for 10 cells for each infection/transfection, and the experiment was performed three times. Data were analysed by the Student's http://rsbweb.nih.gov/ij/) and Adobe Photoshop (Adobe Systems). Images shown are maximum projections of confocal Z stacks with accompanying phase transmission image. For conventional electron microscopy, at 12 h post infection cells were fixed in 0.5% glutaraldehyde-200 mM sodium cacodylate (pH 7.4) for 30 min, washed in 200 mM sodium cacodylate, and postfixed in 1% osmium tetroxide-1.5% potassium ferrocyanide for 60 min at room temperature. The cells were washed in water, incubated in 0.5% magnesium-uranyl acetate overnight at 4°C, washed again in water, dehydrated in ethanol, and embedded flat in Epon. Sections were cut parallel to the surface of the dish. Serial sections were collected onto slot grids, and lead citrate was added for contrast. Grids were viewed on a FEI Tecnai G2 transmission electron microscope. Images were acquired using a CCD camera. For cryoimmuno-electron microscopy primary antibodies were detected using antibodies for appropriate species then protein A-gold 6 nm conjugate.Cells were infected with VACV for 12 h, washed in ice-cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) in 250 mM Hepes pH 7.4 for 10 min on ice, and then in 4 or 8% PFA at room temperature (RT) for 10–50 min. Cells were permeabilised in 0.1 TritonX-100 in PBS for 10 min at RT. Alternatively, cells were for permeabilised for 5 min at RT in PBS containing 0.1% saponin, and the detergent was retained in all subsequent buffers when processing saponin-permeabilised coverslips. Fixed cells were processed for immunofluorescence as described previously http://www.ncbi.nlm.nih.gov/blast). Sequence alignments were performed using Clustal X http://www.psc.edu/biomed/genedoc). The predicted TPR domains 6–12 of F12 were subjected to structural modelling based on the alignment with KLC2 (http://www.pymol.org).BLAST searches ith KLC2  and the Figure S1Homo sapiens KLC1 (HsKLC1). Note VACV WR F12 and HsKLC are similar in length and exhibit a similar oscillating hydrophobicity profile.Kyte-Doolittle hydropathy plots of the VACV WR F12 and (0.83 MB PDF)Click here for additional data file.Figure S2F12 is a cytosolic protein. F12-HA TritonX-114 partitioning. HeLa cells were infected at 5 pfu/cell with vF12L-HA for 12 h. Cells were then collected, lysed and subjected to TritonX-114 partitioning . Protein(0.47 MB PDF)Click here for additional data file.Figure S3http://www.jalview.org/help/html/colourSchemes/clustal.html). The positions of TPRs numbered 1–14 are indicated above the alignment. The F12 TPRs that were aligned with KLC TPRs in Alignment of F12 orthologues. F12 orthologues from different chordopoxvirus genera were aligned using Clustal-X and are displayed using the Clustal colour scheme ((4.95 MB TIF)Click here for additional data file.Figure S4Full length F12 protein is required for IEV transport to cell periphery. HeLa cells were infected at 5 pfu/cell with vΔF12L and at 2 h p.i. were transfected with plasmids expressing full length F12L-HA or the indicated F12 mutants. The cells were fixed at 12 h p.i., permeabilised and stained with mAb against HA (blue), or B5 (red), and phalloidin-Alexa Fluor 488 to label filamentous actin (green). Cells were then viewed by confocal microscopy. The insert shows B5-positive virus particles at the tips of actin tails in cells transfected with the WT F12. Scale bar = 10 µm.(4.38 MB PDF)Click here for additional data file.Figure S5F12 co-localises with kinesin-1. Confocal microscopy showing colocalisation of F12-HA and KHC. HeLa cells were mock-infected or infected with vF12L-HA at 5 pfu/cell. At 12 h p.i. cells were permeabilised and stained with α-HA  and α-KHC (red). The right panels show the merged image. Scale bar = 10 µm.(3.44 MB PDF)Click here for additional data file.Figure S6Mutant F12 and A36 proteins are stable in VACV infected cells. HeLa cells were infected at 5 pfu/cell with vΔF12L or vΔA36R for 2 h. Cells were then were transfected with plasmids expressing HA tagged WT or mutant F12 or A36 proteins . 10 h later the cells were lysed and lysates were analysed by SDS-PAGE and immunblotting with anti-HA mAb. The positions of molecular size markers are indicated in kDa.(0.65 MB PDF)Click here for additional data file.Figure S7Conservation of the WD KBS in A36 chordopoxviruses orthologues. Alignment of the WD kinesin-1 binding site in A36 chordopoxviruses orthologues with the consensus sequence (L-N-W-D-N-+-+). Accession numbers are SWPV 123 (NP_570283), MYXV m125R (NP_051839), EMV (AJ315003), GTPV gp121 (YP_001293318), VACV WR A36 (YP_233041), YLDV 126R (NP_073511), DPV gp136 (YP_002302476).(0.61 MB PDF)Click here for additional data file.Figure S8The A36 WD motif is not required for recruitment of kinesin-1 to IEV or for virion transport to the cell surface. HeLa cells were infected at 5 pfu/cell with vΔA36R-EGFPA5L for 2 h. Cells were then transfected with plasmids expressing A36-WT-HA or A36-AAA-HA for 10 h. Cells were stained with mAbs against HA (blue) and KHC (red), and EGFP were visualized directly. Cells were viewed by confocal microscopy. The merged image is shown on the right. The insets show ×2.5 enlargements of the boxed regions and illustrate that both A36-WT-HA and A36-AAA-HA co-localise with KHC and virus particles. (B) Cells were treated as in (A) and were counterstained with phalloidin to label filamentous actin (red). Numerous actin tails are visible in cells transfected with A36-WT-HA and A36-AAA-HA. Scale bars = 10 µm.(5.65 MB PDF)Click here for additional data file.Video S1Tilt series electron microscopy showing vΔF12L arefully wrapped. Images were acquired from a 90-nm thin section stained with Reynold's lead citrate, 11 images at 10 degree increments were captured +/− 50 degrees tilt angle. Images were aligned with Adobe Photoshop CS4 and made into a movie using GIF Movie Gear and further compressed with Windows Media Encoder. Selected images are shown in (0.25 MB WMV)Click here for additional data file.


LRP5 gene is regulated remains unclear.LRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs.In order to characterize the transcriptional regulation of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.Both KLF15 and Sp1 binding sites contribute to the basal activity of human  LRP5 gene, located on human chromosome 11q13, contains 23 exons encoding a 1615 amino acid single-pass transmembrane receptor that belongs to the low density lipoprotein (LDL) receptor superfamily. LRP5 is highly conserved between species. There is 95% identity between human and mouse LRP5 protein ATP (3000 Ci/mmol) using T4 polynucleotide kinase. The 32P-labeled primer of 106 cpm was added to 50 μg of total RNA from U2OS cells in a final volume of 20 μl. The reverse transcription was performed with AMV reverse transcriptase according to the manufacturer's protocol. The reaction products were analyzed on 6% polyacrylamide/7 M urea gels alongside manual sequencing reaction of the plasmid DNA containing the 5'-flanking region of human LRP5 gene with the same end-labeled primer.When reaching 80% confluence in 75 cmLRP5 and found a BAC clone (PR11-149G19) containing the human LRP5 gene. We therefore amplified the proximal 5' region of human LRP5 (-2381 to +125 bp relative to transcription start site) by PCR using PR11-149G19 clone as the template. The primers (LRP5F and LRP5R) used are listed in Table Utilizing the NCBI GenBank, we identified the genomic sequence of human  clone PR1-149G19 Mutant versions for specific binding sites were prepared by PCR-based mutagenesis from pWT-72, using the common reverse primer LRP5R/Hind III combining with the forward primers -72MT1/MluI, -72MT2/MluI and -72MT12/MluI, respectively [CGCGGATCCATGGTGGACCACTTACTTCCAGTGGACGAG-3') and reverse (5'- CCGCTCGAGTCAGTTCACGGAGCGCACGGAGCGGCTGCTC-3') primers. BamHI and XhoI restriction sites, respectively (bold), were introduced into the primers for subcloning. The amplicons were digested with BamHI and XhoI and inserted into the Drosophila pPac expression vector after excision of the Sp1 coding sequence from pPacSp1. Construct sequences were confirmed by DNA sequencing.pPac, a  Germany). The KLF4 cells per well and 4 × 104 cells per well, respectively, the day before transfection. For each well of cells, 0.4 μg of the LRP5 promoter constructs (deletion and site-directed mutants) were co-transfected with 0.02 μg of pRL-TK plasmid (Promega). The cells were cultured for 24 hours after transfection and then harvested. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). To normalize for transfection efficiency, the promoter activity of each construct was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity. For each construct, more than three independent experiments with U2OS and HEK293 cells were performed in triplicate.Transient transfections were performed with the use of Lipofectamine 2000 reagent (Invitrogen) as previously described. Briefly5 cells/well. Cells were transfected with 0.5 μg of indicated reporter construct  and 0.1 μg of pPacSp1 or pPac KLF15 expression vector or empty vector pPac0 together with pPac-β-gal vector . Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured using Bright-Glo luciferase assay system (Promega) and β-galactosidase activity was measured using Beta-Glo assay system (Promega). Luciferase activities were normalized to β-galactosidase activity, reported as means of three independent experiments, and each performed in duplication.For transient transfection of SL2 cells, one day prior to transfection, cells were plated into 24-well plates at a density of 5 × 10HSD17B5 promoter (5'-TTCTCCACAGACCATATAAG-3' and 5'-TTCCCTGTCACTTGTCTGACT-3') were designed to amplify the promoter region containing Sp1 the KLF15 binding sites [ChIP assays were performed using an EZ-ChIP assay kit  according to the manufacturer's instructions. Briefly, HEK293 cells were treated with formaldehyde and lysed with lysis buffer. The cell lysates were sonicated to generate 200-1000 bp DNA fragments, and cross-linked proteins were immunoprecipitated by incubation with antibodies against Sp1 , KLF15 , and IgG, respectively. A DNA sample from sonicated nuclear lysates that underwent reverse cross-link and phenol/chloroform extraction was used as positive control (input). A DNA sample immunoprecipitated by antibodies against IgG was used as negative control. Immunoprecipitated DNA was detected by PCR amplification using primer LRP5-ChIP-forward (5'-CCGCGGGGACCTGCGCCGCT-3') combining with primer LRP5R. As a positive control, specific primers of ng sites . PCR proThe statistical significance of differences between experimental groups was calculated using the Student's test. Group differences were considered significant if P < 0.05.LRP5: low-density lipoprotein receptor related protein; Sp1: specificity protein 1; KLF15: Krüppel-like factor 15; ChIP: chromatin immunoprecipitation; HSD17B5: human 17-hydroxysteroid dehydrogenase type 5 gene.JL performed luciferase reporter assay and ChIP analysis, and drafted the manuscript. YY and BJ participated in the primer extension and reporter assays. XZ and YZ assisted in RNA isolation and vector construction. YG contributed to the conception and the design of the study and the writing of the manuscript. All authors read and approved the final manuscript.


Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of  This is the first unbiased genome-wide analysis of wildtype AAV integration combined with a thorough bioinformatic analysis of preferred genomic motifs and patterns in the neighbourhood of the integration sites identified. The preference of Rep-dependent AAV integration near multiple consensus Rep-binding sites was lost in the case of AAV vector integration in the absence of Rep expression. Our findings challenge the commonly accepted notion of site-specific AAV targeting to AAVS1 on chromosome 19q13.42. Although AAVS1 contains a canonical Rep-binding site, numerous additional sites including the newly identified hotspots AAVS2 on chromosome 5p13.3 and AAVS3 on chromosome 3p24.3 harbour functional Rep-binding sites suitable for AAV integration. AAV vectors are quickly moving forward in the clinic and Rep-dependent vector targeting strategies are being actively pursued. Detailed information of AAV wildtype versus recombinant AAV vector integration sites and preferences are needed to evaluate the safety profile of AAV vectors in gene therapy. The preferred integration of wildtype AAV-2 in chromosome 19q13.42 is unique and is commonly viewed as a specifically evolved virus-encoded targeting mechanism. Multiple attempts were published that aim to exploit Rep-mediated targeting specificity for chromosome 19q13.42 for the specific integration of gene therapy vectors in silico analysis detected numerous consensus Rep-binding sites in the human genome. Many of these bound Rep in vitroin vivo accessibility for AAV integration has not been explored so far. From an evolutionary standpoint the assumption that AAV latency is ensured by more than one target site or mechanism appeared reasonable.Although AAV has not been associated with disease in humans, it is well established that AAV Rep78/68 induces DNA damage, cell cycle arrest This study was designed to close the knowledge gap between AAVS1-specific and assumedly non-AAVS1-specific wildtype AAV integration and to compare the identified genomic sites to those preferred upon AAV vector transduction. An open survey of chromosomal integration preferences for wildtype AAV-2 was conducted and complemented by the bioinformatic analysis of genomic motifs and patterns in the genomic regions surrounding the integration loci.The genomic structure of latent AAV in infected cells is highly variable. Wildtype AAV-2 was shown to integrate into the host cell genome, as well as persist as extrachromosomal, nuclear episomes Several methods to identify virus-chromosome junctions have been developed to study retrovirus integration, where generally a single proviral copy per chromosomal site is found bona fide AAV-chromosome fusion sequences. The genomic DNA of AAV-infected cells was cleaved with restriction enzymes that lead to sufficiently sized DNA segments to allow unambiguous genomic assignment of the chromosomal junction -PCR was developed which enriched for junction . DNA seqUsing LSM-PCR a total of 1700 cloned PCR fragments were screened for DNA inserts of a minimal fragment size (>500 bp) to insure unambiguous detection of AAV-chromosome junctions. Out of 350 DNA sequence runs a total of 129 unique junction sites could be assigned to the human genome. Of these, 109 fulfilled the criteria outlined in the methods for unambiguous assignment of a single chromosomal site. Junctions were retrieved with non-cut enzymes for AAV-2, PvuII or EcoRV or with DraI, which cuts once in AAV-2 DNA outside of the region covered by the PCR. In addition, 43 wildtype AAV-2 infected Hela-derived single cell clones were generated of which eight harboured AAV-chromosome junctions that fulfilled the criteria outlined in the methods.rep-expression construct and an AAV vector plasmid 47 junction sequences fulfilled our criteria for unambiguous assignment of AAV to a unique chromosomal site  analysis as outlined in the methods. Trimethylated lysine 27 of histone 3 (H3K27me3) is correlated with gene repression (closed chromatin) in vitro ternary complex formation of Rep68 with the AAV-2 ITR and AAVS1 of chr. 19q13.42 A series of publications have shown that fused combinations of two to four GAGC motifs bind to Rep78/68 of AAV-2 Based on these data we hypothesized that AAV-2 wildtype, due to the presence of Rep, prefers integration at chromosomal sites in closer proximity to consensus Rep binding sites than would be expected from control sites. The hypothesis was tested with the three sets of junctions derived from: 1. Infection with AAV-2 wildtype, 2. Cotransfection of plasmids coding for an AAV vector and a constitutive Rep-expression cassette, and 3. Infection with Rep-deficient AAV vectors . The disThe choice of randomly generated genomic control sites was considered optimal for comparative analysis of the three sets of data. Yet, a concern was the choice of restriction endonucleases for the identification of the wildtype AAV-2 integration sites by LSM-PCR. To control a bias introduced by a conceivable non-random genomic distribution of the restriction sites, the average distance of PvuII, EcoRV, or DraI-generated restriction sites to putative Rep-binding sites was compared to the average distances of random sites to Rep-binding sites. PvuII restriction sites were found to be closer to Rep-binding sites than random control sites . This waGAGC GAGC as a minimal Rep-binding site strikingly confirmed our hypothesis that integration of wildtype AAV takes place close to Rep-binding sites with very high significance (p <0.0001). A comparable effect was seen with the data set for AAV vectors in the presence of Rep (p<0.001). Most importantly, the set of integration sites for AAV vectors in the absence of Rep did not show any difference of integration site preference compared to random control sites (GAGC GAGC occurs sufficiently frequent to lead to a mean distance of around 50 kb to the next AAV integration site in the presence of Rep. In the absence of Rep the mean distance to AAV (vector) integration sites rises to around 130 kb  a small effect was seen  the integration preferences of AAV wildtype and Rep-expressing AAV vectors shifted to closer proximity to Rep-binding sites (p<0.0001). This is especially surprising since only 616 sites per human genome are found for GAGY GAGC GAGC (GAGC GAGC GAGC with one or two random mismatches. This led to a significantly decreased mean distance to AAV junctions in spite of the fact that up to 100-fold more genomic hits were found for the motifs (CGAGY GAGC GAG motif (AGAGY GAGC GAG) on the other hand led to a complete loss of association to AAV integration sites. This is surprising in view of the reported in vitro binding of Rep to this motif in vivo. Motifs GCCC GAGT GAGC and GAGT GAGC ACGC are part of the RBS in the viral p5 promoter. The individual motifs are found at very low frequency  in the human genome, so that either no RBS was found in the same contig or the distance to the next RBS was more than several thousands kb. For these reasons we did not proceed with calculations for these motifs. To further exclude the possibility that the calculated associations with Rep binding sites were predominantly based on sequences assigned to the hotspots AAVS1 and AAVS2, the significance of the associations was re-evaluated with data sets omitted for the hotspot sequences , chr. 5p13.3 (AAVS2), and chr. 3p24.3 (AAVS3). But even in the absence of hotspots AAV still shows a highly significant integration preference for Rep-binding motifs at numerous additional sites in the human genome.The bioinformatic calculations with ol sites . With a d 130 kb . To ensuwas seen . ObviousAGC GAGC . To alloe motifs ; E. A siequences . The robThis study represents the first genome-wide survey of wildtype AAV-2 integration in the human genome combined with a thorough bioinformatic analysis of the surrounding genome. We show here that wildtype AAV-2 infection leads to preferential integration in the vicinity of consensus Rep-binding sites (RBS) at defined hotspots as well as at numerous additional genomic sites. In contrast, AAV-2 vectors in the absence of Rep-expression integrate without discernable preference for consensus Rep-binding sites.in vitro as is the RBS of AAVS1 . The 8 kb AAVS2 sequence identified on chr. 5p13.3 represents an intergenic region to the best of current knowledge. It is well known that Rep expression leads to extensive rearrangements of AAVS1 PPP1R12C is essential, since the majority of latently infected cell lines display gene duplications trs) next to the RBS of AAVS2 and AAVS3. In AAVS1 the spatial configuration of RBS and trs resembles that of the AAV-ITR. The trs element lies next to the RBS and serves as a nicking site for Rep trs elements (5′-GTTGG-3′) are 400 and 500 bp away from the RBS, which represents the mean statistical occurrence for this motif. Unfortunately, the consensus nucleotide requirements for a functional trs element are not defined well enough to conduct a meaningful bioinformatic search. Therefore, the presence of nicking sites next to the RBS in AAVS2 or AAVS3 remains open at present.A major difference between the hotspots on chr. 5 and chr. 19 concerns the presence of genes. The junctions identified on chr. 19 span the region of the transcribed gene for protein phosphatase 1, regulatory subunit 12C  supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml).9 