The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in This article can also be viewed as an  The heat shock-70 proteins (Hsp70s) are chaperones involved in crucial cellular functions in all kingdoms of life. Hsp70-family function, structure, and mechanisms have been studied to great detail using cognate HSPA8/Hsc70 The human genome contains over 40 hsp70 sequences. Many of them are pseudogenes, but at least eleven distinct genes located on several chromosomes are translated into protein Several of the human cytosolic Hsp70 isoforms have yet to be extensively characterized. Heat shock 70-like protein-1  is a constitutively expressed, non-inducible cytosolic protein with high abundance in testis The major Hsp70 protein of the endoplasmic reticulum is HSPA5, originally called immunoglobulin heavy chain binding protein (BiP), or glucose regulated protein-78 (GRP78) Despite the large number of NBD crystal structures determined under different nucleotide conditions, the conformational changes that ultimately drive the Hsp70 chaperone machine have never been visualized by X-ray crystallography of NBDs alone. Instead, the NBDs all crystallized in a closed conformation that is interpreted as resembling the ATP-bound state. Only crystal complexes of NBDs and nucleotide exchange factors have yielded structures with a partially opened nucleotide binding cleft E. coli, although the soluble expression levels for HSPA5/BiP were relatively low.A common feature of Hsp70 ATPase domains is that, despite the large conformational changes they are predicted to undergo in their physiological context, their crystal structures are highly similar under different nucleotide conditions. We addressed the question whether this is also true for the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. These share between 67 and 92% sequence identity with the NBD of the major stress inducible isoform, HSPA1A . We used2+- or Mn2+-ions present in the crystallization solution  might act as phosphate donor. This pathway might be employed in situations of high ATP turnover or low ATP concentrations to secure vital cellular functions.A second calcium ion was found at the base of HSPA5 α-helix-6, tethered by the H252 hydroxyl and the E256 and D257 carboxylates. This site has previously been identified in human HSPA1 (1S3X) M1-N387, HSPA1LM1-K386, HSPA2P6-D386, HSPA5D26-D410, and HSPA6E6-D385 were subcloned into expression vector pNIC-Bsa4 by ligation-independent cloning. The resulting expression constructs contained a hexahistidine tag and a TEV-protease cleavage site (MHHHHHHSSGVDLGTENLYFQS) at the N-termini.The cDNAs coding for the full-length human HSPA1A, HSPA1L, HSPA2, HSPA5, and HSPA6 were obtained from the Mammalian Gene Collection . The sequence coding for residues HSPA1Ahttp://www.thesgc.org/structures/). Briefly, each expression construct was transformed into E. coli strain BL21(DE3)R3 pRARE (Novagen) and the cultures were grown in Terrific Broth supplemented with 8 g/l of glycerol at 37°C. At an absorbance at 600 nm between 1 and 2 the temperature was lowered to 18°C, recombinant protein production was induced by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside, and cell growth was continued for 18 h at 18°C. Cells were harvested by centrifugation and the cell pellets were resuspended in 1.5 volumes/wet cell weight of lysis buffer  per 50 ml cell suspension). Before lysis, 4 µl (1000 U) of Benzonase (Novagen) was added per 50 ml cell suspension, and lysis was achieved by sonication. Cell debris was removed by centrifugation and the soluble fractions were filtered through a syringe filter (0.45 µm pore size). Cleared lysates were passed over 1-ml HiTrap Chelating columns  pre-equilibrated with buffer 1 . The columns were washed sequentially with buffer 1 and buffer 1 supplemented with 25 mM imidazole. Bound protein was eluted with buffer 1 containing 500 mM imidazole, loaded onto 16/60 Superdex-200 HiLoad columns , and gel filtration was performed in buffer 2 . Fractions were pooled based on gel filtration profiles and purity, TCEP was added to 2 mM, and the proteins were concentrated to 24.8 mg/ml (HSPA1A), 16.0 mg/ml (HSPA1L), 14.0 mg/ml (HSPA2), 31.0 mg/ml (HSPA5A), and 25.0 mg/ml (HSPA6). Proteins were typically more than 90% pure judged by SDS-PAGE analysis  or mother liquor containing 15%–20% glycerol  and flash-frozen in liquid nitrogen.Crystallization methods and conditions are summarized in A single wavelength dataset for HSPA1A was collected with Cu-Kα radiation (1.54166 Å) on an X8 PROTEUM system equipped with a four-circle Kappa goniostat and a PLATINUM-135 CCD . SAINT (Bruker AXS) and XPREP (Bruker AXS) were used to process the data. Further datasets were collected at synchrotron beamlines at BESSY (BL14-1), Berlin, Germany (HSPA5 and HSPA6), DIAMOND (I03), Oxfordshire, UK (HSPA1L) and ESRF (ID23-1), Grenoble, France (HSPA2). Data sets were indexed, scaled, and reduced using the programs XDS www.rcsb.org .The atomic coordinates and structure factors have been deposited with the Protein Data Bank, Datapack S1http://www.molsoft.com/icm_browser.html.Standalone iSee datapack - contains the enhanced version of this article for use offline. This file can be opened using free software available for download at (ICB)Click here for additional data file.Text S1Instructions for installation and use of the required web plugin (to access the online enhanced version of this article).(0.75 MB PDF)Click here for additional data file.


To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, ΔrelB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the  Mycobacterium tuberculosismazEF has been considered as mediators of bacterial programmed cell death mazEF-mediated cell death is a population-dependent phenomenon requiring a quorum sensing molecule, termed extracellular death factor, which is a linear pentapeptide (NNWNN) important for mazEF-mediating killing activity E. coli strains defective in mazEF showed lower sensitivity to antibiotics than the wild type, indicating that antibiotic addition could induce mazEF-mediated cell death E. coli strains, one wild type (wt) and the other having deletions in five TAS  subjected to short-term stress conditions  showed no significant differences among them Myxococcus multicellular development Chromosomally-encoded Type II toxin-antitoxin systems (TAS), composed of two proteins, are widely spread among Bacteria and Archaea. Typically, they are organized as operons in which the first gene encodes the antitoxin (A) and the second the toxin (T). Both proteins interact to generate a harmless TA complex that auto-regulate their own synthesis. The A protein by itself is metabolically unstable and is constitutively degraded by ATP-dependent proteases, releasing a free and stable T protein that would kill or stop the growth of the cells by disruption of key cellular processes Streptococcus pneumoniae (the pneumococcus): relBE1Spn, relBE2Spn, yefMyoeBSpn, higAB, phd/doc, pezAT, tasAB, and hicABrelBE2SpnyefM-yoeBSpnpezATrelBE1Spn was shown to be non-functional E. coli cells to RelE2Spn toxin resulted in the arrest of cell growth, which was rescued by induction of RelB2Spn antitoxin but only within a time-frame window: long-time exposure to the toxin led to cultures unable to resume growth relBE2Spn TAS in the bacteria lifestyle. We have compared the behaviour of two pneumococcal isogenic strains, wild type (wt) R6 and a relBE2Spn mutant derivative (R6ΔrelB2Spn) Spn toxin could act as a modulator of protein synthesis under stress, but it could also induce cell death when the level of protein synthesis was dramatically reduced. Further, if relBE2Spn played a role in bacterial fitness, then it should contribute to colonization and survival after infection . If this was the case, the relBE2Spn genes should be ubiquitous in the S. pneumoniae population. Thus, the presence and integrity of the relBE2Spn locus was tested in 100 strains from different sources. Unlike E. coli, where several strains lacked the relBE operon relBE2Spn operon exhibited various degrees of polymorphisms in the different isolates, none of the changes impaired the functionality of the relBE2Spn locus. A molecular model of the pneumococcal RelE2Spn protein was constructed based on the three dimensional structure of the RelBE complexes from Pyrococcus horikoshii (PhRelE) Methanococcus jannaschii (MjRelE) Spn kept several residues related to the catalytic activity of ribonucleases, which are also present in MjRelE. However, these residues are missing in EcoRelE and PhRelE proteins EcoRelE Genes for at least eight putative TAS are present in the chromosome of the Gram-positive bacterium 650) and viability  between the two strains, R6 wt and its relBE2Spn mutant derivative, R6ΔrelB2Spn . To perform the experiments, cultures of both strains were grown 24 h, and OD650 and cfu were determined at time intervals. No differences were found between the strains during the entire period in which the cultures passed through exponential (0–2 hrs), stationary (2–8 hrs), and autolysis (8–24 hrs) phases of growth  rather than glucose because of poorer utilization of the latter relB2Spn mutant cells showed no differences although cessation of growth was observed for either strain as compared to sucrose-grown cultures, and no decrease in viability was observed in either sucrose-depleted culture  resulted in an increase of relBE transcription leading to the increase of free RelE due to RelB Lon-dependent proteolysis S. pneumoniae under a SHT-mediated amino acid starvation. The results (Serine hydroxamate (SHT) induces amino acid starvation because it blocks incorporation of Ser residues into proteins by interfering with the load of seryl-tRNA  results  showed t results . In the  results . After t results . SHT wit results , inset. relBE transcriptional induction in E. coli−1 is sufficient −1 (http://www.eucast.org/mic_distributions/). To test whether the mutation of relBESpn had any effect on cell-response to blocking protein synthesis, pneumococcal cultures (wt and ΔrelB2Spn strains) were challenged with two dosages of Erm, 0.1 and 1 µg.ml−1 , followed by OD650 and cfu determination at different times. After the 20 min treatment, growth of both cultures was stopped, concomitantly with a progressive reduction in cfu . In this case, we observed, again, that the mutant cells were more tolerant to Sm-treatment than the wild type strain  stain method. The pneumococcal cultures were stained and examined under the microscope: living cells were stained in green, whereas dead cells were stained in red. Micrographs of the pneumococcal cultures were taken at 0 and after 180 min of Erm treatment. The results showed no