This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors . Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3  were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly  in vitro and in germ cells. Therefore, H2A.B has the novel ability to bind to both RNA and DNA (as well as proteins) thus directly linking chromatin structure with the function of RNA. Taken together, this suggests that a special mechanism of splicing may operate in the testis and brain.The substitution of core histones with their non-allelic variant forms plays a particular important role in regulating chromatin function because they can directly alter the structure of chromatin, and provide new protein interaction interfaces for the recruitment of proteins involved in gene expression. Despite being discovered over a decade ago, the function of H2A.B, a variant of the H2A class, in its proper physiological context (being expressed in the testis and the brain) is unknown. We provide strong evidence that H2A.B has a role in the processing of RNA. It is found in the gene body of an active gene, directly interacts with RNA polymerase II and splicing factors and is located in the nucleus at distinct regions enriched with RNA processing factors (splicing speckles). Most significantly, we show that H2A.B can directly bind to RNA both  Importantly, the structure and function of a nucleosome can be regulated by the substitution of one or more of the major core histones with their variant forms.Histones, the key proteins that compact all eukaryotic DNA into chromatin, have attracted much attention recently because of their impact on all aspects of genome function . HistoneIn vitro biophysical and transcription studies revealed that H2A.Bbd, and the mouse orthologue, which we designated H2A.Lap1 (Lack of an acidic patch) . Gi. Gi19]. in vivo (Based on our findings that H2A.B.3: 1) is bound to chromatin , 2) is lin vivo , 6) co-lin vivo  and 7) rin vivo , we propin vivo . FollowiWild type 28–30 day old male Balb/c mice were used for all testis studies. 6–8 week old mice were used for Hippocampus ChIP and RNA-Seq experiments. Mice were housed according to animal ethics protocol at ANU animal facilities .2; 0.2% Tween-20). Beads were resuspended in 20 μl 1x NuPAGE loading buffer (Life Technologies), containing ß-mercaptoethanol, and eluted proteins were loaded on a 4–12% PAGE for electrophoretic separation. The gel was fixed with mass spectrometry-compatible protein stain Instant Blue (Expedeon). Each whole gel lane (H2A.B.3-IP or H2A.Z-IP) was divided in 10–12 segments. The gel segments were dehydrated with acetonitrile (60μl per gel piece) and then dried using a SpeedVac SC100 (Savant). For mass spectrometry analysis, gel slices were destained, reduced and alkylated following the procedure described by Shevchenko et al.[4HCO3, and 40 ng of trypsin (Promega) was then added to each gel slice. Samples were incubated for 16 h at 37°C. Each digest solution was removed to a new microfuge tube and the gel slices treated with the following solutions sequentially for 30 min each: 80 μl 0.1% (v/v) formic acid, 67% (v/v) acetonitrile and 80 μl 100% acetonitrile. The pooled digest and peptide extraction solutions for each sample were then dried  before resuspending in 20 μl of 0.1% (v/v) formic acid.The preparation of mononucleosomes from seminiferous tubules, as prepared for ChIP-Seq experiments, was carried out as described recently by us. 10–15μg of anti-H2A.B.3 or anti-H2A.Z antibody was covalently bound to magnetic Dynabeads using the Dynabeads antibody coupling Kit (Life Technologies) and incubated with 100–200μg of formaldehyde crosslinked nucleosomes  in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS; 0.5% sodium deoxycholate, Roche protease inhibitor cocktail for 4 hours at 4°C with rotation. The immunoprecipitated histone variant protein complexes were washed twice in high-salt wash buffer , and twice with wash buffer . Up to the 15 most abundant ions (>5000 counts) with charge states of >+2 were sequentially isolated and fragmented via collision induced dissociation (CID) with an activation q = 0.25, an activation time of 30 ms, normalized collision energy of 30% and at a target value of 10 000 ions; fragment ions were mass analyzed in the linear ion trap. Peak lists derived from LC-MS/MS were generated using Mascot Daemon/ExtractMSn.exe  and submitted to the database search program Mascot [Mus musculus sequences only. Peptide identifications were considered to be high confidence if they were statistically significant (p<0.05) according to the Mascot expect metric.Proteolytic peptide samples were separated by nano-LC using an UltiMate 3000 HPLC and autosampler system , and ionized