i.u./ml were prepared on HeLa cells as described before 6 HeLa cells were seeded overnight on 10 cm diameter dishes and infected with AAV-2 at a MOI of 500. Cells were harvested at 96 hours post infection (p.i.) for the extraction of genomic DNA. The period of cell growth after infection was minimized to reduce the chances of selection of particular integration sites during cell proliferation. Alternatively, AAV-infected HeLa cells were seeded to microtiter plates at a dilution of 60 cells per plate and grown up as single-cell clones without drug selection.Viral stocks of wildtype AAV-2 with infectious titers of 5×10Plasmid pTAV2-0 covers the AAV-2 wildtype genome (GenBank accession number AF043303), pRVK the 4 kb fragment of the AAVS1 locus on chromosome 19 (GenBank accession number S51329), and pAAVS1-TR covers an AAV-ITR/AAVS1 junction 600 nm of 0.6 to 0.8. Production of MBP-Rep78 was induced with 0.3 mM IPTG for 3 h at 30°C. Cells were harvested by centrifugation and lysed by sonication for 2 min (30% duty cycle) in lysis buffer of 50 mM phosphate pH 7.8, 300 mM NaCl, 1% (v/v) Triton X-100, 0.1 mM PMSF. Cell debris was removed by centrifugation at 6500×g for 20 min at 4°C. The supernatant was adsorbed to amylose resin (New England Biolabs) in a batch process and the resin was washed extensively (5 washes with about 100 volumes of the resin) with lysis buffer. The adsorbed proteins were eluted with lysis buffer containing 10 mM maltose and analyzed for purity by SDS-polyacrylamide gel electrophoresis.MBP-Rep78 encoding Rep78 fused to maltose-binding protein was expressed und purified essentially as described 32P- labeled double-stranded oligonucleotide probes was detected by altered mobility of the probes in nondenaturating polyacrylamide gels essentially as described previously 2, 40 mM NaCl, 1 mM DTT, 2% glycerol, 12.5 µg/ml BSA, 0,01% Nonidet P40 and 5 µg/ml salmon sperm DNA. The following oligonucleotides were used:Binding of MPB-Rep78 fusion protein to GCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA;AAV-ITR (nucleotide position 85–133): TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCAAV-ITR complementary strand: TGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCGGGCGGGCChr. 19q13.42 (AAVS1): GCCCGCCCGCCCAGCGAGCGAGCGAGCGCCGAGCCCCAACCGCCGCCAChr19 (AAVS1) complementary strand: AGCTGGACCCCACGCTCGCTCACTCACTCTCCCCTCACCGCTTTGTChr. 5p13.3 (AAVS2): ACAAAGCGGTGAGGGGAGAGTGAGTGAGCGAGCGTGGGGTCCAGCTChr. 5 (AAVS2) complementary strand: GCTTCCCAAGGGGAATGAATGTGCGCTCGCTCACTCACTCACTCCTCACChr. 3p24.3 (AAVS3) GTGAGGAGTGAGTGAGTGAGCGAGCGCACATTCATTCCCCTTGGGAAGCChr.3 (AAVS3) complementary strand: CCTCGCTCCCTCCCTCTCCCCTCACCGCTTTGTAGCTGGACCCCACChr. 5MUT (AAVS2 mutated): GGAGGGAGCGAGGGTGGGGTCCAGCTACAAAGCGGTGAGGGGAGAGChr.5MUT (AAVS2 mutated), complementary strand: TCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAV p5 (nucleotide position 245–292): CAAAATGGAGACCCTGCGTGCTCACTCGGGCTTAAATACCCAGCGTGAAAV p5 complementary strand: CAGAGCAGCAGCACAGACGCTAGCAGATCTCCTGCGACCGGAGATGTGrandom control: CACATCTCCGGTCGCAGGAGATCTGCTAGCGTCTGTGCTGCTGCTCTGrandom control, complementary strand: Total genomic DNA was extracted by SDS/proteinase K digestion followed by repeated phenol/chloroform extractions and ethanol precipitation, as described before 5′GTA ATA CGA CTC ACT ATA CGG CAC GCG TGG TCG ACG GCC CGG GCT GGT 3′) and “linkershort” . Equal amounts of “linkerlong” and phosphorylated “linkershort” (100pmol each) were annealed and ligated to restriction enzyme-digested genomic DNA.A linker-based strategy described in 5′-GTA ATA CGA CTC ACT ATA CGG C; Tm = 58.4°C) and “P linker nested” . Two AAV-2-specific primer sets were used. The first primer set covered the AAV p5 promoter: “AAV2p5” , primer “AAV2p5 nested” . The other primer set is located in the cap gene region, as described before 5′-GTC TGT TAA TGT GGA CTT TAC TGT GGA CAC; Tm = 65.4°C, AAV-2, nt 4320–4349) and “CAPgsp2” 5′-GTG TAT TCA GAG CCT CGC CCC AT; Tm = 64.2°C, AAV-2 nt 4357–4379).PCR-primers: The linker-primers were “P linker outside” with biotin attached to its 5′ end , 2.5 U proofreading hot-start polymerase (Herculase) in reaction buffer, as provided by the supplier (Stratagene). Of the preceding linker-ligation reaction 1–5 µl was added to a final volume of 50 µl. PCR conditions were as follows: 3 min at 98°C, followed by 10 cycles of 40 sec at 98°C, 30 sec at 65°C, and 4 min at 72°C, followed by 20 cycles of 40 sec at 98°C, 30 sec at 65°C, and 4 min + 10 sec per cycle at 72°C, terminated by an extension period of 10 min at 72°C. Biotin-labelled PCR products were further enriched on streptavidin-labelled Dynabeads M-280, as outlined by the supplier (Invitrogen). Subsequent nested PCR used conditions identical to the first round but with pairs of the nested PCR primers, as outlined above. Finally, to add overhangs of multiple As, PCR products were incubated with 1 U Taq polymerase (New England Biolabs).Products of LSM-PCR reactions were separated on agarose gels. To ensure sufficient chromosomal fragment lengths, PCR bands of a calculated minimal length (>500 bp) were excised and purified by the QIAEX II Gel extraction kit . TOPO-TA cloning was performed as described http://genome.ucsc.edu/cgi-bin/hgBlat) The genomic positions of AAV integration sites in the human genome  were determined using the BLAT tool from the UCSC Genome Browser web site  were discarded in view of the inability to unambiguously assign the surrounding genome for subsequent bioinformatic analysis (see below).Duplicate AAV-chromosomal fusion sequences  were counted only once.rep-deficient AAV vector integration sites in diploid human cells http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucgss) and reanalyzed using the UCSC March 2006 human genome build. The analysis led to a total of 450 junction sequences that fulfilled all of the above inclusion criteria for bioinformatic comparisons. For the subsequent data analysis we implemented software in C++ using the software library SeqAn In addition to the LSM-PCR derived sequences, the original DNA sequence files of 157 chromosomal junctions For different Rep binding motifs, we computed the average distance of virus integration sites to the closest occurrences of Rep binding motifs within the genome. We supposed that the observed integration events were independent from each other and the sample size was high enough for assuming the distance to be normally distributed. To assess whether these distances differ significantly from expectation, several background models were generated:For the background model “random”, we assumed that the probabilities for the observation of virus integrations were equally distributed among all conceivable positions in the genome. A program was implemented that computed the exact mean and standard deviation of this background distribution.Since the integration site analysis required a suitable restriction enzyme site in the neighbourhood of the integrated virus three background-models for the restriction enzymes DraI, EcoRV or PvuII were generated. These models served as a corrective tool for an eventual bias of a non-uniform distribution of the respective restriction enzyme sites in the genome. For each AAV integration site observed, the distance to the closest restriction site was determined individually. Then, 1000 control sites per integration site were generated that displayed the same distance to randomly chosen restriction sites.all relevant background models. Data sets of AAV vectors were analyzed with the “random” background model. We applied a Z-test for determining statistical significances for the distances of integration sites to Rep binding sites. For comparing integration sites from AAV wildtype infection sites against those from rep-deficient AAV vector infection we applied the Student's t-test.The generation of both, the data analysis and the background model was confined to those genomic contigs that contained at least one Rep binding motif, since otherwise the distance to the “closest Rep binding motif” would not be defined. A given set of AAV integration sites was considered to be significantly closer to Rep binding motifs than expected by chance, if the significance was calculated for AAV integration sites were examined for the occurrence of various genomic features using tables available in the UCSC database. For the determination of significant divergences from expectations, we compared the actual integration sites with a set of 100,000 randomly chosen control sites in the human genome using a two-tailed binomial test.http://www.wip.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSM325898) http://www.bcgsc.ca/data/histone-modification) were mapped to the human genome using Bowtie Chromatin immunoprecipitation sequencing (ChIP-Seq) data were used to define the state of histone modifications in genomic regions of AAV integration. H3K27me3 domains determined by Cuddapah et al. were used as markers for closed chromatin  Cleavage sites of restriction enzymes used to digest genomic DNA of wildtype AAV-2-infected Hela cells and the numbers of occurrences per human genome are shown. (B) The mean distances of restriction enzyme cleavage sites to Rep binding sites were compared to those of random control sites to Rep Binding Sites. Calculations are displayed for consensus RBS that yielded significant proximity of integration sites to RBS as displayed in (0.13 MB TIF)Click here for additional data file.Figure S2GAGC GAGC GAGC one mismatch allowed; (E) GAGC GAGC GAGC two mismatches allowed. In order to analyze only the Rep-binding sites outside of integration hotspot regions, sites within the hotspots of chr. 19 (AAVS1) and/or chr. 5 (AAVS2) were omitted in separate calculations.Bioinformatic analysis of AAV-2 wildtype integration sites. Distances of integration sites from Rep Binding Sites were calculated and the z-score was assessed in relation to the following control sites: (A) GAGC GAGC; (B) GAGT GAGC; (C) GAGY GAGC GAGC; (D) (0.25 MB TIF)Click here for additional data file.