significant differences between both strains when the cultures were challenged with 0.1 µg.mle strain . We concrelBE2Spn could confer a selective advantage to the pneumococcus, then a conservation of this locus in the bacterial chromosome of most, if not all, isolates should be expected, in spite of hyper-recombination typical for this species relBE2Spn and the yefM-yoeBSpn loci , was tested by PCR using the oligonucleotide pairs relB2p/relE2c and yefMN/yoeBC, respectively. The first pair was previously used to amplify the relBE2Spn locus of R6 strain c primer annealing partially into the region encoding RelE2Spn toxin  exhibited changes compared to strain R6 in the chromosomal regions flanking the relBE2Spn operon, whereas the region around the yefM-yoeBSpn locus was kept intact.We reasoned that if the expression of pn toxin . In theselBE2Spn . Howeverdetected . The sizhttp://spneumoniae.mlst.net/). Spanish strains were isolated from blood and sputum in the years 2002 and 2006. The Polish clinical isolates proceeded from the National Medicines Institute pneumococcal collection, and amounted to 58 isolates representing 37 serotypes and 52 different STs relBE2Spn locus and its flanking regions by amplification with different oligonucleotides spanning the appropriate regions (relBE2Spn operon in another 31 strains whose sequences are available at the NCBI Genome Project (http://www.ncbi.nlm.nih.gov/) and at the Sanger Institute (http://www.sanger.ac.uk/Projects/S_pneumoniae/), thus making a total of 100 strains analyzed . Two other polymorphisms in which minor amino acid changes occurred were also found , whereas some strains, like CipR-25 .The results of the global PCR analyses of the chromosomal structure of tegories : Type I BE Types  showed n CipR-25 , exhibitrelBE2Spn promoter region in the different types was not essentially modified, synthesis of the relBE2Spn mRNA (and hence the expression of these two proteins) could be affected. To detect the relBE2Spn mRNA in several clinical isolates, primer extension analyses were carried out. Total RNA was isolated from selected strains belonging to the three genomic types: i) from Type I, strains R6, and CipR-25, the latter containing the A/G change at the position -28 in the relBE2Spn proposed regulatory region; ii) from Type II, strains CipR-31, CipR-67, and 2115, the latter harbouring also the same change in the proposed regulatory region, and iii) from Type III, CipR-51 and CipR-14. In all strains, a primer extension product was detected , whereas change T34I was relatively frequent (around 35% of the sequenced isolates). To verify whether these changes affected the toxic activity of RelE2Spn, and to elucidate their possible physiological consequences, we tested toxicity on E. coli cells based on two criteria previously used for the pneumococcal yefM-yoeB operon Spn, and ii) ability of RelE2Spn to interact with the cognate RelB2Spn antitoxin in cultures harbouring uncoupled genes (relB2Spn and relE2Spn) cloned in two different plasmids under inducible promoters. In the latter conditions, induction of the antitoxin should neutralize the toxic effect of RelE2Spn thus permitting bacterial growth. To this end, DNA fragments containing genes relE2Spn, wt or mutants harbouring the D39G, T34I, and, as a control, relE2ter (encoding a truncated and inactive RelE2Spn protein) were cloned into plasmid pFUS2 araBAD promoter (PBAD), which is inducible by arabinose and repressed by glucose. The resulting plasmids (Spn), pE71 (D39G RelE2Spn), pE81 (from strain 8651) or pE600 (from strain k-600), both harbouring the T34I RelE2Spn mutation and pE2ter (truncated RelE2 protein). In addition, the relB2Spn wt gene was cloned in plasmid pNM220 lac promoter; the resulting plasmid was termed pB2wt , pE81(T34I), or pE600(T34I)  > relE2T34I (nearly two orders) > relE2D39G (twofold). The toxicity of the RelE2Spn toxin could be counteracted by its cognate antitoxin, encoded in the pB2wt plasmid. Cells, containing the different pairs of plasmids were streaked on plates supplemented with 0.4% arabinose (induction of toxin synthesis) with or without 2 mM IPTG (induction of antitoxin synhtesis). Transformants containing, in addition to pB2wt, plasmids encoding the toxin  were able to grow only on plates supplemented with IPTG while control cells harbouring pB2wt and either pFUS or pE2ter did not show growth differences in the presence or absence of IPTG  . Additio00(T34I) . This re of IPTG .S. pneumoniae from which these bacteria spread to other body parts and to new hosts; hence an increase in pneumococcal persistence during colonization may influence its virulence and epidemicity relBE2Spn was identified in the chromosome of S. pneumoniae and shown to be functional, in contrast to the pneumococcal homologue termed relBE1SpnrelBE2Spn operon exhibited the same growth profile and response to sugar starvation as the wt bacteria did  the wt strain exhibited an exponential growth period after recovery which was longer than that of the mutant. These results indicated that the pneumococcal relBE2 system, under amino acid starvation, could help the bacteria to divert the scarce resources to essential processes, thus improving its survival potential. Treatment with Erm (or with Sm) also resulted in a different response in the two pneumococcal strains used. The wt showed a higher and quicker reduction in cfu than the mutant .Thus, activation of the pneumococcal relBE2Spn operon subsequent to antibiotic treatment appeared to induce an extreme interruption of the protein synthesis, leaving the bacteria unable to recover viability or even inducing cell death. Then, lack of the relBE2Spn operon in S. pneumoniae would lead to antibiotic tolerance a role that coincides with the one proposed for the E. coli mazEF TAS The human upper respiratory tract is a natural environment for eria did . Differer of cfu  and 2, br of cfu  and 2: ie mutant  and S2 ae mutant . The higrelBE2Spn operon is not essential, at least under the laboratory growth conditions used  showed that more than 40% of them lacked this TAS (not shown). In E. coli, the homologous relEB TAS have been lost in several strains E. coli strains showed decay in the chromosomally-encoded ccdAB TAS and a molecular evolution analysis of these data suggests that this TAS does not seem to retain any role in E. colirelBE2Spn locus showed the existence of three different genetic organizations, although transcription of the operon was not affected by these rearrangements  Methanococcus jannaschii (MjRelBE) PhRelE toxicity  were conserved in RelE2Spn . Curiously, this R-distribution was not fully conserved in the pneumococcal RelE1Spn  appeared to be located close to the toxicity-related R residues. According to the structure of toxins Kid and MazF Spn mRNA cleavage activity, thereby diminishing RelE2Spn toxicity , residues H43, Y31 and Y57, and two acidic residues (E38 and D39)  . These rrelBE2Spn locus could provide a mechanism for S. pneumoniae to cope with unfavourable conditions, allowing the bacteria to efficiently survive and colonize humans. Further, the results show the importance of TAS as targets for designing new antimicrobials, which is especially true for bacteria like S. pneumoniae because of their high recombination rates and, being naturally competent, horizontal transfer. As a consequence, the appearance of new polymorphic and antibiotic resistant strains poses a serious threat for infection management.Our results indicate that the E. coli cultures were grown in TY medium R, 150 µg.ml−1), or kanamycin resistance . S. pneumoniae cells were grown in AGCH medium R, 2 µg. ml−1). All cultures were grown at 37°C. The agar plates were incubated at 37°C in air. SHT , Erm (used at 0.1 or 1 µg. ml−1), and Sm (used at 20 µg.ml−1) both of them blocking bacterial ribosomes, were purchased from Sigma. We used Erm instead Cm because the mutant strain, R6ΔrelB2Spn, harbours a chromosomally-integrated copy of the cat gene , 7153 (pE71), 8151 (pE81), or K-600 (pE600) and amplified with primers relE2N (5′-CGCG GATCCGATGCATGATTTAGGCTTGAAGGATGAATA-3′) and relE2tga (5′-CGTGGTACCTCAATAAATATCTCTCCGATGACCAACTTC-3′). The resulting 290-bp PCR products were digested with EcoRI and KpnI before ligation into the equivalent sites of pFUS2. Plasmid pE2ter was randomly isolated during construction of pE71 and contains a mutation in the relE2sequence changing the E38 residue for a termination codon, yielding a truncated RelE2 protein. Plasmid pB2wt was constructed by amplification of a chromosomal DNA fragment encoding gene relB2Spn with its own ribosome binding site using the primers relB2BamHI (5′-CGGGATCCGTGTTACCATTAAAAAAGGGAGCACA AAG-3′) and relLCC (5′-CGGGGTACCATCGCGAATTCTAAAACGTCTTGTT GGAACTAATTTATAC-3′). The resulting 310-bp DNA fragment was digested with BamHI and EcoRI before ligation into the equivalent sites of pNM220. All plasmids were rescued by transformation of competent E. coli cells.Plasmids used in this work were constructed as follows: pE2wt, pE71, pE81, and pE600: The S. pneumoniae R6 and R6ΔrelB2Spn were exponentially grown in AGCH complete medium to OD650 = 0.2 at 37°C. Then, half of each culture was exposed to the different experimental conditions: carbon starvation, or addition of SHT or Erm (or Sm). Growth of treated and untreated cultures was followed by OD650 and the viability (number of cfu) was measured by plating serial dilutions of each culture on AGCH plates. After SHT-, Erm, or Sm-treatment, cultures were washed twice with pre-warmed AGCH and suspended in the same volume of complete AGCH drug-free medium. OD650 and viability was then tested for at least 180 minutes. In the carbon starvation experiments exponentially growing cultures (to OD650 = 0.2) were washed twice in pre-warmed AGCH medium and finally suspended in the same volume of AGCH medium supplemented with 0.2% yeast extract and with or without sucrose. Cell growth and viability were measured as above. All the experiments were performed at least three times.Cultures of ΔrelB2Spn were exponentially grown at OD650  = 0.25 in AGCH complete medium at 37°C and Erm was added at two concentrations (0.1 µg.ml−1 or 1 µg.ml−1). After 180 min, cells were harvested by centrifugation, washed twice with buffer  and stained with the LIVE/DEAD BacLight Bacterial viability kit (Invitrogen) according to the manufacturer's instructions. Cells were visualized using a multidimensional AF6000 LX LEICA microscope and filter cube L5 for green fluorescence or N5 to detect red fluorescence.Cultures of R6 and R6Chromosomal DNAs were extracted using the Bacterial Genomic DNA Isolation Kit (NORGEN) and chromosomal DNA (50–100 ng) was added to PCR reactions performed using Phusion