using positive ion mode electrospray following experimental procedures described previously. Single Hypotonic spreads of male germ cells were prepared essentially as described. Fixed cRound spermatids were isolated by sedimentation on a BSA gradient as described elsewhere . Subcell7 cells were fixed for 12 min with rotation at room temperature in 10 ml of fresh medium HBSS in the presence of 1.2% (v/v) formaldehyde. Mononucleosomes were obtained by digesting purified nuclei with 2–4 units of MNase I (NEB) at 37C.Mononucleosomes were prepared from testicular tubules using Micrococcal nuclease I (Mnase I) digestion as described . To prep-ΔΔCt.Chromatin immunoprecipitations were performed with affinity purified H2A.B.3, H2A.Z and H3K36me3 antibodies  as described by us previously. Two biological replicates were performed for each ChIP-Seq experiment. Libraries were prepared for the first biological replicate using the Sample Prep Kit (Illumina) according to the manufacturer's protocol for single end sequencing of DNA. The second biological replicate used the ChIP-Seq Library Prep (New England Biolabs) with barcodes and paired end adaptors. Quality and concentration of the libraries were assessed on Agilent Technologies 2100 Bioanalyzer and using qPCR with adaptor specific primers (ABI Prism 7900HT) according to Illumina's recommendations. DNA was sequenced on HiSeq 2000 or HiSeq 2500 (rapid run mode) (Illumina) using 100 base pairs single—or paired—end reads. Mapped reads for all input and ChIP-Seq experiments (and RNA-Seq plus RNA-IP Seq experiments) are shown in 2 at 254 nm produced a high background of non-specific protein-RNA interactions masking specific protein-RNA interactions. We therefore reduced the irradiation to 75mJ/cm2 at 254 nm. A single cell suspension prepared from testicular tubules was washed 3 times with cold DPBS-GL (DPBS (Gibco) supplemented with 5.6mM D-glucose, 5.4mM DL-lactate, 0.5mM DTT, 0.2mM PMSF, EDTA-free protein inhibitor cocktail (Roche), pH-7.1). These cells were then resuspended in 6 ml at a density of 4-6x106 cells per ml in the same buffer and UV-irradiated with 75mJ/cm2 at 254 nm . Cells were then cross-linked with 1.2% (v/v) formaldehyde. To prepare chromatin for RNA-IP-seq, MNase I digestion was substituted by sonication . Purified nuclei from germ cells were re-suspended in 2ml of sonication buffer  SDS, 0.1% (w/v) sodium deoxycholate, 0.2 mM PMSF, Roche EDTA-free protein inhibitor cocktail) and sonicated for 15 minutes  at 4°C using a Bioraptor Sonicator (Diagenode) to obtain ~ 300 base pair-long chromatin fragments. After sonication, the sheared chromatin was cleared by centrifugation at 10000 g for 5 minutes, dialysed against 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 4% (v/v) glycerol and stored at -80°C. 300μg of sheared testis chromatin was used for the RNA-IP procedure to detect H2A.B.3 associated RNA [The RNA-IP procedure to detect H2A.B.3 associated RNA was carried out as described  with somated RNA  with the™ DNase (Life Technologies). RNA-Seq libraries  were prepared using NEBNext mRNA Library Prep kit using oligo-dT enrichment module (New England Biolabs) following manufacturer’s recommendations. Resulted RNA—Seq libraries were sequenced on HiSeq 2000 sequencer (Illumina) using 100 base pairs paired-end reads.Total RNA was isolated using TRIzol (Invitrogen) and the RNeasy Mini kit (Qiagen). Samples were treated with TURBO2 at 254 nm to crosslink RNA/protein complexes in a UV-irradiation oven, Stratalinker 1800, (Stratagene). 3-4x107cells were lysed in 2 ml of lysis buffer  for 30 min on ice. Chromatin was mechanically sheared by passing nuclei through a 31G syringe. To remove insoluble material, the lysates were centrifuged at 10,000 g. Half of the supernatant was subjected to RNase I treatment  for 30 min at 37°C, while the other half was treated in the same way but without the addition of RNase I. The degradation of RNA was monitored by the Qubit HS RNA assay. Both untreated and RNAase I-treated lysates were immunoprecipitated with H2A.B.3 antibodies exactly as described for IP-MS.Total germ cells from 28–30 day mice testis were UV-irradiated once with 75mJ/cm2; 1% NP-40, 0.1% SDS, 0.5% Na deoxycholate, pH 7.4) and twice with wash buffer ), immunoprecipitated complexes were dephosphorylated on beads using PNK and than the RNA was 5' end labeled with P32 gamma-ATP. Eluted labeled complexes were electrophoresed through SDS-PAGE and transferred onto a nitrocellulose membrane. The same membrane was uses for a western analysis using anti-H2A.B3 and anti-H2AZ antibodies.To distinguish IPs performed with formalin and UV-crosslinked cells from UV-only-crosslinked cells, we use the term CLIP (UV-cross-linking immuno precipitation) for the latter samples only. For CLIP assays, germ cell lysates were prepared in an identical manner as just described. 