In the current investigation we examined the influence of genetic background on susceptibility to V2O5-induced inflammation and evaluated whether V2O5 functions as a tumor promoter using a 2-stage (initiation-promotion) model of pulmonary neoplasia in mice.Elevated levels of air pollution are associated with increased risk of lung cancer. Particulate matter (PM) contains transition metals that may potentiate neoplastic development through the induction of oxidative stress and inflammation, a lung cancer risk factor. Vanadium pentoxide , and chemokines, transcription factor activity, and MAPK signaling were quantified in lung homogenates. We found that treatment of animals with MCA followed by V2O5 promoted lung tumors in both A/J (10.3 ± 0.9 tumors/mouse) and BALB (2.2 ± 0.36) mice significantly above that observed with MCA/PBS or V2O5 alone (P < 0.05). No tumors were observed in the B6 mice in any of the experimental groups. Mice sensitive to tumor promotion by V2O5 were also found to be more susceptible to V2O5-induced pulmonary inflammation and hyperpermeability (A/J>BALB>B6). Differential strain responses in inflammation were positively associated with elevated levels of the chemokines KC and MCP-1, higher NFκB and c-Fos binding activity, as well as sustained ERK1/2 activation in lung tissue.A/J, BALB/cJ (BALB), and C57BL/6J (B6) mice were treated either with the initiator 3-methylcholanthrene  or corn oil followed by 5 weekly aspirations of V2O5, an occupational and environmentally relevant metal oxide, functions as an in vivo lung tumor promoter among different inbred strains of mice. Further, we identified a positive relationship between tumor promotion and susceptibility to V2O5-induced pulmonary inflammation. These findings suggest that repeated exposures to V2O5 containing particles may augment lung carcinogenesis in susceptible individuals through oxidative stress mediated pathways.In this study we demonstrate that V Althoug ≤10 μm) ,10. PM2.and wood . Most adand wood . Using m[et. al.  predicteThe mechanism(s) contributing to increased lung cancer risk by PM have not been fully characterized, although it has been suggested that pulmonary inflammation mediated by particle-induced oxidative stress may play an important role ,14. Gene2O5) is the most common commercial form of vanadium [2O5 is released into the environment during oil and coal combustion and from metallurgical works [2O5. Coughing, wheezing, chest pain, bronchitis, and asthma-like symptoms as well as impaired lung function occurred in humans exposed to high levels of V2O5-containing dust [2O5 particles increased bronchoaveolar polymorphonuclear neutrophils (PMNs) and impaired pulmonary function [Vanadium pentoxide  resulted in a significant increase (~50%) in the incidence of alveolar/bronchiolar neoplasms in both male and female B6C3F1 mice [2O5, long-term continuous exposure was required before tumors developed and no dose response was observed, which suggests V2O5 may be promoting spontaneous tumors. In addition, different mouse strains were not assessed, which can greatly influence pulmonary responses to environmental pollutants [Occupational and ambient exposure to vanadium has been associated with an increase in biological markers for oxidative DNA damage ,32, howecer risk ,34. In vll lines . In a Na3F1 mice . Althougllutants  as well llutants ,38.2O5 on pulmonary neoplasia among different inbred strains of mice. Using a two-stage (initiation-promotion) model, we hypothesized that inflammation induced by sub-chronic V2O5 administration would promote tumorigenesis in susceptible strains. Three strains of mice were included in this study that display altered susceptibility to chemical carcinogenesis: A/J mice are sensitive, BALB are intermediate, whereas B6 mice are resistant to most short term chemically-induced carcinogenesis protocols (eg. not initiatable using MCA) [2O5 functions as an in vivo lung tumor promoter in both A/J and BALB mice. Further, we demonstrate a positive relationship between tumor promotion and susceptibility to V2O5-induced inflammation, involving the induction of the chemokines KC and MCP-1, the transcription factors NFκB and c-Fos, as well as sustained activation of ERK1/2 in pulmonary tissue.This study was conducted to further evaluate the role of Ving MCA) -39. Thesing MCA) -42. Resuad libitum and were assessed daily for health status. All mice were allowed one week to acclimatize prior to treatment. Animal use was conducted in AAALAC-accredited facilities and in accordance with the regulatory guidelines of the Michigan State University All University Committee on Animal Use and Care.Male A/J, BALB/cJ (BALB), C57BL/6J (B6) mice were purchased from Jackson Laboratories  at 5-6 weeks of age. Animals were housed in plastic, filter-capped cages containing hardwood bedding and maintained in temperature (23 ± 2°C) and humidity (40-60%) controlled rooms with a 12 hour light/dark cycle. Animals were given standard laboratory chow  and spring water 2O5 was suspended in sterile-filtered Dulbecco's phosphate buffered saline , sonicated for 20 minutes, then further diluted to a working concentration of 2 mg/mL. Prior to aspirations mice were anesthetized using 3% isoflurane in 1-2 L/min oxygen, and V2O5 (4 mg/kg body weight) was administered following the methods of Foster et. al. [2O5 suspensions were prepared fresh prior to use and administered to animals within two hours of preparation.Pulmonary administration of vanadium pentoxide  was performed by oropharyngeal aspiration as previously described . Briefly et. al. . This do et. al. ,29. Cont2O5 would promote pulmonary carcinogenesis using a two-stage (initiation-promotion) model. Mice were injected ip. (10 μg/g body weight) with the carcinogen MCA  dissolved in corn oil or with corn oil alone. Beginning one week later, mice were treated with 5 weekly aspirations of either V2O5 (4 mg/kg) or PBS as described above. To assess tumor promotion, animals were sacrificed 20 weeks following MCA treatment; the lungs were perfused with saline then inflated and fixed in Tellyesniczky's fixative for 48 hrs. Tumors were enumerated using an Olympus SZX7 stereomicroscope  and sized with digital calipers . Using this protocol, pulmonary inflammation was additionally assessed in A/J mice 21 days following the last aspiration as described below.The experimental designs utilized in this study are depicted in Figure 2O5 (4 mg/kg) or PBS. At selected time intervals  following the last dose, bronchoaveolar lavage fluid (BALF) was collected to quantify differences in cellular infiltrates and protein content, a marker of hyperpermeability, as described previously [To assess strain differences in inflammation,  -47, an aA neutrophil-specific marker (sc-59338) and ABC detection kit (sc-2019) were purchased from Santa Cruz Biotechnology . Left lungs were fixed in 10% NBF for 24 hrs, processed using standard histological procedures, embedded, then cut into 5 μm sections. Strain differences in pulmonary neutrophil infiltration were evaluated using peroxidase biotin-streptavidin immunohistochemistry, and bound enzyme was visualized using the chromagen 3-3'-diaminobenzidine (DAB). Tissues were then lightly counterstained in Gill's hematoxylin.g for 10 min at 4°C, and protein was quantified using the DC protein assay . For chemokine analysis 25-50 μg of RIPA extracted protein was used in accordance with manufacturer's instructions. Absorbance was measured at 450 nm using a VersaMax microplate reader . All data are presented as pg/mg protein.ELISA kits for keratinocyte-derived chemokine , macrophage inflammatory protein-2 , and monocyte chemoattractant protein 1  were purchased from R&D systems . Protein was prepared by homogenizing lungs in ice-cold RIPA buffer  containing protease inhibitors . Homogenates were centrifuged at 13,000 × Nuclear protein was prepared from the left lung of mice using the TransAM nuclear extraction kit  and quantified with the DC protein assay . Strain differences in binding of NFκB (p65 subunit) and AP-1 (c-Fos) were then measured from 8 μg of nuclear protein using TransAM Transcription Factor ELISA kits . Absorbance was measured at 450 nm using a VersaMax microplate reader .Primary antibodies specific for MAPKs were purchased from Cell Signaling  and secondary antibodies from Pierce . Protein was prepared from right lungs as described above. Samples (100 μg protein) were resolved on 12.5% SDS polyacrylamide gels. Following transfer, PVDF membranes were blocked for 1 hr at room temperature in 5% nonfat dry milk, and then incubated with primary antibodies to detect phosphorylated ERK1/2, JNK1/2, or p38 overnight at 4°C. After washing, blots