high fidelity DNA polymerase (Finnzymes) and, as primers, the following oligonucleotides:N: 5′-CGCGGATCCGCTTGTACAAGTTCCTGACAATTTC-3′yefMC: 5′-CTGGAATTCCGGTAGAGACTTGAGAAAAAGCCTA-3′yoeB5′-CGGAATTCCGATCAGGTTCTTACGCTTGGCG-3′rel2p5′: 5′-CAGATAC CGCAACACCATTGACAG-3′relB2p: N: 5′-TGCTCCCGGGCTATTACATTAAAAGTTTCTGAA GCTG-3′relB2C: 5′-CGCGAATTCCTTCCCAAGTAATGGGT TCAACTCC-3′relB2N: 5′-CGCGGATCCGATGCATGATTTAGGCTTGAAGGATGAATA-3′relE2tga: 5′-CGTGGTACCTCAATAAATATCTCTCCGATGACCAACTTC-3′relE25′-CCTCACGACTAATCCGTTGCAG-3′SP1222: ter: 5′-GCATCTGCTAA AGAATTACAAGCATCATTG-3′ldhC. The sequence of this DNA fragment was determined using primers relB2p, relB2C, and two specific IS1167 primers: 2167N (5′-GTCATAGTAAGGACTAAACATA TCC-3′) and 2167C (5′-GAAAAGCGATCAAACAACTCATTAG-3′).To analyse the IS1167 transposon sequence, a 1750-bp PCR-DNA fragment including this element was obtained using as template chromosomal DNA isolated from strain 2167 and, as primers, relB2p and relB2aroE, gdh, gki, recP, spi, xpt and ddl was performed as described by others http://spneumoniae.mlst.net/was used to assign allele numbers and STs.MLST based on sequencing of fragments of seven housekeeping genes, relBE2Spn specific primer  malX specific primer mal1, 5′-GTGTAACAGTTCCAAGCACCG-3′). The 3′-ends of primers were located 56 nt or 48 nt from the nucleotide A of the ATG initiation codon of the relB2Spn gene or in the malX ATG initiation codon, respectively Total RNA was isolated from R6 and from different pneumococcal isolates with Aurum total RNA minikit (BIO-RAD). For RNA extraction, 1.5–3 ml of the bacterial cultures in late exponential phase were centrifuged and the cells were resuspended in 100 µl of lysis buffer . The cell suspension was incubated 10 min at 30°C and further preparation was done according to the manufacturer's instructions. Primer extension assays were performed as described Spn toxin was constructed using Geno3D molecular modelling program  and the 2.3 Å resolution X-ray crystallographic structure of the P. horikoshii OT3 aRelBE complex M. jannaschii MjRelBE complex http://www.pymol.org).The three-dimensional model of RelE2http://www.ncbi.nlm.nih.gov/NCBI Genome Project: http://spneumoniae.mlst.net/Pneumococcal MLST database: http://www.eucast.org/mic_distributions/European Committee on Antimicrobial Susceptibility Testing: http://www.pymol.org.PyMOL: http://www.sanger.ac.uk/Projects/S_pneumoniae/Sanger Institute: Geno 3D: http://geno3d-pbil.ibcp.frFigure S1Growth profile of S. pneumoniae cells wt and mutant R6relB2Spn under normal or carbon-starvation conditions. Pneumococcal strains R6 (triangles) or relB2Spn (circles) were grown in AGCH complete medium . In carbon-starvation conditions (panels C and D), cells were exponentially grown in AGCH complete medium to an OD650  = 0.2, twice washed, and suspended in the same medium with (open symbols) or without sucrose (filled symbols). Growth of the cultures was followed by measurement of OD650 nm  or by determination of the number of cfu .(1.35 MB EPS)Click here for additional data file.Figure S2S. pneumoniae cells from strains R6 (circles) or R6relB2Spn (triangles) were grown exponentially in complete AGCH medium to an OD650  = 0.1−0.14. Then, Sm (20 (mu)g.ml−1) was added, and incubation was continued for 180 min more. Growth was followed by measurement of OD650 nm of the cultures untreated (open symbols) or treated (closed symbols) with Sm (A). At indicated times appropriate dilutions of cells were plated and incubated as in Inhibition of protein synthesis mediated by Sm treatment. (1.11 MB EPS)Click here for additional data file.Figure S3Primer extension analysis using total RNA from different S. pneumoniae clinical isolates. RNA samples from R6 (1) and the following clinical isolates: CipR25 (2), 2115 (3), CipR67 (4), CipR31 (5); CipR14 (6), and CipR51 (7) were annealed with [32P]-labelled specific primers mal1  or with the relRNA oligonucleotide (r) to detect relBE2Spn mRNA. Ct Indicates a G+A Maxam and Gilbert sequencing reaction, used as DNA size marker.(0.53 MB EPS)Click here for additional data file.Figure S4RelE2Spn three dimensional structural model. Location of R41, R56, R63, R83, and D39 residues is depicted. Other residues  supposedly involved in the catalytic mechanism are displayed in cyan.(1.06 MB EPS)Click here for additional data file.Table S1(0.04 MB XLS)Click here for additional data file.


Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters  of the avian-like N1 NA glycoproteins were highly consistent with their IC50 values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 106 EID50 dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues  or in close proximity to the NA enzyme active site . NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate  inhibitors (oseltamivir and zanamivir) will be crucial for disease control. However, the emergence of NA inhibitor–resistant virus mutations can significantly limit the effectiveness of treatment. We used reverse genetics techniques to study the impact of specific point mutations in the NA gene on the pathogenicity of clade 2.2 H5N1 influenza viruses in a ferret animal model. Seven recombinant H5N1 viruses  were viable and genetically stable in MDCK cells. Three of the NA mutations were of particular significance: (1) E119A conferred resistance to both oseltamivir carboxylate and zanamivir and increased virus virulence in ferrets; (2) H274Y conferred a high level of resistance to oseltamivir carboxylate; and (3) N294S conferred moderate level of resistance to oseltamivir carboxylate and increased virus virulence in ferrets. Strategies to limit the emergence of drug resistance are urgently needed. The highly pathogenic avian H5N1 influenza viruses remain of concern as a pandemic threat. Of the 486 cases of human H5N1 infection confirmed between March 2003 and March 2010, ∼60% were lethal Although vaccination remains the primary method of influenza control, effective antiviral drugs are the best option when vaccine availability or efficacy is limited in vitro oseltamivir carboxylate (the active methabolite of oseltamivir) susceptibility of A/Turkey/15/06 (H5N1) virus (clade 2.2) and A/Vietnam/1203/04 (H5N1) virus (clade 1) and different treatment efficacy in mice inoculated with these viruses  in vitro susceptibility of H5N1 influenza viruses to oseltamivir carboxylate in vivo. These findings demonstrate the need for systematic evaluation of the impact of natural NA gene variations on anti-NA drug susceptibility and on other biological properties of H5N1 influenza viruses.In the event of a pandemic, the effectiveness of therapeutic and prophylactic oseltamivir will depend not only on the correct dosage and duration of treatment but also on the susceptibility of the targeted virus strain. Our group in vitro and clinically tend to be NA subtype–specific: E119A/G/D/V, R292K, and N294S in the N2 and N9 subtypes and H274Y and N294S in the N1 subtype in vitro susceptibility of seasonal and H5N1 influenza viruses to NA inhibitors together with recent crystal structure data and conformational studies of influenza N1 enzyme identified several additional conserved or semiconserved NA residues  that may also confer resistance Oseltamivir and zanamivir were designed to bind only to highly conserved residues within the active site of influenza A and B virus NA protein Early studies suggested that seasonal influenza viruses resistant to NA inhibitors may be less infective and transmissible in ferrets than their wild-type counterparts In the homogeneous clade 2.2 A/Turkey/15/06-like (H5N1) genetic background, we studied the role of single point NA mutations near or within the enzyme active site on NA inhibitor susceptibility, NA enzyme kinetics, viability, genetic stability, and pathogenesis in ferrets. Seven substitutions were stable in the N1 NA protein and five reduced virus susceptibility to oseltamivir carboxylate or to both NA inhibitors. Infection of ferrets with the recombinant H5N1 viruses caused mild disease of various duration, although NA inhibitor-resistant variants with the E119A and N294S mutations were more virulent than the wild-type virus.We used the eight-plasmid reverse genetics technique to generate 11 recombinant A/Turkey/15/06-like (H5N1) viruses carrying different NA mutations , that weP<0.01) (The recombinant wild-type (WT) H5N1 influenza virus and seven mutants  were successfully rescued. Direct sequencing of their HA and NA genes revealed that all mutations were correctly incorporated and no additional changes were present in either gene (data not shown). All of the viruses grew to comparable titers and formed homogeneous plaques in MDCK cells , although viruses with the Y252H, H274Y, and N294S NA mutations formed significantly smaller plaques than did the WT virus (P<0.01) .in vitro, we serially passaged each virus three times in MDCK cells  as compared to that of WT virus. Virus carrying the E119A mutation was moderately more resistant to oseltamivir carboxylate  and markedly more resistant to zanamivir  than was WT virus  and slightly less susceptible to zanamivir  than WT virus. In contrast, the Y252H NA change conferred increased susceptibility to oseltamivir carboxylate   .m), which reflect NA affinity for the MUNANA substrate, their inhibition constants (Ki) for oseltamivir carboxylate and zanamivir, and their relative NA enzymatic activity (Vmax)  than did other NAs studied. The Ki values of all NA glycoproteins were consistent with their IC50 values  . NA prot0 values , suggestmax values for each of the recombinant H5N1 viruses (max ratios in relation to that of WT NA glycoprotein (P<0.01). The other five NA mutations studied significantly reduced the NA activity of A/Turkey/15/06 (H5N1) influenza virus   . Thus, t6 EID50 of each virus and observed four different patterns of clinical outcome (Limited information is available on the pathogenicity of H5N1 influenza viruses carrying various drug-resistant NA mutations in ferrets  outcome . RecombiP<0.01). Virus shedding started at 1 day p.i. for mutant viruses and continued until 6 days p.i. Interestingly, on day 1 p.i. virus titers in nasal washes were significantly lower for animals infected with recombinant V116A and K150N viruses than those in animals inoculated with WT virus (P<0.05), but remained significantly higher on day 6 p.i. (P<0.05)  was seen in ferrets infected with mutant V116A and K150N viruses . Despite(P<0.05) .P<0.05)  was observed in ferrets inoculated with H5N1 viruses carrying I117V and Y252H NA mutations: only slight temperature elevation , mild clinical features (RII≈0.05)  and minoP<0.05) .P<0.01) (Virus with the H274Y NA mutation exhibited virulence comparable to that of recombinant WT