0.25ml of lysate was diluted in 1 ml of 50mM Tris, 100mM NaCl, 5mM MgCl2, 1mM DTT for RNase treatement with 400 units of RNase I (Ambion AM2294) for 30 min at 37°C. Treated lysates were immunoprecipitated with equal amount of anti-H2A.B.3 and anti-H2A.Z antibodies (10μg each), bound to protein A/G dynabeads (Thermo Fisher), for 2 hours. Following washing, twice with high salt wash buffer , 1 mM DTT, 0.03 mg/ml heparin). The binding reactions were analysed on 5% acrylamide 1x TB gels. Gels were stained with SYBR Gold Nucleic Acid Gel Stain and visualised using a Typhoon FLA 9000. Biotinylated peptides corresponding to the N-terminal tails of histones H2A, H2A.Z, H2A.B, and H2A.B.3 were purchased from GL Biochem at >90% purity. A Cy3-labelled 25 nt RNA (Cy3-CAGCGACUCGGGUUAUGUGAUGGAC) was purchased from Sigma-Aldrich. The biotinylated peptides (30 pmol) were immobilised on steptavidin-coated M-280 Dynabeads (LifeTechnologies) and incubated with 150 pmol of the Cy3-labelled RNA in binding buffer for 1 hr at room temperature. The beads were then washed three times with binding buffer. Following the final wash, the beads were resuspended in Novex TBE-urea Sample Buffer (Life Technologies), heated at 70°C for 2 min and then run on 15% Novex TBE-urea gels (Life Technologies). Cy3 fluorescence was visualised on a Typhoon FLA 9000.Histone H2A-H2B (and variant) dimers were produced using standard protocols for refolding of histone complexes . Either interest  using a Antibodies used were as follows. Anti-H2A.B.3, Anti-H3Chip-Seq reads were adaptor trimmed and mapped to the genome using Bowtie2. Paired RNA-IP data was mapped with Tophat  using boTo determine if exons versus introns gained H2A.B.3 preferentially, the association between mean H2A.B.3/input ratio (in RPM) and log expression from RNA-Seq data (RPM) was calculated for a 1kb flanking window around all intron-exon boundaries. At each base position relative to the intron-exon boundary, a linear model was fit to the H2A.B.3/input ratio versus gene log expression, using the mean H2A.B.3/input ratio value in a sliding 20 bp window around each bp, and the slope of the fitted model plotted. When overlapping RNA-IP data with H2A.B.3 ChIP-Seq data over gene bodies and promoters (-1000 bp upstream of TSS), a minimum coverage of 30 reads per base was used, which was above background reads. To determine the percentage of H2A.B.3 expression compared to the total H2A pool, the mean TPM was determined for all different H2A subtype genes expressed.S1 Fig(a) The normalised distribution of input (0 to 0.15 RPM) at the intron—exon boundary for all genes separated into 100 groups in the testis as a heat map. (b) The normalised distribution of H2A.B.3 (0 to 0.15 RPM) at the intron—exon boundary for all genes separated into 100 groups in the testis as a heat map. (c) The normalised distribution of H3K36me3 (0 to 0.15 RPM) at the intron—exon boundary for all genes separated into 100 groups in the testis as a heat map. (d) The normalised distribution of H2A.Z (0 to 0.15 RPM) at the intron—exon boundary for all genes separated into 100 groups in the testis as a heat map.(PDF)Click here for additional data file.S2 Fig(a) A testis H2A.B.3 meta-intron plot where the length of all introns are normalised to the same size. (b) A testis H2A.B.3 meta-exon plot where the length of all exons are normalised to the same size. (c) The individual line represents the normalised H2A.B.3 reads aligned between -1 and +1 kb from the exon—intron boundary for all exons in the testis.(PDF)Click here for additional data file.S3 FigParaffin-embedded mouse brain sections were indirectly immunostained with H2A.B.3 and H2A.Z antibodies. The primary antibody signal was amplified with the TSA signal amplification system (Perkin Elmer). Representative images of the hippocampus and surrounding regions are shown.(PDF)Click here for additional data file.S4 Fig(a) Normalised testis H2A.B.3 ChIP-Seq reads (mean reads per base pair per million reads mapped (RPM)) aligned between -1 and +1 kb from the TSS ranked according to their level of expression. (b) Normalised hippocampus H2A.B.3 ChIP-Seq reads ranked according to their level of expression aligned with the TSS. (c) Normalised testis input Seq reads aligned between -1 and +1 kb from the TSS ranked according to their level of expression. (d) Normalised hippocampus input Seq reads ranked according to their level of expression aligned with the TSS. (e) The normalised distribution of H2A.B.3 (0 to 0.15 RPM) at the TSS for all genes separated into 100 groups in the testis as a heat map. (f) The normalised distribution of H2A.B.3 (0 to 0.15 RPM) at the TSS for all genes separated into 100 groups in the hippocampus as a heat map. To detect localised biological functions over nucleosomes in the H2A.B.3 ChIP-Seq and input data sets, gene set enrichment analyses were performed of gene symbols ranked by the mean coverage over a fixed window size of 50 bp at successive distances from the TSS. This enrichment was computed for H2A.B.3 and input using gene sets corresponding to GO terms for the testis (g) and the hippocampus (h). Shown are those GO terms where H2A.B.3 was most highly enriched.