were incubated in secondary antibody linked to horseradish peroxidase for 1 hr at room temperature, and bands were detected using chemiluminescence. Images were captured using BioRad ChemiDoc illumination system . Following detection, membranes were stripped in Restore stripping buffer  then reprobed for total MAPK using procedures described above. Densitometry of bands were quantified with BioRad Quality One software and phosphorylated proteins were normalized to the respective total MAPK prior to statistical analyses.P < 0.05), comparisons of means were analyzed using the least significant difference (LSD) method. All data are presented as mean ± SEM.All statistical analyses were conducted using SAS statistical software . Time- and strain- dependent changes in BALF protein, cellularity, chemokines, nuclear transcription factor activity, protein densitometry, and tumor multiplicity/size were analyzed using an analysis of variance (ANOVA). When statistical differences were detected (2O5 functions primarily as a lung tumor promoter in both A/J and BALB mice following a low dose of MCA given as an initiating agent (Table P < 0.05). For both strains, tumor multiplicity was higher in V2O5-treated mice compared to MCA-treated, PBS controls (P < 0.05). Additionally, a significant difference in tumor multiplicity and size was observed between BALB and A/J in the MCA group . In A/J and BALB mice, tumors were further evaluated by histopathological analyses in a subset of animals. A majority of the tumors were found to be solid adenomas (80%) and the remaining papillary (20%), consistent with previous studies using MCA [Strain differences in tumor multiplicity and size are presented in Table up Table . In the sing MCA .2O5 dose . The peak protein response was at 6 hr in BALB and B6 mice compared to 1 day in the A/J strain. Furthermore, protein levels in A/J mice remained significantly elevated above other strains at 3 days.To determine whether chronic inflammation was associated with tumor promotion, we evaluated strain differences in BALF cellularity and protein content up to 21 days following the final Vose Fig. . BALF pr2O5 on BALF cellularity are depicted in Fig. P < 0.05; Fig. P < 0.05).The effects of V2O5 dose  and least sensitive (B6) strains 1 day following the final Ve Figure . Figures2O5 dose to determine any synergistic effect of MCA on inflammation , due to the extra weekly dose of V2O5 used to maximize promotion.In a separate study, we assessed the inflammatory cell profile in the most sensitive strain (A/J) 21 days following the last V2O5 and not to MCA is more strongly associated with lung tumor promotion in our model.To further confirm that strain differences in tumor promotion were not due to differences in inflammatory responses to MCA, an additional control experiment was conducted . In BALB mice, chemokine levels increased to a similar extent at 6 hr, but levels declined sharply by 1 day. Comparably, only modest increases (~1.5-fold) were seen in B6 mice. Strain responses were more variable and less pronounced for MIP-2 . No corresponding changes were observed in B6 mice at the time points assessed .Nuclear transcription factor activity and MAPK signaling (see below) were evaluated in the most sensitive (A/J) and resistant (B6) strains Fig. . Sub-chrsed Fig. . Nuclear2O5 treatment. By one day, phospho-p38 returned to basal levels in both strains . No significant changes in JNK1/2 were observed between or within strains at any of the time points examined  and BALB (2.2 ± 0.36) mice exposed to the carcinogen MCA followed by 5 weekly aspirations of V2O5. The effect of V2O5 was limited to tumor promotion, as no significant increase in tumor numbers were observed in animals exposed to V2O5 alone. Susceptibility to promotion paralleled relative strain sensitivity to V2O5-induced inflammation: A/J mice were most sensitive and BALB were intermediate. B6 mice were found to be most resistant to V2O5-induced inflammation, however were used as a control since they are not initiated by the low dose of MCA administered in this study [In the current investigation, we provide evidence that Vis study .Mpo), colony stimulating factor (Csf)3, CC chemokine receptor (Ccr10), and Ccl2 (Mcp-1) [2O5, our findings suggest that that genetic (host) factors contributing to V2O5-induced pulmonary inflammation are also strongly associated to lung tumor promotion.Differences between the two susceptible strains of mice (A/J and BALB) are not unusual based on past genome mapping studies demonstrating distinct genes responsible for tumorigenesis in these specific strains ,55. Whil (Mcp-1) ,55. Alth2-, H2O2, and ·OH) in target cells [Vanadium is thought to mediate pulmonary inflammation through generation of multiple reactive oxygen species , as well as drafted the manuscript. DMW assisted in experimental design. AKB conceived of the study design and methodology utilized, assisted in V2O5 exposures and euthanasia, enumerated pulmonary tumors, and helped to draft the manuscript. All authors read and approved the final manuscript.EAR performed VTable S1. Pulmonary inflammation and hyperpermeability in B6, BALB, and A/J mice treated with corn oil or MCA (10 μg/g) and then aspirated with 4 weekly doses of PBS.Click here for fileFigure S1. The MAPKs ERK 1/2 and p38 are not significantly altered between B6 or A/J mice treated with the carcinogen MCA (10 μg/g) and then aspirated with 4 weekly doses of PBS. Homogenates were prepared from the right lungs of mice treated with either MCA or oil and then 4 weekly aspirations of PBS (n = 3-5/group). Phosphorylated and total levels of MAPK in lung homogenates were analyzed from 75 μg protein by Western blotting. Representative images and mean band intensities are representative of 2-3 independent experiments. No significant differences were observed for either of the MAPK tested (P > 0.05).Click here for file


Prostaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production.Cells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates.BEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity.Results from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production. BEND coduction ,15. Simioduction , protein2 (PLA2) catalyzes the release of arachidonic acid, the rate-limiting step in PG biosynthesis, most studies on the regulation of uterine PG production have focused on expression and activation of downstream enzymes such as cyclooxygenases, which catalyze the committed step, and PG synthases. The reports  phosphatidylcholine (cold AA-PC) and 1-palmitoyl-2-[arachidonoyl-1-14C] phosphatidylcholine (14C-AA-PC) or 1-palmitoyl-2-[linoleoyl] phosphatidylcholine (cold LA-PC), 1-palmitoyl-2-[linoleoyl-1-14C] phosphatidlycholine (14C-LA-PC) were used to assay for PLA2 activity. The substrates were dried under nitrogen and resuspended by sonication in assay buffer  to a final optimum concentration of cold to radiolabeled substrate as determined previously. Calcium-dependent activity (primarily PLA2G4A) assays were performed with AA-PC and 14C-AA-PC in the presence of 5 mM CaCl2 and calcium-independent activity (primarily PLA2G6 and PLA2G4C) assays were performed using the same substrates but in the absence of CaCl2 and the presence of EGTA (5 mM). Assays optimized for identification of PLA2G6 utilized LA-PC and 14C-LA-PC in both the presence and absence of Ca++ and presence of 400 μM Triton-X and 0.8 mM ATP which enhance the activity of this enzyme . Base ou et al.  observedu et al.  observed results , it is s++ concentrations [2α production in PDBu-stimulated cells [Stimulation of BEND cells with PDBu results in the phosphorylation and activation of several components of mitogen activated protein kinase (MAPK) pathways ,24. It itrations . Importatrations ,27. Paretrations , demonsted cells . It is sed cells  and revied cells .The mechanism by which IFNT exerts negative regulation on PG production in BEND cells is unclear. Guzeloglu, et al,  investig2 than PGF2α, whereas luminal endometrial epithelial cells produce greater quantities of the latter. PDBu stimulation of BEND cell PG production did not affect PLA2G6 expression or activity and the PLA2G6 inhibitor, bromoenol lactone, did not diminish BEND cell PG production. Conversely, stimulation of PGF2α production with oxytocin in luminal endometrial epithelial cells increased PLA2G6 expression and activity and this response was attenuated by bromoenol lactone. In BEND cells, the PLA2G4A inhibitor, PYR-1, diminished PG production in unstimulated cells and abolished PDBu stimulation of PG production whereas it had little effect on luminal endometrial epithelial cell PG production . Together, these results indicate that PG production by the two cell types may be regulated by different PLA2 isotypes. Another possible explanation for the discordant results may be that stimulation with a phorbol ester may result in activation of signal transduction pathways different from those activated by oxytocin.Results from the present study with BEND cells differ from results of our previous study with bovine luminal endometrial epithelial cells . Similar2 enzymes as key regulators of PG production may lead to novel methods of regulating prostanoid production.PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production. Identification of PLAThe authors declare that they have no competing interests.2 and Western blot assays. PKT supervised the assays and helped draft the manuscript. All authors read and approved the manuscript.JDG designed the study, overall, and drafted the manuscript. MPR performed the PG assays. WG and ME performed the PLA


Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected  in vitro and in vivo.Although most patients infected with HIV who have persistent viral replication will experience a decline in CD4 T cell number, this is not always the case. In a small subset of patients in whom ART fails to suppress viral replication, CD4 T cell counts do not fall, for unknown reasons. We identified that these patients have an increased frequency of selected protease mutations, which we call discordance associated mutations (DAMs). While wild type protease rapidly induces cell death, protease containing DAMs have an impaired ability to induce cell death, due to a selective defect in cleavage of caspase 8. Furthermore, viruses containing DAMs replicate as efficiently as wild type, yet fail to induce infected cell death. These results demonstrate an unanticipated role of protease in determining the immunologic outcome of HIV infection  Prior to the development of effective treatments for HIV infection, the overwhelming majority of HIV-infected patients experienced an inexorable depletion of CD4 T cells, resulting first in immunodeficiency, followed by development of one or more AIDS defining illnesses and then death. The advent of effective therapies, and the knowledge of how best to use them, led to a reversal of this pathway for a majority of patients. Following initiation, the desired response to antiretroviral therapy is a reduction in the level of viral replication, reflected by reduced plasma viremia, and an increase in CD4 T cell number.The study of exceptional circumstances has lead to some of the most valuable advances in our understanding of HIV pathogenesis. For example, studying a subset of long term non-progressors identified the CCR5Δ32 mutation as a factor in disease progression, which in turn has lead to the development of chemokine receptor antagonists as therapies for HIV infection. Another exceptional circumstance in HIV infection is the situation of immunologic response and virologic non response to antiretroviral treatment. Such discordance has been intensely studied and found to occur in 10–20% of cases of patients initiating therapy in vivo, and is of direct relevance to HIV pathogens. Further, lymph nodes from HIV-infected patients with detectable viral replication also express Casp8p41, which colocalizes with both infected and apoptotic cells in vivo. In the current study, we evaluated whether protease mutations which occur in patients with discordant antiretroviral responses differ in their ability to generate Casp8p41 and to cause cell death, compared to patients with concordant antiretroviral responses.Expression of HIV-1 protease in cells is intrinsically cytotoxic and this property was exploited to screen and develop the protease inhibitor class, which has become an important treatment for HIV-1 infected patients. Indeed, HIV protease has degenerate substrate specificity and various host cellular proteins are targets for HIV-1 protease which are then cleaved following HIV infection including: actin, macroglobulin, myosin, spectrin, vimentin, desmin, DNA fragmentation factors, filamin, eukaryotic translation initiation factor-4γ, fibronectin, microtubule–associated proteins and other proteins 10 copies/mL. Our rationale was that if protease mutations were unrelated to the generation of a discordant response, then the frequency of protease mutations should be similar between discordant patients and those with concordant failures. Conversely, if specific protease mutations predispose to the development of discordant response, then those mutations should occur with a significantly higher frequency in discordant compared to concordant virologic failures. Indeed in discordant patients a high frequency of mutations in protease were seen in selected sites that differed from that seen in patients with concordant failures. Differences were significant for mutations at amino acids 10 (p = 0.03) 54 (p = 0.001), and 82 (p = 0.008)  . We termWe next tested whether DAMs reduce the ability of HIV protease to kill host cells relative to non-DAM mutations. For this we chose the DAMs I54V, V82A, and the non-DAMs K20R, L63P, D30N and L90M. A YFP-PR fusion construct was developed containing the p6 and RT cleavage sites which normally flank protease. Following expression, this YFP fusion construct auto-processes to generate mature 11 kd protease . Within We have previously shown that expression of HIV-1 PR causes cell death which is associated with loss of mitochondrial transmembrane potential, caspase 3 activation and nuclear fragmentation The putative impairment in cytotoxic activity of I54V and V82A were next assessed by an independent measure of death, generation of active caspase 3, and consequent caspase 3 activity. Consistent with previous findings, WT protease expression generated a significant amount of active caspase 3, and of caspase 3 activity . MirroriIn order to examine the relevance of these differences to primary CD4 T cell death, we next transfected primary CD4 T cells with the same constructs and analyzed death by TUNEL positivity. YFP positive primary CD4 T cells containing I54V or V82A protease had significantly fewer apoptotic cells than YFP positive primary CD4 T cells containing WT, K20R, L63P, D30N or L90M protease . AltogetOur previous publications indicate that HIV PR mediated killing requires procaspase 8 cleavage with subsequent generation of Casp8p41 We next assessed the ability of HIV protease to cleave procaspase 8 to produce Casp8p41. We also compared the ability of these protease constructs to cleave procaspase 8 relative to their ability to cleave gag-pol, in order to understand whether the reduced Casp8p41 production was due to a selective inability of protease to cleave that substrate, or a global reduction in catalytic activity. Two different 12 amino acid peptides were constructed reflecting the 12 amino acids surrounding the protease cleavage sites within caspase 8 and gag-pol. These peptides were generated containing fluorescence resonance energy transfer peptides; a DABCYL fluorescence acceptor group at the N terminus, and a C terminal EDANS fluorescence donor group. In this system, the DABCYL group acts to quench the EDANS fluorophore. Upon cleavage into two separate fragments by HIV-1 protease (at the Phe-Phe in Casp8p41 or at the Tyr-Pro in gag-pol), the fluorescence of EDANS is unrepressed and peptide cleavage is monitored by increasing fluorescence emission.We tested WT HIV protease or the following point mutations: D25G (active site dead), T26S , D30N, F53L, or L90M; or the DAMs I54V and V82A produced in E-coli. Each protease preparation was reacted against either the Casp8p41 substrate or the gag-pol substrate. Of interest, the two mutations (I54V and V82A) that are over represented in the discordant subjects had reduced cleavage of the Casp8p41 substrate compared to WT protease or D30N, F53L, or L90M, suggesting that these mutations have a selective impairment in the ability to generate Casp8p41, and therefore, an impairment in the ability to induce Casp8p41-dependent death . In ordeWe have presented evidence that the DAM mutations I54V and V82A, have an impaired ability to cleave procaspase 8 relative to gag-pol. This would predict that viruses containing the mutations should have preserved viral replication, yet a selective impairment in Casp8p41 production and reduced killing of HIV-infected cells. Therefore, we performed HXB2 virus infections of the human T cell lymphoblastoid line MT4, and measured cell viability, Casp8p41 content, and apoptosis by TUNEL positivity in the HIV-infected cells, as well as viral replication kinetics. Infection of MT4 cells with WT HXB2 virus resulted in a time-dependent decline in total cell viability, such that by Day 8 post-infection total cell viability approaches 60%, whereas uninfected control cell viability remained >95% . InfectiWe next specifically analyzed cell death induced in HIV-infected cells, as well as in uninfected bystander cells by co-staining with TUNEL and P24 antigen, and comparing death in the infected  population. Reasons for less cell death in the uninfected cells are unclear, but might involve an altered cytokine milieu which is associated with reduced priming of cells towards an apoptosis susceptible phenotype. A possible pathway by which this might occur is through HIV infected cells releasing paracrine factors when they die, which promotes an activated or apoptosis susceptible population of bystander cells. In such a scenario, when infected cell death is reduced, bystander death would likely also be reduced. Soluble factors which might contribute to such an effort on bystander cells include mitochondrial DNA, which is recognized by innate mechanisms as a damage associated molecular pattern, which leads to activation, inflammation and AICD It has previously been suggested that Protease mutations might impact the ability of HIV to kill CD4 T cells, through unknown mechanisms The mutation L10I is also of interest, in that it caused an intermediate degree of procaspase 8 cleavage generating less Casp8p41 than WT protease, but more