virus . In contP<0.01) . The rec6 EID50 of the WT, E119A, and N294S viruses (P<0.01)  . Thus, i10EID50/gram tissue) and liver (∼1.7–2.9 log10EID50/gram tissue) (P<0.01). Of the three H5N1 viruses, the N294S recombinant yielded significantly higher virus titers in the trachea   .We further histologically examined the tissues of inoculated ferrets to investigate the lesions associated with virus replication in infected organs and to obtain more information about the differences in virulence between recombinant WT, E119A, and N294S viruses. Histological changes were detected only in the lungs of infected animals. Bronchopneumonia and bronchiolitis with epithelial necrosis and/or regeneration characterized by bronchiole hypertrophy and hyperplasia were observed in all ferrets on day 4 p.i. . HoweverDifferences in the type and extent of the alveolar pathology were also apparent on histopathologic examination. Of the three H5N1 viruses, the E119A mutant caused very severe and extensive alveolitis with necrosis of the alveolar pneumocytes and interstitial septal walls resulting in the loss of the alveolar architecture and leading to large coalescing spaces of edema fluid, fibrin, inflammatory and hemorrhagic red blood cells . In the in vivo, we extracted RNA directly from nasal wash samples obtained on day 6 p.i. and sequenced the NA and HA genes of the dominant virus populations. No amino acid substitutions were identified in the NA gene of viruses isolated from ferrets inoculated with the WT, E119A, or N294S recombinants. A single F291L mutation in the HA gene was identified in 1 of 3 ferrets inoculated with either E119A or N294S mutants.Additionally, to assay the genetic stability of the recombinant viruses Taken together, our results indicated that the introduced E119A and N294S NA mutations were stably maintained in A/Turkey/15/06 (H5N1) virus background in ferrets. Further, increased virulence and severity of disease caused by H5N1 viruses carrying E119A and N294S NA changes were associated with higher virus titers and more pronounced local inflammatory response in the lungs compared to WT virus.Oseltamivir-resistant H1N1 influenza viruses emerged recently and became predominant during the 2007–2008 season in the absence of drug-selection pressure in vitro passages in the presence of zanamivir and has resulted in reduced NA activity Our most important finding is that the E119A NA substitution in the clade 2.2 A/Turkey/15/06 (H5N1)-virus background is viable and genetically stable and confers cross-resistance to oseltamivir carboxylate and zanamivir. Structural analysis showed that the glutamate at 119 is one of two conserved amino acid residues that interact with oseltamivir’s carboxylate group to allow a strong, specific bond between the enzyme active site and the inhibitor In the present study, V116A and I117V mutants showed a low (∼6-fold greater than WT) level of resistance to oseltamivir carboxylate. This finding is consistent with those of Hurt et al in vitro  in vivoWe also confirmed previous findings that the presence of histidine at position 252 in recent H5N1 isolates is associated with increased affinity of their NAs for oseltamivir carboxylate P<0.01). The molecular basis of this finding is unknown. However, the possibility that after exposure to one NA inhibitor the chance exists that occurring drug-resistant H5N1 mutant could be cross resistant to both antiviral drugs and will be more virulent that the WT virus (like E119A H5N1 recombinant virus) should prompt to re-assess the suitability of single-drug usage. None of the NA mutations studied resulted in decreased viral virulence in the ferret animal model, consistent with our group’s previous finding that NA inhibitor-resistant mutations did not impair the virulence of clade 1 A/Vietnam/1203/04 (H5N1) virus in mice max and Km) on virulence in ferrets. None of the recombinant mutants exhibited an increased affinity for the substrate and a higher activity compared to WT NA, which could possibly indicate their overall better fitness. However, recombinant H5N1 viruses carrying I117V and Y252H NA mutations possessed the highest relative NA activity, but ferrets infected with these viruses experienced a mild, brief illness. Additional studies are needed to identify the impact of NA activity and affinity on the duration of influenza disease in vivo.We studied the pathogenicity and virulence of the NA-mutant H5N1 influenza viruses in ferrets, an acceptable animal model to evaluate influenza virus disease manifestations Our data demonstrate the significance of continued characterization of all H5N1 isolates for susceptibility to NA inhibitors in order to identify novel NA markers of altered susceptibility. We believe that this knowledge is essential for planning appropriate management strategies for a future pandemic. It is noteworthy that alignment of the NA genes of currently circulating highly pathogenic H5N1 influenza A viruses identified the NA mutations studied here in ∼0.1%–1.4% of isolates , raising concern about the drug sensitivity of a possible pandemic strain. Future studies should focus on the establishment of novel antiviral strategies to minimize the emergence of resistance.All animal experiments with recombinant H5N1 influenza viruses were performed in biosafety level 3+ facilities at St. Jude Children’s Research Hospital . All animal studies were approved by the St. Jude Children’s Research Hospital Animal Care and Use Committee and were conducted according to applicable laws and guidelines.Madin-Darby canine kidney (MDCK) and human embryonic kidney (293T) cells were obtained from the American Type Culture Collection and maintained as previously described Eight plasmids were constructed from the DNA sequences of the 8 gene segments of wild-type A/Turkey/15/06 (H5N1) virus for the reverse-genetics generation of recombinant wild-type virus. Recombinant virus was generated by DNA transfection of 293T cells 3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexene-1-carboxylic acid) and zanamivir (4-guanidino-Neu5Ac2en) were provided by Hoffmann-La Roche, Ltd.The NA inhibitors oseltamivir carboxylate (oseltamivir) -α-D-N-acetylneuraminic acid . All H5N1 viruses were standardized to an equivalent dose of 107.5 PFU/ml. The reaction was conducted at 37°C in a total volume of 50 µl, and the fluorescence of released 4-methylumbelliferone was measured every 92 sec for 45 min in a Fluoroskan II instrument (Labsystems) using excitation and emission wavelengths of 355 and 460 nm, respectively. To measure the inhibitory effect of oseltamivir carboxylate or zanamivir on NA activity, H5N1 viruses were preincubated for 30 min at 37°C in the presence of various concentrations of the drugs (0.00005–5 µM). The kinetic parameters Michaelis-Menten constant (Km), maximum velocity of substrate conversion (Vmax), and inhibitory constant (Ki) of the NAs were calculated by fitting the data to the appropriate Michaelis-Menten equations by using nonlinear regression in the commercially available GraphPad Prism 4 software .Modified fluorometric assay was used to determine the NA activity of the recombinant H5N1 viruses 50) was determined by plotting the dose-response curve of inhibition of NA activity as a function of the compound concentration. Values are the mean of 2–3 independent determinations.Recombinant H5N1 viruses were standardized to equivalent NA activity and incubated for 30 min at 37°C with NA inhibitors at concentrations of 0.00005–100 µM with MUNANA (Sigma-Aldrich) as a substrate. After 1 h, the reaction was terminated by adding 14 mM NaOH and fluorescence was quantified in a Fluoroskan II (Labsystems) fluorometer. The concentration of NA inhibitor that reduced NA activity by 50% relative to a control mixture with no inhibitor . Clinical signs of infection, relative inactivity index Pathogenicity was tested in 4- to 5-month-old male ferrets obtained through the ferret breeding program at St. Jude Children’s Research Hospital or from Marshall Farms . All ferrets were seronegative for influenza A H1N1, H3N2, and H5N1, and for influenza B viruses. Groups of three ferrets were inoculated intranasally under light isoflurane anesthesia with 1010EID50/ml. Inflammatory cell counts and protein concentrations in nasal washes were determined as described previously To monitor virus shedding, nasal washes were collected from ferrets on days 1, 2, 4, 6, and 8 post-challenge. Virus was titrated in 10-day old embryonated chicken eggs by injecting 0.1 ml of serial 10-fold dilutions of the sample (three eggs per dilution) and expressed as log10EID50/gram tissue) was determined in 10-day old embryonated chicken eggs.Two animals inoculated with WT, E119A, or N294S viruses were euthanized by intracardiac injection of Euthanasia V solution on day 4 post-inoculation, and tissue samples (∼0.5 g each) were collected from lungs (4 lobes tested separately), nasal turbinate, trachea, spleen, liver, and small intestine. Samples were homogenized in 1 ml sterile PBS with antibiotics, and the virus titer  were collected at the time of necropsy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin and studied by light microscopy.Serum samples were collected from ferrets 3 weeks after inoculation, treated with receptor-destroying enzyme, heat-inactivated at 56 °C for 30 min, and tested by hemagglutination inhibition (HI) assay with 0.5% packed chicken red blood cells by a standard method as described previously Viral RNAs were isolated from virus-containing cell culture fluid after transfection, after three passages in MDCK cells or from ferret nasal washes by using the RNeasy Mini kit (Qiagen). Samples were reverse-transcribed and analyzed by PCR using universal primers specific for the HA and NA gene segments, as described previously m, Vmax, Ki), virus titers in ferret organs and nasal wash samples, differences in fevers, weights, total number of cells and protein concentrations in nasal wash samples, mean survival time, and relative inactivity index of ferrets inoculated with wild-type and mutant viruses were compared by analysis of variance (ANOVA). Virus yields and plaque size of recombinant H5N1 viruses after the first and the third passage in MDCK cells were compared by unpaired two-tailed t-test. The probability of survival was estimated by the Kaplan-Meier method and compared between groups of ferrets by using the log-rank (Mantel-Haenszel) test The virus yield, plaque size, NA inhibitor susceptibility, NA enzyme kinetic parameters  ferrets. The mean s.d. of all data points for change in body temperature and weight was ∼±4.5%. *, P < 0.05, **, P < 0.01 compared to WT virus (one-way ANOVA).(0.48 MB TIF)Click here for additional data file.