(PDF)Click here for additional data file.S5 Fig(f) Pearson correlation of the log coverage, across 50 base pair windows, was calculated between hippocampus H2A.B.3 ChIP-Seq reads and H3K36me3 ChIP-Seq reads for each base pair relative to the intron—exon boundary. (g) Normalized hippocampus H3K36me3 ChIP-Seq reads ranked according to the incorporation of H2A.B.3  aligned with the intron—exon boundary. 95% confidence bands are shown in grey.Input nucleosomes, nucleosomes immunoprecipitated with H2A.B.3 or H3K36me3 affinity purified antibodies, and poly (A)-transcripts obtained from adult hippocampal neurons were sequenced yielding 100 base pair paired-end reads. (a) The individual lines represent the normalised H2A.B.3 and total input reads aligned between -1 and +10 kb from the TSS in the hippocampus. (b) The individual line represents the normalized input nucleosome reads (mean reads per base pair per million reads mapped (RPM)) aligned between -1 and +1 kb from the intron—exon boundary for all exons in the hippocampus ranked according their expression level . The colour-map panel shows the relationship between colour and the gene expression rank. (c) Normalized hippocampus H2A.B.3 ChIP-Seq reads ranked according to their expression level aligned with the intron—exon boundary. (d) At each base position relative to the intron exon-boundary, a linear model was fitted to the mean H2A.B.3/input ratio versus gene log expression across all intron—exon boundaries, and the slope of the fitted model plotted for the hippocampus. (e) Normalized hippocampus H3K36me3 ChIP-Seq reads ranked according to their expression level aligned with the intron—exon boundary. (PDF)Click here for additional data file.S6 Fig(a) Normalised testis H2A.B.3 ChIP-Seq reads ranked according to the incorporation of H2A.B.3 at the TSS  aligned with the intron—exon boundary. (b) Normalised hippocampus H2A.B.3 ChIP-Seq reads ranked according to the incorporation of H2A.B.3 at the TSS  aligned with the intron—exon boundary.(PDF)Click here for additional data file.S7 Fig(a) Normalized testis H2A.B.3 ChIP-Seq reads ranked according to their expression level aligned with the intron—exon boundary of constitutive exons. (b) Normalized hippocampus H2A.B.3 ChIP-Seq reads ranked according to their expression level aligned with the intron—exon boundary of constitutive exons.(PDF)Click here for additional data file.S8 FigT method was used to calculate relative gene expression; mouse β-actin was used as an endogenous reference gene. 10do: 10 day old testis ; 12do: 12 day old testis ; 19do: 19 day old testis ; 24do: 24 day old testis , 28do: 28 day old testis , 65do: adult mice testis.Total RNA was extracted from whole mouse testes at various stages of spermatogenesis. cDNA was synthesised using random and poly(dT) priming. Comparative C(PDF)Click here for additional data file.S9 FigFollowing the RNA—H2A.B.3 IP procedure, the released RNA was sequenced to yield 100 base pair paired end reads. A H2A.B.3 RNA plot ranked according to expression aligned with the TSS.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


H9N2 avian influenza virus (AIV) of genotype S frequently donate internal genes to facilitate the generation of novel reassortants such as H7N9, H10N8, H5N2 and H5N6 AIVs, posing an enormous threat to both human health and poultry industry. However, the pathogenicity and transmission of reassortant H5 viruses with internal gene cassette of genotype S H9N2-origin in chickens and mice remain unknown. In this study, four H5 reassortants carrying the HA and NA genes from different clades of H5 viruses and the remaining internal genes from an H9N2 virus of the predominant genotype S were generated by reverse genetics. We found that all four H5 reassortant viruses showed attenuated virulence in both chickens and mice, thus leading to increased the mean death times compared to the corresponding parental viruses. Consistently, the polymerase activity and replication ability in mammalian and avian cells, and the cytokine responses in the lungs of chickens and mice