than I54V and V82A (data not shown). Furthermore, expression of L10I caused less protease induced death than WT protease, more than I54V and V82A, albeit neither difference reached significance. This observation suggests that there are likely degrees of impairment in cell death afforded by different mutations.in vitro; it seems very unlikely, therefore, that well less than 0.1% of susceptible target cells would be infected in vivo. Fourth, we have analyzed Casp8p41 content in cells from HIV-infected patients both within the peripheral blood and lymphoid tissues, and have consistently demonstrated as many at 1–3% of CD4 T cells in the peripheral blood of HIV-infected patients containing HIV-specific marker Casp8p41 in vivo and that some of these die through Casp8p41-dependent mechanisms. The in vivo role of infected cell killing is further supported by recent data whereby HIV infects and kills CD4 T cells in vivoin vivo. Data contained in the current report suggests however that patients harboring selected protease mutations might have a higher proportion of infected cells that do not die through this Casp8p41 mechanism.It is generally agreed that the proportion of infected CD4 T cells Identification of Casp8p41 as a mechanism by which infected, but not uninfected, cells die following HIV infection, along with the current observation that HIV PR resistance is associated with impaired CD4 T cell killing, allows a potential explanation for observations that arose during a period when PI drug resistance was common. Physicians who treated HIV-infected patients during that era consistently observed impaired CD4 T cell losses, despite ongoing viral replication Patient genotypes were abstracted from the clinical records of patients attending the immunodeficiency clinic of the University of Ottawa. Patients were selected in 2002 based upon a rising viral load over a period of three or more months while receiving three or more antiretroviral agents. When CD4 counts rose over that period, they were classified as discordant virologic failures, as we have previously described HIV (HXB2) viral stocks containing either the WT protease gene or mutations coding for single amino acid substitutions  of the protease gene were produced as follows: Mutant PR coding sequences were generated from a pGEM vector containing the HIV-1 LAI (clone HXB2) PR and RT coding sequence, using the QuikChange Site-Directed Mutagenesis Kit  and high-performance liquid chromatography-purified primers (Genset Oligos). Genotyping of the HIV coding sequences in the plasmids upon site-directed mutagenesis confirmed the presence of the desired change(s). HXB2 proviral clone 2. To transfect HeLa cells, exponentially growing cells were transfected with DNA plasmids isolated from CsCl gradients and delivered into cells with Fugene 6 at a ratio of 1 µg DNA: 2 µl Fugene 6. For Western blot analysis, 50 µg of cytosolic proteins were fractionated on a 15% polyacrylamide gel, then transferred onto PVDF membrane  for 3 hours at 1000 mA using transfer buffer . The membranes were blocked by incubation in TBS buffer  containing 2% BSA overnight at 4°C and washed five times with TBS buffer. Then the membranes were blotted for 1 hour at room temperature with a primary antibody against HIV protease (AIDS Reagent Program). The blots were washed five times with TBS and developed with an HRP linked secondary antibody. The blot was developed using SuperSignal  following the manufacturer's protocol.Primary CD4 T cells were harvested from peripheral blood, and separated by negative selection using RosetteSep (Stem cell technologies). Transfection of primary human Cd4 T cells was accomplished using Amaxa Nucleofector. HeLa cells were purchased from ATCC . Cells were maintained in Dulbecco Modified Eagle Medium (DMEM) or RPMI1640  supplemented with 10% (v/v) FBS, 1% penicillin and streptomycin, and 2 mM L-glutamine, and incubated at 37°C in a humidified atmosphere containing 5% COYFP-HIV-1 PR was constructed by inserting a PCR product developed from the WT HxB2 provirus plasmid encompassing the PR gene plus 69 base pairs upstream and 60 base pairs downstream of the PR gene into the EcoRI/BamHI sites of pEYFP-C1 . The PR sequence was confirmed within the EYFP open reading frame by DNA sequencing at Mayo Clinic's Molecular Core Facility. The PR mutants, L10I, K20R, D25G, T26S, D30N, I54V, L63P, V82A and L90M were created on the pEYFP-C1PR plasmid with the Quick-Exchange Site-directed Mutagenesis Kit . The primers used are as follows:5′-GGGCAACTAAGGGAAGCTCTA-3′, anti-sense: 5′-TAGAGCTTCCCTTAGTTGCCC-3′; D25G sense: 5′-AGGAAGCTCTATTAGGTACAGGAGCAGATGA-3′, anti-sense: 5′-TCATCTGCTCCTGTACCTAATAGAGCTTCCT; T26S sense: 5′-GAAGCTCTATTAGATAGTGGAGCAGATGATACA-3′; anti-sense: 5′-TGTATCATCTGCTCCACTATCTAATAGAGCTTC-3′; I54V sense: 5′-GGAATTGGAGGTCTTATCAAAGTAAGA-3; anti-sense: 5′-TCTTACTTTGATAAGACCTCCAATTCC-3′; I54V sense: 5′-ATTGGAGGTTTTGTCAAAGTAAGACAG-3′, anti-sense: 5′-CTGTCTTACTTTGACAAAACCTCCAAT-3′; L63P sense: 5′-GTAAGACAGTATGATCAGATACCCATAGAAATCTGTGGAC-3′; anti-sense: 5′-GTCCACAGATTTCTATGGGTATCTGATCATACTGTCTTAC-3′; V82A sense: 5′-GTAGGACCTACACCTGCCAACATAATTGGAAG-3′; anti-sense: CTTCCAATTATGTTGGCAGGTGTAGGTCCTAC-3′; L90M sense: 5′-TAATTGGAAGAAATCTGATGACTCAGATTGGTTG-3′;and anti-sense: 5′-CAACCAATCTGAGTCATCAGATTTCTTCCAATTA-3′. All primer synthesis and mutation confirmation was carried out in Mayo Clinic's Molecular Core Facility.K20R sense: 6 cells in 1 ml PBS at 37°C for 20 minutes. TMRE and YFP double positive cells were analyzed by flow cytometry at 20,000 events per sample. To measure apoptosis, the In Situ Cell Death Detection kit  was used to stain 3′ DNA strand breaks following the manufacturer's protocol. Briefly, CD4 T cells were fixed in 2% paraformaldehyde, and permeabilized in 0.1% sodium citrate containing 0.1% Triton X-100 for 5 minutes. Fixed cells were resuspended in 50 µl of TUNEL reaction buffer containing PE-dUTP and 5 U TdT, and analyzed by flow cytometry. To determine the expression of Casp8p41, 10∧6 peripheral blood lymphocytes were fixed with 2% paraformaldehyde in PBS then permeabilized with PBS plus 0.1% NP-40 on ice for 2 minutes and then incubated with Alexa 647- or PE-labeled Casp8p41 antibody Mitochondrial membrane potential (MMP) of the YFP and YFP fusion transfected cells was measured with the mitochondrial specific red fluorescent dye, tetramethylrhodamine, ethyl ester, perchlorate (TMRE) HeLa cells were seeded onto glass coverslips and transfected with plasmids coding for YFP-HIV protease WT or mutants. TUNEL staining for detection of apoptosis was done according to the manufacturer's protocol . DAPI was added to identify nuclei. Laser-scanning confocal microscopy was performed using a Zeiss LSM-510 .Caspase 3/7 activity in HIV-1 PR WT and mutant transfected HeLa cells was measured with a Caspase 3 Fluorometric Assay Kit  following the manufacturer's instructions. Briefly, the cells were harvested at 6–8 hours after transfection, lysed, and equal amounts of protein were added to each reaction in a 96 flat well microplate. The fluorescent signals were read on a Microplate Fluorescent Reader  using excitation and emission wavelengths of 340 and 450 nm, respectively.To assess the effect of the various mutations on the proteolytic activity of HIV protease, a fluorometric assay was devised to compare the relative rates of cleavage of a consensus gag-pol site with the caspase 8 site. The fluorogenic peptides (gag-pol: Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg; caspase 8: Arg-Glu(EDANS)-Pro-Lys-Val-Phe-Phe-Ile-Gln-Ala-Lys(DABCYL)-Arg) were synthesized in the Mayo Protein Core Facility such that fluorescence would be quenched until the internal peptide is cleaved. Measurements were performed in 96-well plates with a SPECTRAmax GeminiXPS spectrofluorometer . For each enzymatic reaction, 50 µl of cell lysates containing HIV protease was added to 150 µl of protease assay buffer  containing 2.5 µM of either gag-pol or caspase 8 substrate peptide prewarmed to 37°C. Fluorescence intensity readings at 490 nm (excitation 340 nm) commenced immediately and data was continuously recorded every 2 minutes for 60 minutes.5′- AGAAGAGAGGATCCGATGTGGGGTAGA -3′ and reverse primer 5′- TTCTTC TGTGAATTCTCATTGTTTAAC - 3′. The PCR product was digested with BamH1 and EcoR1 , and ligated into the protein expression vector pET-21b  using T4 DNA ligase , which had been restriction digested with BamH1 and EcoR1. The ligation product was transformed into E-coli stain BL21(DE3)pLysS  cells. The insert was confirmed by DNA sequencing, using dye terminator cycle sequencing at Mayo DNA sequencing core facility. Proteases were expressed by an induction of 1.0 mM isopropyl beta-D thiogalactoside and bacterial pellet lysed directly using HIV-1 PR assay buffer, in order to avoid protease degradation with appropriate control cells lysate. The lysate were subjected to protease assay, as described.The PCR approaches were utilized to generate HIV-1 PR wild type or mutant sequences from PR expression vector pEYEP-C1 using forward primer Values of continuous variables are expressed as means +/− standard error. Comparisons of continuous variables were made using T tests. The prevalence of resistance mutations in discordant versus concordant patients was compared using the Fisher's Exact test. A P value of ≤0.05 was considered statistically significant.