The cellular cytidine deaminase APOBEC3G (A3G), when incorporated into the human immunodeficiency virus type 1 (HIV-1), renders viral particles non-infectious. We previously observed that mutation of a single cysteine residue of A3G (C100S) inhibited A3G packaging. In addition, several recent studies showed that mutation of tryptophan 127 (W127) and tyrosine 124 (Y124) inhibited A3G encapsidation suggesting that the N-terminal CDA constitutes a viral packaging signal in A3G. It was also reported that W127 and Y124 affect A3G oligomerization.Here we studied the mechanistic basis of the packaging defect of A3G W127A and Y124A mutants. Interestingly, cell fractionation studies revealed a strong correlation between encapsidation, lipid raft association, and genomic RNA binding of A3G. Surprisingly, the presence of a C-terminal epitope tag affected lipid raft association and encapsidation of the A3G W127A mutant but had no effect on wt A3G encapsidation, lipid raft association, and interaction with viral genomic RNA. Mutation of Y124 abolished A3G encapsidation irrespective of the presence or absence of an epitope tag. Contrasting a recent report, our co-immunoprecipitation studies failed to reveal a correlation between A3G oligomerization and A3G encapsidation. In fact, our W127A and Y124A mutants both retained the ability to oligomerize.Our results confirm that W127 and Y124 residues in A3G are important for encapsidation into HIV-1 virions and our data establish a novel correlation between genomic RNA binding, lipid raft association, and viral packaging of A3G. In contrast, we were unable to confirm a role of W127 and Y124 in A3G oligomerization and we thus failed to confirm a correlation between A3G oligomerization and virus encapsidation. InhibiIn vitro studies using purified recombinant NC and A3G found that the two proteins do not competitively bind RNA but instead form an RNA-protein ternary complex [The antiviral effect of A3G generally requires encapsidation of the deaminase into viral particles. Interestingly, the antiviral effects of A3G are not limited to HIV-1 but extend to other retroviruses including murine leukemia virus, mouse mammary tumor virus, simian immunodeficiency virus, equine infectious anemia virus, and hepatitis B virus (for review see ). Packag complex .Several reports have investigated domains in A3G required for packaging into HIV-1 virions. We and others have recently reported that mutations in the A3G catalytic domain 1 (CD1) can impair A3G packaging ,22. CharHere we further characterized the role of W127 and Y124 for the packaging of A3G into HIV-1 virions and for A3G oligomerization. Consistent with previous reports we found that packaging of A3G-HA was severely affected by the W127A mutation. Similarly, packaging of Myc epitope-tagged A3G-Myc W127A was severely restricted suggesting that the packaging defect imposed by the W127A mutation is not epitope tag specific. Of note, the effect of the W127A mutation on virus encapsidation was much less severe in the context of untagged A3G. This is surprising and implies that the effects of mutations around position W127 are sensitive to and exacerbated by changes at the C-terminus of the protein. In contrast, mutation of Y124A imposed a severe packaging defect irrespective of the presence or absence of an epitope tag. A3G-HA was previously found to associate with cellular raft structures . Interesvif-defective molecular clone pNL4-3Vif(-)[The 4-3Vif(-) was used4-3Vif(-). For the4-3Vif(-). Mutatio4-3Vif(-).2 flasks to about 80% confluence. Cells were transfected using LipofectAMINE PLUS  following the manufacturer's recommendations. A total of 5 to 6 μg of plasmid DNA per 25 cm2 flasks (~5 × 106 cells) was used. Total amounts of transfected DNA were kept constant in all samples of any given experiment by adding empty vector DNA (pUC18 or pcDNA3.1(-)MycHis) as appropriate. Unless stated otherwise, cells were harvested 24 h post-transfection.HeLa cells were propagated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). For transfection, HeLa cells were grown in 25 cmA3G was identified using a polyclonal rabbit serum against a synthetic peptide comprising the 17 C-terminal residues of A3G . Serum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. Tubulin was identified using a monoclonal antibody to α-tubulin . For immunoprecipitation of tagged and untagged A3G, polyclonal ApoC17 antibody was used. Raft associated marker protein caveolin was identified by polyclonal anti-caveolin antibody . Transferrin receptor (TfR) was included as a non raft marker protein and was identified using a TfR-specific monoclonal antibody .Virus stocks were prepared by transfection of HeLa cells with appropriate plasmid DNAs of pNL4-3Vif(-) in the presence of tagged and untagged variants of wild type and mutant  A3G as indicated in the text. Virus-containing supernatants were harvested 24 h after transfection. Cellular debris was removed by centrifugation  and clarified supernatants were filtered (0.45 μm) to remove residual cellular contaminants. For determination of viral infectivity, unconcentrated filtered supernatants were used for the infection of LuSIV indicator cells. For immunoblot analysis of viral protein, virus-containing supernatants (7 ml) were concentrated by ultracentrifugation through 4 ml of 20% sucrose in phosphate-buffered saline (PBS) as described previously .5) in a 24-well plate in a total volume of 1.2 to 1.4 ml. LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 long terminal repeat [To determine viral infectivity, virus stocks were normalized for equal levels of reverse transcriptase activity and used to infect LuSIV cells (5 × 10l repeat . These c7 cells), and mixed with an equal volume of sample buffer . Proteins were solubilized by boiling for 10 to 15 min at 95°C, with occasional vortexing of the samples to shear cellular DNA. Residual insoluble material was removed by centrifugation . For immunoblot analysis of virus-associated proteins, concentrated viral pellets were suspended in a 1:1 mixture of PBS and sample buffer and boiled. Cell lysates and viral extracts were subjected to SDS-polyacrylamide gel electrophoresis; proteins were transferred to polyvinylidene diflouride membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  and visualized by enhanced chemiluminescence .For immunoblot analysis of intracellular proteins, whole-cell lysates were prepared as follows. Cells were washed once with PBS, suspended in PBS . Cell extracts were clarified at 13,000 × g for 3 min, and the supernatant was incubated on a rotating wheel for 1 h at 4°C with protein A-Sepharose coupled with anti-ApoC17 antibody. Immune complexes were washed three times with 50 mM Tris, 300 mM NaCl, and 0.1% Triton X-100, pH 7.4. Bound proteins were eluted from beads by heating in sample buffer for 5 min at 96°C and analyzed by immunoblotting.2PO4, Na2HPO4, pH 7.2) as described [HeLa cells were transfected with 2.5 μg each of vectors expressing untagged or C-terminally Myc tagged A3G proteins in various combinations. Cells were lysed in 600 ml lysis buffer  was co-transfected into HeLa cells with A3G vectors (4 μg) as indicated in the text. RNA was then extracted from the immunocomplexes as above.Total cellular RNA was extracted from untransfected and transfected HeLa cells using the RNeasy RNA extraction kit  following the manufacturer's instructions. To isolate RNA form immune complexes, beads were washed three times with RNA-protein binding buffer . RNA was then extracted using the RNeasy RNA extraction kit. For isolation of genomic RNA precipitated with the A3G complex, qRT-PCR was performed using the one tube SYBR green method as per manufacturer instruction . Briefly, each 16 μl reaction mixture contained 0.08 μl of Reverse Transcriptase, 0.04 μl of RNase inhibitor, 300 μM of forward and 50 μM of reverse specific primers, 8 μl of 2× SYBR green PCR Master Mix, 2.6 μl of RNase-free water, and 5 μl of template RNA. RNaseA treated RNA from A3G wt samples were used as a negative control. The reactions were performed on a AB Biosystems 7300 Real Time PCR System (AB Biosystems) using the following conditions: 48°C for 30 min followed by 95°C for 10 min and then 40 cycles of 95°C for 15 s and 60°C for 1 min with a dissociation protocol. The target sequences amplified by the SYBR green method used the following primer pairs: 7SL RNA, forward (5'-CCCGGGAGGTCACCATATT-3'), reverse (5'-CTGTAGTCCCAGCTACTCG-3'); HIV-1 genomic RNA, forward (5'TCAGCATTATCAGAAGGAGCCACC-3'), reverse (5'-TCATCCATCCTATTTGTTCCTGAAG-3').vif-defective HIV-1 virions and exhibit strong antiviral activity. In contrast, HA-tagged A3G W127A and W127L mutants were reported to be packaging incompetent [vif-deficient NL4-3. Cells and virus containing supernatants from transfected cultures were prepared for immunoblot analysis as described in Methods and probed with antibodies to A3G and an HIV-1-positive patient serum  are efficiently incorporated into ompetent ,23,26,32rum Fig. . A3G andrum Fig. . Resultsrum Fig. , lane 1,vif-defective proviral DNA (pNL4-3Vif(-)) and total amounts of transfected DNA were kept constant. Surprisingly, epitope-tagged A3G W127A variants were much less efficiently packaged into virions than their untagged counterparts (Fig. To address potential effects of epitope tags on packaging of A3G, we performed a side-by-side comparison of untagged and epitope tagged A3G wt, A3G W127A, as well as A3G Y124A. For the Y124A mutant the presence or absence of a C-terminal Myc tag was investigated; A3G W127A constructs encoding either a C-terminal Myc tag or an HA tag were analyzed. Amounts of transfected A3G vectors were adjusted as described in the legend to figure Consistent with their poor packaging efficiency, tagged and untagged Y124A mutants as well as epitope tagged A3G W127A mutants exhibited only modest