were also decreased when compared to their respective parental viruses. Moreover, these reassortants transmitted from birds to birds by direct contact but not by an airborne route. Our data indicate that the internal genes as a whole cassette from genotype S H9N2 viruses play important roles in reducing the pathogenicity of the H5 recombinants in chickens and mice, and might contribute to the circulation in avian or mammalian hosts. Avian H5 and H9N2 influenza viruses are two major subtypes that circulate in poultry populations globally . In 1996Similarly, the low pathogenic avian influenza virus (LPAIV) of H9N2 subtype has evolved into more than 102 genotypes and become endemic in most poultry raising areas since its first emergence in chickens in 1994 in China . Among tIn fact, H9N2 and H5 AIVs co-exist in poultry in many regions in China, thus providing an ideal environment for the generation of novel AIVs by reassortment. Recently, various novel reassortant viruses whose HA gene originated from the A/goose/Guangdong/1/96-like (Gs/GD-like) lineage and the internal genes from H9N2 viruses were isolated in humans and birds . We prevIn this study, four H5 reassortant viruses carrying the HA and NA genes from two subclade 2.3.2.1c and two subclade 2.3.4.4 of H5 viruses, respectively, and the remaining internal genes from an H9N2 virus of the predominant genotype S were generated by reverse genetics. Viral replication, polymerase activity and pathogenicity of these viruses in chickens and mice were evaluated. Additionally, transmission capacity of four H5 reassortant viruses in birds was also determined. Our study may help to further understand the specific effect of the internal gene cassette of genotype S H9N2 AIV on H5 viruses and provide valuable information for the control and prevention of the resultant recombinant viruses.All experiments involving live viruses were carried out in a biosafety level 3 (BSL3) facility at Yangzhou University in accordance with the institutional biosafety manual.Animals were housed in negative-pressure isolators with HEPA filters in a BSL3. The protocols for animal experiments were approved by the Jiangsu Administrative Committee for Laboratory Animals , and complied with the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals.Four H5 HPAIV strains including A/chicken/Anhui/QD1/2014 [QD/1 (H5N1)], A/goose/Jiangsu/QD5/2014 [QD/5 (H5N8)] , A/chick2. For virus inoculation experiments, DMEM medium supplemented with 1% FBS was used.Chicken embryo fibroblasts (CEF), and chicken fibroblast cell line (DF-1) cells were cultured in DMEM (Invitrogen) medium with 4% fetal bovine serum (FBS) (Gibco). Madin-darby canine kidney (MDCK), and human embryonic kidney (293T) cells were grown in DMEM containing 10% FBS. All cells were incubated at 37°C with 5% COTable 1).Four reassortant viruses were generated by reverse genetics as described previously . The rea50) and 50% tissue culture infectious dose (TCID50), were determined in chicken embryos and in MDCK and CEF cells as previously described , and the virus titers were determined by TCID50 assay in MDCK cells as described above. Each experiment was performed in triplicate.Madin-darby canine kidney or CEF Cells were infected with the viruses at a multiplicity of infection (MOI) of 0.01 TCIDA minigenome assay was used to detect luciferase activities as reported previously . Brieflyin vivo, groups of eleven 6-week-old SPF chickens were inoculated intranasally with 106.0 EID50 of each virus. Three inoculated birds from each group were euthanized at 2 dpi, and gross lesions were recorded. At this time point, tissue samples , trachea and cloacal swabs were collected for virus isolation. Another eight virus-infected chickens were monitored for mortality and morbidity for 14 days. Mean death time (MDT) was determined as the mean time (d) for the virus to kill chickens within the observation period. For histopathological evaluation, the lung specimen from each group was fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 5 μm. Three tissue sections from each lung, and five sections per bird were stained with hematoxylin-eosin (H&E). This experiment was performed by three trained pathologists blinded to the treatment groups. The tissue pathological changes were observed and scored with an Olympus microscope . Criteria for grading lung pathological changes as described previously (The highly pathogenic H5 AIV has the multiple basic amino acid at the HA cleavage site or possesses an intravenous pathogenicity index (IVPI) greater than 1.2, and one of the IVPI methods to determine pathogenicity in chickens is used according to the World Organisation for Animal Health (OIE) criteria . To