Fifteen known type 2 diabetes (T2D) gene variants were assessed for their associations with T2D status in 228 T2D families from the Framingham Heart Study (FHS) Original, Offspring, and Children Cohorts. Bayesian approach was used to test single-single-nucleotide polymorphism (SNP) association followed by logistic regression. Bayesian and logic regression approaches were used to test multiple SNP association searching for the best combinations of variants followed by logistic regression reconfirmation. The significant variants for T2D risk were also tested for their main and interacting effects on triglyceride (TG)/high-density lipoprotein (HDL) ratio change derived from four point measures across time. This slope phenotype was made available using mixed model growth curve approach from 155 T2D families in the FHS Offspring Cohort.CDKN2B rs10811661 (p = 0.042), TCF7L2 rs4506565 (p = 0.004), and JAZF1 rs864745 (p = 0.04) were individually associated with risk of T2D . CDKN2B and TCF7L2 were found with significant main  and interacting (p = 0.05) effects for increased (OR = 3.0) risk of T2D. CDKN2B and JAZF1 were found with significant main (p = 0.0002 and 0.034) and interacting (p = 0.001) effects on increased (β = 0.42) TG/HDL ratio longitudinal change. These interacting effects were independent of effects of age and sex with effect sizes of 0.3-0.4% for risk of T2D or TG/HDL ratio longitudinal change.CDKN2B and TCF7L2 interacting effect for T2D risk and CDKN2B and  JAZF1 interacting effect on TG/HDL ratio increase over time among  T2D families in the FHS. These interacting effects were consistent  in conferring risk of T2D or progressive insulin resistance with  modest effect sizes.  These synthetic approaches allowed for successful detection of   Fifteen replicated gene variants for type 2 diabetes (T2D) have been assessed . While m2, average TG was 83.7, 108.5, 134.0, and 135.5 mg/dl, average HDL was 51.6, 52.6, 51.6, and 54.5 mg/dl, and average TG/HDL ratio was 1.9, 2.5, 3.1, and 3.0, at Visit 1, 3, 5, and 7, respectively. Every study subject provided written informed consent. The study was approved by Boston University Institutional Review Board.All participants in the FHS were Caucasians. Their characteristics were described elsewhere . In brieAffymetrix 100 k single-nucleotide polymorphisms (SNPs) and genotype annotation resources were described elsewhere . Identic2, and sex. BIMBAM (Bayesian imputation-based association mapping) [p-value . This was followed by logistic-GEE  single-SNP test under general and additive assumptions. BIMBAM output corrected p for multiple testing. BIMBAM was further used to identify the best multiple-SNP combination with the highest BF. LOGREG [Covariates included age, agemapping)  was used. LOGREG  was alsoCDKN2B rs10811661 (p = 0.0011), TCF7L2 rs4506565 (p = 0.0043), and JAZF1 rs864745 (p = 0.0402). Multiple SNP test results are also given in Table CDKN2B rs10811661 and TCF7L2 rs4506565 was significantly detected using the logistic-GEE model . For the slope trait, single-SNP test results were non-significant for CDKN2B rs10811661 (p = 0.4621), but significant for TCF7L2 rs4506565 (p = 0.0379) and JAZF1 rs864745 (p = 0.0349) in the mixed sandwich estimator additive model. Interaction between CDKN2B rs10811661 and TCF7L2 rs4506565 variants were significant , and it was associated with high slope values, which may be expressed as an increase in insulin resistance across ages.Single-SNP test results are presented in Table TCF7L2 intron 3 variant for T2D is in line with a previous FHS genome-wide association study report [CDKN2B near/promoter and JAZF1 intron 1 variants for T2D risk was also found. We thought the T2D families in FHS may contain an enriched genetic component for SNP association assessment of T2D gene variants. Currently, functions of these two genes are yet not clear, but as is the case for TCF7L2 and other confirmed T2D genes [β-cell failure, insulin resistance, and lipid toxicity. Our finding of interactions among the three variants for T2D risk was interesting and novel, which may help disentangle underlying gene pathways and further our understanding of T2D etiology and pathophysiology.Our finding of Correction for multiplicity to minimize the false-positive declaration rate seems to be less sensitive for this analysis for two reasons. First, we aimed to identify and evaluate the best combination of SNPs from confirmed T2D variants. Second, the FHS T2D status information was obtained at the initial visit of the study, and 'new' T2D cases may have remained in the 'non-diabetic' control group after decades of follow-up. This would bias our association findings towards reduction of type 1 error. The results of BIMBAM were consistent with those of logistic-GEE analyses for the single-SNP main effect; likewise, BIMBAM and logic regression results for multiple-SNP joint/interacting effect were also consistent. These synthetic approaches apparently worked well in association detection for both main and joint effects of candidate gene variants with prior knowledge.This 500 k SNP genotype data allowed us to examine 15 of the 18 confirmed exact variants (genotypic imputation was not applied). While reproducibility may be cohort-specific and sensitive to sample heterogeneity and size, it was a bit surprising that we could only identify three meaningful variants. It is very possible that this was due to the presence in the control groups of persons with diabetes who had not received a clinical diagnosis. We did not use a transmission-disequilibrium test-like FBAT program for this analysis simply because we wanted to maximize the sample size by using all available family information. We did not correct the data for potential effect of body max index because obesity coexisting with T2D is very likely rooted in the same pathway genes among most T2D patients, at least in most Western countries.TCF7L2 variant was only associated with β-cell function failure but not insulin resistance [TCF7L2, together with JAZF1 variants, may indirectly impart insulin resistance through joint modulation of lipid metabolism.We were excited to find that the identified interacting variants also affected the TG/HDL ratio. Using the ROC/AUC (receiver operator characteristic/area under the curve) model, we found that it was an imperfect predictor of T2D with best sensitivity and specificity of approximately 75%. Applying a mixed growth curve approach to model longitudinal multiple measures of the TG/HDL ratio proved useful. The derived slope phenotype represented the TG/HDL ratio change across decades of observation time. This approach is advantageous in handling missing data points, as well as remote values, especially in intervention and interaction studies. Genetic heritability of the slope phenotype was 20%, and these three variants were individually and interactively associated with an increase of the slope phenotype. Previously, it was thought that the sistance . Our curTCF7L2, CDKN2B, and JAZF1 genes were independently and interactively associated with increased T2D risk and the TG/HDL ratio change across ages. For the remaining T2D risk conferring gene variants, we did not find significant association evidence in this analysis.In conclusion, using the FHS T2D family data and different statistical approaches, we found variants in the AUC: Area under the curve; BF: Bayes factor; BIMBAM: Bayesian imputation-based association mapping; GAW16: Genetic Analysis Workshop 16; GEE: Generalized-estimating equations; HDL: High-density lipoprotein; TG: Triglyceride; T2D: Type 2 diabetes; FHS: Framingham Heart Study; ROC: Receiver operator characteristic; SNP: Single-nucleotide polymorphism;The authors declare that they have no competing interests.PA, MF, IB, and MP drafted the manuscript. SK, AA, and SL performed the statistical data analysis. All authors read and approved the final manuscript.