antiviral activity in the context of Vif-defective viruses (Fig. A3G was reported to associate with lipid rafts, presumably on intracellular membranes . Since lPrevious reports suggested that A3G packaging into HIV-1 virions requires interaction with RNA ,32,38,39We previously reported that mutation of C97 in the N-terminal enzymatically inactive deaminase domain of A3G affected oligomerization of the protein but did not abolish packaging or antiviral activity . In contSince our own packaging studies revealed an impact of C-terminal epitope tags for packaging of A3G W127A, we decided to analyze the correlation between A3G dimerization and packaging competence. To that end we cotransfected A3G wt, A3G W127A, or A3G Y124A mutants in various combinations using untagged or Myc-tagged wild type or mutant A3G constructs. Proteins were immunoprecipitated by a Myc-specific monoclonal antibody followed by immunoblotting with a polyclonal A3G-specific antibody Fig. , lane 2.The mechanism of A3G encapsidation into HIV-1 virions has attracted significant attention since it offers a potential target for therapeutic interference with virus replication. There is increasing evidence that encapsidation of A3G into virions requires interactions with the viral nucleocapsid domain in the Gag precursor and involves RNA although the nature of the RNA involved in A3G packaging, i.e. cellular versus viral, remains under investigation ,38,41-43Our current study focused on two amino acid residue, W127 and Y124, in A3G to demonstrate that the requirements for A3G packaging are complex and can be influenced by the presence or absence of terminal epitope tags. Our finding that untagged A3G W127A was packaged 10 times more efficient than Myc- or HA-tagged A3G W127A was surprising and unexpected since the interfering epitope tags were not located adjacent to residue W127 but were more than 250 amino acids away at the C-terminus of the protein. There is currently no structure of full-length A3G available. However, computer modelling suggests that W127 is located at the surface of the protein . It is tOur data reveal an interesting correlation between A3G's propensity to associate with lipid raft structures, viral RNA interaction, and packaging competence. The reason for the inability of the epitope tagged A3G W127A mutants as well as tagged and untagged Y124A mutants to associate with lipid rafts and with viral RNA remains to be investigated. Since A3G is not a membrane protein it is likely that its affinity to raft structures is due to an interaction with other raft associated host factors. Our floatation studies were done in the absence of viral proteins, ruling out the possible contribution of Gag protein in the raft targeting of A3G. Thus, the lack of raft association could be indicative of altered intracellular localization or trafficking of A3G or could be due to a conformational change resulting in the loss of protein-protein interaction.The authors declare that they have no competing interests.MAK conceived the study, performed the molecular and biochemical studies, and drafted the manuscript. RG, EM, and RCW assisted with biochemical studies and helped with data analysis. SK assisted with A3G mutagenesis and biochemical analyses. KS coordinated and supervised the project and wrote the final manuscript.


MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the  MET oncogene, not only in primary bladder tumors but also in premalignant urothelium across entire bladders with tumors. Our study has important implications for tumor biology, cancer detection, and treatment, and it also answers the long-standing question of whether hypomethylation of retrotransposons induces ectopic gene expression and influences disease susceptibility in humans, a phenomenon first described in agouti mice.A surprisingly large portion of our transcriptome originates within repetitive elements, most commonly LINE-1s. However, the mechanism of activation has not been definitively shown. We directly demonstrate for the first time the causal relationship between DNA hypomethylation and transcriptional activation of LINE-1 promoters. Hypomethylation of specific LINE-1 promoters can alter the transcriptome, including activating an alternate transcript of the  Ectopic induction of the agouti gene results in altered coat color, obesity, and an increased incidence of tumors Aberrant DNA methylation is involved in the initiation and progression of carcinogenesis and includes both hypermethylation of CpG islands at gene promoters and global hypomethylation. While a small portion of hypomethylation occurs at gene promoters, resulting in overexpression of certain oncogenes ArabidopsisA recent study has revealed that more than 30% of transcription start sites in the human genome are located within repetitive elements, with just over 7% in L1s MET oncogene is active across entire bladders with cancer. The clinical implication of our finding is that surgical excision of the tumor would leave behind large areas of the bladder that remain epigenetically altered and express a potential oncogene. We also provide evidence that an active L1 acquires H2A.Z and nucleosome free regions upstream of TSSs, which has only been described previously at single copy genes, and undergoes chromatin remodeling from an inactive tetranucleosomal structure to an active dinucleosomal structure.Until recently it has not been possible to study the promoters of individual L1s since the sequences are too similar to design primers for one particular locus MET oncogene (L1-MET) MET transcripts by sequencing EST clones obtained from a bladder carcinoma cell line (GenBank accession no. BF208095) and placenta (BX334980). Both transcripts have start sites located in the L1 promoter, share the same reading frame as MET  (−5) but not as dramatic as L1-MET hypomethylation and that the methylation pattern can be quite different between global L1s and a specific L1, such as in the cell lines LD137, T24, and RT4 (To examine the methylation status at a specific L1 we designed bisulfite-specific PCR primers with one located in the L1 promoter and the other in the surrounding intronic region of the host gene . The L1-4×10−10) . We also4×10−10) . We foun and RT4 . This reMET anti-sense promoter contains its own exons 1 and 2, referred to as L1-MET exon 1 and L1-MET exon 2  and two non-tumorigenic urothelial cell lines, UROtsa MET was also not expressed in normal tissues except for placenta (data not shown). Therefore L1-MET hypomethylation correlated with the expression of the alternate transcript , allowing the promoter to be methylated without affecting the plasmid backbone. We created two plasmids, differing only the orientation of the L1-MET promoter, allowing us to measure either the L1 transcriptional activity or the L1-MET activity transcriptional activity  has previously been used to obtain single molecule resolution of nucleosome positioning at unmethylated CpG island promoters MET promoter in the UROtsa immortalized urothelial cell line was completely occupied by nucleosomes, revealing that the methylated L1-MET promoter exists in a tetranucleosomal structure .The endogenously methylated L1-tructure . GpCpG sf either . We werethylated . HoweverMLH1 bidirectional promoter has demonstrated that while each transcription start site loses the nucleosome directly upstream when active (−1 nucleosome), the nucleosome directly downstream is always maintained (+1 nucleosome) MET and global L1s (MET (data not shown) and nucleosome eviction at the L1-MET promoter   curves for L1-MET revealed an extraordinary degree of both sensitivity and specificity for detecting bladder tumors (AUC of 0.97) and premalignant tissue (AUCs of 0.89)  . Hypomet1-RAB3IP  [35]. Thof 0.89) . Since apatients , a noninMET was not correlated with hypomethylation of the L1-MET promoter, since the expression of MET is regulated by its endogenous promoter and not by the specific L1 promoter  mice revealed that hypomethylation of a retrotransposon could induce ectopic expression of a gene and influence disease susceptibility et al. The consequences of global hypomethylation at repetitive elements in cancer has long been the subject of speculation regarding the generation of genomic instability and potential activation of oncogenes. Ever since studies on viable yellow agouti  according to the manufacturer's instruction. See The non-tumorigenic human urothelial cell lines UROtsa and NK2426 and the normal fibroblast cell line LD419 have been described previously Tumor tissue samples were collected from the patients undergoing cystectomy or TURBT for bladder cancer. Normal bladder epithelium was obtained from 12 patients undergoing radical prostatectomy for prostate cancer (aged from 50 to 80) and 7 autopsy patients aged from 34 to 82, 5 of which were from non-cancer related deaths and 2 from deaths due to cancers other than bladder). All of these collections took place at Norris Cancer Hospital in IRB-approved protocols with patients' consent. Hematoxylin and eosin (H&E) sections marked with the location of the adjacent urothelium or tumor were used to guide in microdissection. DNA was bisulfite treated as previously described Methylation-sensitive single nucleotide primer extention (MS-SNuPE) was performed as previously described Expression was determined by quantitative RT–PCR as described previously MET and L1 promoters were cloned into the pCpGL luciferase vector MET promoter cloned was 555 bp, with 535 bp within the L1 and 20 bp within the MET gene (ch7:116364010–564). These experiments were performed as described previously The L1-ChIP was performed as described previously SssI. DNA was isolated, bisulfite converted, and PCR fragments were cloned for sequencing of individual molecules. In order to examine endogenously methylated promoters and increase the resolution of this method, chromatin from 250,000 cells was treated with the enzyme M. CviPI, which methylates GpC sites M-SPA was performed as described previously MNase digestion and sucrose density gradient centrifugation were performed as described previously Significant differences in methylation and expression levels in normal, corresponding normal, and tumor tissues were determined using a Mann-Whitney test.Figure S1Specific L1s with alternate transcripts located in intron of genes. Black boxes represent exons of the host gene while red boxes represent a specific L1. The black arrow represents the transcriptional start site of the host gene while the red arrow represents the alternate transcriptional start site within the potentially active L1 promoter. GenBank accession numbers for representative alternate transcripts are followed by the number in parentheses of similar transcripts transcribed from the individual L1. All L1s are antisense to their host genes, yielding alternate transcripts that are sense with their host genes.