deteeviously : Grade 04.0 EID50 of each virus, as the direct inoculation group. The three chickens were placed in the same cage with the inoculated chickens at 30 min after inoculation of the infected group, as the physical contact group. The three chickens was placed in a cage directly adjacent to the direct inoculation group with a distance of 50 cm between cages, as the aerosol contact group (Transmission experiments were designed as previously described . Brieflyct group . Orophar50) of the viruses, groups of 5 (6-week-old) female BALB/c mice were inoculated intranasally with serial dilutions of virus and observed the morbidity and mortality for 14 days as previously described .To determine the 50% mouse lethal dose .The Independent-Samples Table 2). The CK/S, Y7/S were successfully rescued in one time, whereas Q1/S and Q5/S were generated in two and three experiments, respectively (Table 2). To investigate the properties of the rescued reassortants and the H5 parental viruses, EID50 and TCID50 were assessed. As shown in Table 2, the reassortants  and the parental viruses  infected efficiently both in MDCK, CEF cells and chicken embryos. Viruses grew to similar titers in MDCK and CEF cells and chicken embryos, from 106.0 to 107.8, 106.4 to 108.6, 107.2 to 108.8, respectively. However, Q5/S showed lower infectivity in CEF cell with a TCID50 of 104.5. Overall, these results indicate that the internal gene cassette from the genotype S H9N2 have high genetic compatibility with the surface genes of the tested H5 viruses.Four H5 reassortants harboring the surface genes from CK/10 (H5N1), YB/7 (H5N1), QD/1 (H5N1), QD/5 (H5N8) viruses and the internal genes from CZ/73 (H9N2) were successfully generated by reverse genetics  and CEF  cells at several time points as compared to their respective parental viruses . Of noted, the recombinants CK/S and Q1/S presented significantly lower viral titers at all the tested time points both in MDCK and CEF cells when compared with the corresponding parental viruses. These data show that the internal gene cassette of the genotype S H9N2 virus decreases the replication ability of the H5 viruses in mammalian and avian cells.To evaluate whether the internal gene cassette of the genotype S H9N2 virus affected replication of the H5 viruses in avian and mammalian cells, we compared kinetics of replication of the viruses in both CEF and MDCK cells. As a result, the recombinants presented significantly lower replication activity in both MDCK  were 364 and 250% higher than those of CZ/73 (H9N2) in DF-1 and 293T cells. Activity of QD/5 (H5N8), CK/10 (H5N1) and YB/7 (H5N1) RNP complexes were 168% (P < 0.01), 151% (P < 0.01), and 107% (P < 0.05) higher than that of CZ/73 (H9N2) in DF-1 cells, respectively. Additionally, QD/5 (H5N8), CK/10 (H5N1), and YB/7 (H5N1) RNP activities were 138% (P < 0.01), 108% (P < 0.05), and 103% (P < 0.05) higher than that of CZ/73 (H9N2) in 293T cells, respectively. Collectively, CK/10 (H5N1), YB/7 (H5N1), QD/1 (H5N1), and QD/5 (H5N8) complexes showed higher RNP activities than that of CZ/73 (H9N2). Our findings suggest that the internal gene cassette of the genotypes S H9N2 virus significantly decreases the RNP activity of the H5 recombinants.To investigate the relationship between RNP activities and the replication ability of these reassortants, we determined the activities of the combined RNP complexes from the CK/10 (H5N1), YB/7 (H5N1), QD/1 (H5N1), QD/5 (H5N8), or CZ/73 (H9N2) virus gene segments by measuring the luciferase activity in 293T and DF-1 cells. As shown in Table 3, the IVPI of CK/S, Y7/S, and Q5/S were 0.1 lower than that of their parental viruses, and Q1/S showed a decrease of 0.5 in relative to QD/1. The IVPI result suggested that the H5 recombinants had lower virulence in chickens when compared to their parental viruses. Intriguingly, MDTs of the group infected with parental viruses were about 2 days, while the chickens infected with four H5 reassortants were over 5 days. Especially, the Q1/S virus resulted in a mortality of 50% and an MDT of 8.75 days, whereas the QD/1 virus brought about a higher mortality (100%) and a shorter MDT (2.13 days) (Table 3). Therefore, MDTs and survival rates were significantly different (P < 0.001) between the H5 reassortants and the parental viruses.To examine the pathogenicity of these H5 viruses in chickens, we determined the replication and IVPI of the viruses in chickens. The IVPIs of all the viruses were over 2.4, indicating that they were highly pathogenic in chickens. As shown in 6 EID50 of the viruses. Three chickens from each group were killed at 2 dpi, and their organs were collected for virus titration. At this time point, the parental