(0.56 MB TIF)Click here for additional data file.Figure S2MET. (A) The functional domains of MET include the signal peptide (SP), sema domain at the N-terminus, the PSI domain, IPT repeats, the transmembrane domain (TM), and the kinase domain at the C-terminus. The structure of truncated MET proteins 1 and 2 are shown, encoded by transcripts derived from placenta (GenBank accession no. BX334980) and a bladder carcinoma cell line (BF208095), respectively. (B) The two L1-MET transcripts, truncated L1-MET-1 (T-MET-1) and truncated L1-MET-2 (T-MET-2), were cloned into a pMEV expression vector with 2 HA tags fused at the N-terminal. Hela cells were transfected with either the empty pMEV vector, pMEV T-MET-1, or pMEV T-MET-2 and protein was extracted after 48 hours. The expression of truncated MET-1 (90 kDa) and truncated MET-2 (60 kDa) was detected by western blot using an HA antibody. (C) Results of 5′RACE reveal the start site for L1-MET within the L1 element. The transcriptional start site of L1-MET was confirmed by 5′RACE in the T24 cell line which expressed L1-MET. The underlined sequence is located inside of the LINE-1. (D) RT–PCR analysis of reactivation of L1-MET by 1 or 3 µM of 5-Aza-CdR treatment for 24 hours (day 3 after treatment). β-actin expression level was used as a control.The truncated MET protein encoded by L1-(1.22 MB TIF)Click here for additional data file.Figure S3ACVR1C correlates in cell lines. (A) Map of alternate transcripts from L1-ACVR1C. Exons are represented by black boxes while the specific L1s are represented by red boxes. The lower tick marks represent each CpG site. The left bent arrow indicates transcriptional start sites and ATGs indicate translational start sites. Green arrows indicate the primers used to amplify the pyrosequencing product and the black arrow in between indicates the location of the pyrosequencing primer for L1-ACVR1C. (B) L1-ACVR1C methylation (red bars) and L1 methylation (black bars) was analyzed by pyrosequencing in 6 normal tissues, one normal bladder fibroblast cell line (LD419), one non-tumorigenic urothelial cell lines (UROtsa), and 10 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1-ACVR1C from two technical duplicates. (C) Expression of L1- ACVR1C was measured using real-time RT PCR in one normal bladder fibroblast cell line, one normal urothelial cell line, and 10 bladder carcinoma cell lines. Values are also the average from two technical duplicates. Red bars indicate the methylation status of L1-ACVR1C, which is also represented in (B), and green bars represent the level of expression relative to GAPDH.Methylation and expression of L1-(0.86 MB TIF)Click here for additional data file.Figure S4RAB3IP correlates in cell lines. (A) Map of alternate transcripts from L1-RAB3IP. Exons are represented by black boxes while the specific L1s are represented by red boxes. The lower tick marks represent each CpG site. The left bent arrow indicates transcriptional start sites and ATGs indicate translational start sites. Green arrows indicate the primers used to amplify the pyrosequencing product and the black arrow in between indicates the location of the pyrosequencing primer for L1-RAB3IP. (B) L1-RAB3IP methylation (red bars) and L1 methylation (black bars) was analyzed by pyrosequencing in 6 normal tissues, one normal bladder fibroblast cell line (LD419), one non-tumorigenic urothelial cell lines (UROtsa), and 10 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1-RAB3IP from two technical duplicates. (C) Expression of L1-RAB3IP was measured using real-time RT–PCR in one normal bladder fibroblast cell line, one normal urothelial cell line, and 10 bladder carcinoma cell lines. Values are also the average from two technical duplicates. Red bars indicate the methylation status of L1-RAB3IP, which is also represented in (B), and green bars represent the level of expression relative to GAPDH.Methylation and expression of L1-(0.88 MB TIF)Click here for additional data file.Figure S5Chromatin remodeling occurs at active L1 promoters. (A) DNA methylation at specific and global L1s (with p16 as a control) was determined by pyrosequencing in the immortalized urothelial cell line UROtsa and bladder carcinoma cell line T24. The specific L1s had less methylation in the cancer cell line. Chromatin immunoprecipitation was performed using antibodies for (B) H3K4me3; (C) acetylated H3; and (D) H2A.Z. The values of the ChIP assay are the average of three experiments with technical duplicates. Error bars represent the standard deviation. The presence of active histone marks was associated with absence of DNA methylation at the specific L1s in the cancer cell line.(0.67 MB TIF)Click here for additional data file.Figure S6MET promoter. Nucleosome positioning in an active fully unmethylated L1-MET promoter in T24 bladder carcinoma cells reveals a dinucleosomal structure, as determined by both M. SssI, a CpG methyltransferase and M. CviPI accessibility.Chromatin remodeling occurs an active L1-(1.29 MB TIF)Click here for additional data file.Figure S7ACVR1C was analyzed by pyrosequencing in normal tissues (N), corresponding normal tissues (CN), and bladder tumors (T). Values are an average of two CpG sites. (B) Expression of the alternate transcript from L1-ACVR1C and (C) the host gene ACVR1C, and the control gene GAPDH was measured by real-time RT–PCR. *** represents p<0.001, ** represents p<0.01, and * represents p<0.05. (D) Methylation status of L1-RAB3IP was analyzed by pyrosequencing in normal tissues (N), corresponding normal tissues (CN), and bladder tumors (T). Values are an average of two CpG sites. (E) Expression of the alternate transcript from L1-RAB3IP and F. the host gene RAB3IP, and the control gene GAPDH was measured by real-time RT–PCR. *** represents p<0.001, ** represents p<0.01, and * represents p<0.05 as determined by the Mann-Whitney test. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA. the results show a consistent trend.Methylation and expression status of specific L1s correlates in bladder tissues. Horizontal lines represent the mean. (A) Methylation status of L1-(0.58 MB TIF)Click here for additional data file.Figure S8MET methylation distinguish between normal bladder tissue (N) and corresponding normal bladder tissues (CN), N and bladder tumors (T), and CN and T. (B) ROC curves using L1-ACVR1C methylation, and (C) ROC curves using L1-RAB3IP methylation. *** represents p<0.001 and * represents p<0.05.ROC curves for specific L1s. (A) ROC curves using L1-(0.65 MB TIF)Click here for additional data file.Figure S9MET. Bladder tissues (N) from age-matched patients without bladder cancer (n = 10) and urine (N) from age-matched healthy volunteers (n = 10) showed low levels of L1-MET hypomethylation. However, urine (n = 20) from patients with TCC showed high levels of L1-MET hypomethylation, which was specific to the bladder since it was not detected in their white blood cells (WBC) (n = 20). Unmethylated levels (Y axis) indicate the Percent of fully Unmethylated Reference (PUR) values.Detection of L1-MET hypomethylation in urine sediments of patients with bladder cancer. Bisulfite-specific primers and a probe were designed for the MethyLight assay that amplified only completely unmethylated strands of L1-(0.30 MB TIF)Click here for additional data file.Figure S10ACVR1C and (C) L1-RAB3IP was measured by pyrosequencing. The green line represents the mean methylation of 12 normal samples from cancer-free patients. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA. the results show a consistent trend.Methylation of specific L1s across the bladder. (A) Tissue samples were taken from five patients of their tumors  and at increasing distances from the tumor (0.5 to 2 cm) in the surrounding normal-appearing tissue in multiple directions . Additionally, distant normal-appearing samples were taken at least 5 cm from the tumor . (B) Methylation at L1-(1.62 MB TIF)Click here for additional data file.Figure S11MET. Biphasic distribution of L1-MET methylation status in corresponding tissue from a patient with bladder cancer is revealed by plotting the number of DNA strands by the percent of CpG sites methylated.Bisulfite sequencing of L1-(0.18 MB TIF)Click here for additional data file.Figure S12Ms-SNuPE and pyrosequencing yield similar methylation results. While both Ms-SNuPE and Pyrosequencing are quantitative assays, Pyrosequencing is much more high throughput. Therefore, we developed a Pyrosequencing assay for the rest studies. (A) We measured 4 CpG sites by Pyrosequencing assay in contrast with the 2 CpG sites by Ms-SNuPE. (B) We randomly chose 66 samples previously analyzed by Ms-SNuPE to perform Pyrosequencing on and the results are very similar from both assays (R = 0.91).(0.42 MB TIF)Click here for additional data file.Table S1Primer sequences.(0.08 MB DOC)Click here for additional data file.