viruses could be detected in the lungs, brains, spleens, kidneys from three birds, whereas Y7/S, CK/S could be detected in the lungs, spleens, kidneys from three birds  and Q1/S, Q5/S could only be detected in the lungs from two out of three birds . In addition, parental viruses were recovered successfully in the trachea and cloacal swabs of the infected birds. Viral titers were higher in the trachea than in the cloacal swabs at this time point. However, we failed to detected virus from trachea and cloacal samples of the H5 reassortants-infected birds at this time point . Moreover, a significantly higher virus load was observed in the lung, brain, spleen, and kidney of the parental viruses-infected chickens compared to those of the H5 reassortants .To examine the replication of the viruses in chickens, groups of three 6-week-old SPF chickens were intranasally inoculated with 10Figures H, the lungs of the H5 parental viruses-infected chickens showed severe bronchopneumonia, characterized by severe bronchopneumonia and interstitial pneumonia, bronchial epithelial cell desquamation, lymphocytes infiltrates around lung lobular, interstitial congestion, erythrocytes within alveolar spaces. By contrast, mild bronchopneumonia and mild desquamation of bronchial epithelial cells were seen in the lung of the H5 recombinants infected-birds. Scores of microscopic lung lesions are shown in Figure 4Q. Taken together, these results suggest that the internal gene cassette of the genotype S H9N2 virus decreases pathogenicity of the H5 viruses in chickens.We further systematically compared the virus-induced lung histological changes between the parental viruses and the recombinants. As shown in Table 4). As a result, during the observation period, all chickens from the physical contact group that were placed in the same cage with the CK/S or Y7/S-infected chickens, died within 9 dpi. Viruses were detected in oropharyngeal swabs of the CK/S or Y7/S of the physical contact birds at 3, 5, and 7 dpi and the mean titers were from 103.25 to 104.0 EID50, 103.33 to 103.92 EID50, respectively (Table 4). The physical contact birds housed with the Q1/S survived on 9 dpi, shed with titers detected at a range from 103.25 to 103.67 EID50 and showed seroconversion at 14 dpi. One of three physical contact birds housed with the Q5/S died on 9 dpi and the rest of the physical contact birds seroconverted on 14 dpi (Table 4). The virus shedding was tested from oropharyngeal swabs of the Q5/S of the physical contact birds with titers detected at a range from 103.08 to 103.88 EID50 on 9 dpi (Table 4). However, no virus from airborne transmission chickens of four reassortants could be detected in oropharyngeal during the observation period except the CZ/73 (H9N2) group (Table 4). All together, these results indicate that four reassortants are transmitted to direct contact group but fail to be transmitted efficiently by aerosol way.Because the MDTs of the H5 reassortants were longer than the parental viruses, we then investigated the transmission of four H5 reassortants in chickens. All reassortants viruses were detected in oropharyngeal swabs from the directly inoculated birds . In contrast, the H5 reassortants CK/S, Q1/S, and Q5/S viruses were moderately pathogenic for mice, with MLD50 values of 105.5, 105.8, and 106.0 EID50, respectively. Moreover, Q1/S showed the greatest attenuation, with a 105.3-fold decrease in MLD50. In addition, mice infected with these parental viruses resulted in >25% body weight loss and 100% mortality in mice in a dose of 106.0 EID50 early before 6 dpi (Table 5). By contrast, mice infected with Q1/S, Q5/S, and CK/S viruses lead to two, two, three of five observed mice died, but the surviving mice recovered from inoculation by 8 dpi and experienced 14.8–17.4% weight loss, and the mean survival time (MST) 9.4–11.2 days (Table 5). The mortality rates of 40, 40, and 60 was observed in mice infected with Q1/S, Q5/S, and CK/S . Furthermore, Y7/S resulted in 19.6% weight loss, 100% mortality and the 6.2 days of MST (Table 5). Taken together, the internal gene cassette of the genotype S H9N2 virus significantly decreases the virulence of the H5 recombinants in mice.To investigate the pathogenicity of these H5 viruses in mouse models, we then determined the MLDin vivo. All the parental viruses, and the reassortants  could be detected in the lungs, brains, spleens, and kidneys, whereas Q1/S, Q5/S could only be detected in the lungs . Additionally, significantly higher virus yields in the lungs of CK/10, QD/1, and QD/5 viruses-infected mice were observed compared to those of CK/S, Q1/S, and Q5/S viruses on 3 and 5 dpi . In addition, virus titers in the brains, spleens, and