Two hundred and twenty patients with progressive advanced breast cancer were given primary endocrine treatment (PET) according to menstrual status. Pre-menopausal patients received ovarian irradiation (O) and post-menopausal tamoxifen 10 mg bd (T). Patients were randomised to receive either no additional treatment or prednisolone 5 mg bd (P). Similar results were observed in each menstrual subgroup. In 194 evaluable patients, the response to PET + P was 49% and to PET alone 30% (P less than 0.01). P increased the median duration of response from 9 to 14 months (P less than 0.002) and the median time to disease progression from 5 to 9 months (P less than 0.001). Response to P after O or T alone occurred in only 2/62 (3%). Median survival in patients randomised to receive P at the outset of PET was prolonged by 4 months (P less than 0.05). The addition of P significantly improves the response to O or T in the treatment of advanced breast cancer.


This study was performed to characterize a gene-addition transgenic mouse containing a BAC  clone spanning the human CYP2C18&19 genes (tg-CYP2C18&19).Hemizygous tg-CYP2C18&19, 11 week old mice were compared with wild-type littermates to obtain information regarding clinical status, clinical pathology and anatomical pathology. After one week of clinical observations, blood samples were collected, organs weighed, and tissues collected for histopathology.p ≤ 0.05), adrenal glands (p ≤ 0.05) and brown fat deposits (p ≤ 0.001) while the heart weight was higher (p ≤ 0.001). In female tg-CYP2C18&19, the tissue weights were lower for brain (p ≤ 0.001) and spleen (p ≤ 0.001) compared to wild-type females. Male tg-CYP2C18&19 had increased blood glucose levels (p ≤ 0.01) while females had decreased blood triglyceride levels (p ≤ 0.01).In males, the tissue weights were lower in tg-CYP2C18&19 than in wild-type mice for brain (Despite the observed alterations, tg-CYP2C18&19 did not show any macroscopic or microscopic pathology at the examined age. Hence, these hemizygous transgenic mice were considered to be viable and healthy animals. CYP2C19 stereo-selectively metabolises the S-enantiomer of the anticonvulsant mephenytoin to the metabolite 4-hydroxy- (S)-mephenytoin [in vitro. The 5-hydroxylation of R-omeprazole is selectively performed by CYP2C19 [The human cytochrome P450 enzymes from the 2C subfamily (CYP2C) are fairly well characterized and are known to metabolise many clinically important drugs. Four members belonging to the CYP2C family are found in man, namely CYP2C8, CYP2C9, CYP2C18 and CYP2C19 , the ant CYP2C19 , certain CYP2C19 , the β-b CYP2C19 , the anx CYP2C19  and the  CYP2C19 .). Cyp2c29 was the first mouse Cyp2c member identified [In contrast to the relatively small human CYP2C family, the mouse Cyp2c family is one of the largest and most complex, with 15 members published to date [entified , Cyp2c44entified , Cyp2c50entified . Six addentified -19.Murine CYP2C enzymes are involved in the metabolism of arachidonic acid, but the products formed differ between the isoforms. Human CYP2C19, on the other hand, is inhibited by the presence of arachidonic acid .CYP2C18 and CYP2C19 genes are expressed at the mRNA and protein levels, and the inserted CYP2C19 genes have been shown to be functional in vitro in metabolism studies using the CYP2C19 substrates S-mephenytoin and R-omeprazole)[The transgenic mouse presented in this paper contains, in addition to all mouse Cyp2c enzymes, human CYP2C18 and CYP2C19. The inserted human eprazole).The aim of the present study was to characterize the humanized CYP2C18&19 mouse model as a basis for upcoming pharmacokinetic and toxicological studies. Hemizygous humanized CYP2C18&19 mice (tg-CYP2C18&19) were compared with wild-type littermates to obtain information regarding clinical status, body weight, clinical pathology, anatomy and morphology of this particular mouse model.CYP2C18 and CYP2C19 genes was purified. BAC DNA was injected into C57BL/6JOlaHsd (C57BL/6) eggs. Founders were identified by genotyping of DNA extracted from tail or ear biopsies.The transgenic CYP2C18&19 mice characterized in this article were generated as previously described). In brie® Tissues, Qiagen). The gDNA was amplified in a 20 μL reaction mixture containing 10 μL HiFi PCR MasterMix , primers  and 1 μL of gDNA. The four different specific primer pairs used are listed in Table For PCR detection of the inserted gene segment, gDNA was extracted from tail or ear biopsies either by using established protocols  or commeThe hemizygous tg-CYP2C18&19 and the C57BL/6JOlaHsd (C57BL/6) wild-type littermates used were generated by crossing hemizygous tg-CYP2C18&19 males with C57BL/6JOlaHsd (C57BL/6) wild-type female mice. Wild-type littermates were used as controls. Attempts were also performed to generate homozygous mice by crossing hemizygous tg-CYP2C18&19 males and females, but the offspring died within a few days from birth.For logistic reasons, the male and female groups were housed at different sites, but all mice were kept under conventional conditions and had free access to standard rodent diet  and tap water. The animal husbandry and experimental conditions were approved by the Swedish Animal Welfare Agency.All mice were observed for one week prior to necropsy at an age of approximately 11 weeks. During that week, any adverse clinical signs observed on ocular inspection were recorded and the body weight gain and food consumption were measured. Individual body weights were recorded four and six days before necropsy for males and three and six days before necropsy for females. Six males and six females from each genotype (wild-type and tg-CYP2C18&19) were examined.The total body weight of each mouse was determined, to the nearest 0.1 g, prior to necropsy and animals were killed by exsanguination of the common carotid artery under enflurane and nitric oxide anaesthesia. During necropsy, the organs were examined macroscopically and weights of a standard set of tissues Table  were meaForty-eight tissues Table  from eac® 120 Haematology System  using standard methodology. Blood chemistry parameters were analysed using a Cobas Integra 400 analyser , and appropriate kits. Parameters measured in haematology and blood chemistry are listed in Table Blood samples for haematology (EDTA tubes) and blood chemistry (lithium heparin tubes) were collected from the orbital plexus under enflurane and nitric oxide anaesthesia, prior to necropsy. Animals were not fasted at the time of blood sampling but the genotype groups were necropsied with every second animal being wild-type and every second being transgenic in order to minimize daytime variations in glycogen content between the groups. At necropsy, one femur was taken for bone marrow analysis. Haematology analysis was performed with an ADVIAThe bone marrow differentials were determined by flow cytometry as described by Saad et al  and the th/75th percentile (outer boundaries of each box) and the 10th/90th percentiles (whiskers above and below each box). In addition the minimum and maximum are marked with dots.The statistical comparisons between the transgenic and wild-type groups were performed, using a computerized statistical program . All variables were compared with a t-test. Organ weights were compared both as absolute weights and as relative weights . Box plots in figures were generated using Sigma Plot, 2001 and the plots show the median (line within each box), the 25in vivo part of the study. In the female groups most of the mice gained weight. Two wild-type females and two tg-CYP2C18&19 females decreased in weight, but the weight loss in all cases was ≤ 0.6 g. No significant differences in food consumption were recorded.All male mice either retained or increased their weight during the At necropsy, all mice were in good nutritional condition. On macroscopical examination, small white foci were found in the eyes of one wild-type female and two tg-CYP2C18&19 females. These macroscopical changes, and the recorded weight differences between groups, did not correlate to any changes on the histopathological level. Minimal perivascular infiltration of neutrophils was found on microscopic examination in the epididymal fat in one tg-CYP2C18&19 male and minimal alveolar histiocytosis was present in one tg-CYP2C18&19 female. All these changes were considered to belong to the spontaneous background pathology observed in laboratory mice of the C57BL/6JOlaHsd strain.Weight distributions for all tissues within the different genotype and sex groups are shown in Table p ≤ 0.05 and p ≤ 0.001 for males and females respectively). The adrenal glands (p ≤ 0.05) and brown fat deposits (p ≤ 0.001) were smaller, while the heart weight was larger (p ≤ 0.001) in the tg-CYP2C18&19 males than in wild-type males. The spleen weight was lower in female tg-CYP2C18&19 than in wild-typefemales (p ≤ 0.05). All other organ weight comparisons  showed no significant differences between the genotypes.In both male and female mice, the brain weight was lower in tg-CYP2C18&19 than in wild-type mice (p ≤ 0.05) between the genotypes are shown in Figure Comparisons of all clinical pathology variables (between the genetic and sex groups) are shown in Table p ≤ 0.01). The level of circulating triglycerides in the blood was decreased in female tg-CYP2C18&19 compared to the wild-type females (p ≤ 0.01).The blood glucose levels were altered in male tg-CYP2C18&19 mice, which had higher levels than wild-type males .in vivo and extrapolation of results obtained from studies with this model may be more predictive to humans than when using traditional animal models.In the present study a gene-addition transgenic mouse, containing a BAC spanning the human CYP2C18&19 genes, has been characterized. Some alterations in organ weight and clinical pathology parameters were observed. Despite the alterations, no pathological changes were observed macroscopically or histologically and these hemizygous tg-CYP2C18&19 mice were considered to be viable and healthy. Hopefully, this model could be used to investigate the roles of CYP2C18 and CYP2C19 The authors declare that they have no competing interests.SL carried out the pathological evaluation of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. SE and YT helped to draft the manuscript. R F-S helped to draft the manuscript and funded the study. SE, YT and R F-S have all been supervisors to SL during the course of the study.