kidneys of CK/10, YB/7 viruses-infected birds were dramatically higher than those of CK/S, Y7/S viruses on 3 and 5 dpi (P < 0.01) . Notably, Q1/S and Q5/S viruses showed no replication in the brains, spleens, and kidneys. Collectively, these results suggest that the internal gene cassette of the genotypes S H9N2 virus significantly decreases the replication of the H5 recombinants in mice.We next evaluated the replication ability of the viruses Figures P, the parental H5 viruses of this group resulted in severe interstitial pneumonia and bronchopneumonia at 5 dpi, characterized by edema, hemorrhages, desquamation, necrosis, and extensive infiltration of inflammatory cells. By contrast, mild to moderate bronchopneumonia was observed in the H&E-stained lung tissues of mice infected with the reassortant H5 viruses of this group. Scores of lung histopathological lesions are shown in Figure 4R. Collectively, these results indicate that the internal gene cassette of the genotypes S H9N2 virus dramatically decreases the pathogenicity of the H5 recombinants in mice.Hematoxylin-eosin staining of lung tissues was performed for histopathological evaluation. As shown in Figures F, the parental viruses elicited higher levels of interleukin 6 (IL-6), interferon α (IFN-α), interferon β (IFN-β), and interferon γ (IFN-γ) compared to the recombinant-infected lungs. However, the recombinants and the parental viruses elicited similar cytokine responses in terms of interleukin 8 (IL-8) expression . In addition, significantly higher levels of interleukin 18 (IL-18) genes were induced by QD/1 than Q1/S, QD/5 than Q5/S, whereas there were similar levels in this gene induction between CK/10 and CK/S, YB/7 and Y7/S.To better understand the role of cytokine expression in the induction of the different lung pathological manifestations caused by the recombinants and the parental viruses, we then systematically investigated the production of some representative cytokines and chemokines in the lungs of the virus-infected chickens and mice. In chickens, we analyzed six antiviral proinflammatory cytokines from the lungs of the virus-infected chickens at 2 dpi. As shown in Figures L). Thus, these results indicate that internal gene cassette of the genotype S H9N2 virus dramatically decreases cytokine and chemokine response in both chickens and mice lungs.In mice, 6 antiviral proinflammatory cytokines were analyzed in the virus-infected mouse lungs. Significantly higher levels of cytokines tumor necrosis factor α (TNF-α), IL-6, IFN-γ, IFN-α, monocyte chemotactic protein 1 (MCP-1), interferon-inducible protein 10 (IP-10) were detected in the lungs of the parental viruses-infected mice than in those inoculated with the recombinants at 3 and 5 dpi  Epidemic strains, phylogenetic analysis of the HA genes revealed that CK/10 (H5N1) and YB/7 (H5N1) belong to subclade 2.3.2.1c while QD/1 (H5N1) and QD/5 (H5N8) belong to subclade 2.3.4.4, both of which are currently the predominant subclades of H5 HPAIVs circulating in China. The CZ/73 (H9N2) virus belongs to genotypes S, which is the predominant genotype in the field throughout China. (ii) In previous studies, we have collected a very large number of biological materials of these viruses . (iii) RFigure 1). Furthermore, a remarkably lower luciferase activity of the H9N2-derived RNP complexes was also detected in both 293T and DF-1 cells in contrast to either of the H5NX-derived ones . Since RNP compatibility and activity are closely related to viral reassortment, replication, and pathogenicity , which may facilitate the longer virus shedding and circulation to pose a persistent threat to poultry. Different from H5 HPAI field isolates that generally fail to transmit between chickens in aerosol just with few variants showing contact transmission (Table 4), transmission between birds by aerosol route was not detected except the CZ73 (H9N2) group (Table 4), indicating that the envelop gene segments of H9N2 may contribute more critically to the acquisition of aerosol transmission. However, since all of the parental H5 HPAI viruses were lethal to chickens within 2 days even at the low inoculation dosage of 103.0 EID50 (data not shown), we failed to include those viruses in the transmission experiment. Therefore, whether the internal gene cassette of H9N2 subtype AIVs enhance the contact transmissibility of reassortant H5 viruses as inferred by our results yet needs further investigation.Despite highly pathogenic to chickens as evidenced by the IVPI value, each of four H5 reassortants showed obviously longer MDTs than the corresponding parental H5 viruses . Of note, the attenuated H5 reassortants could easily infected mice, implying that these viruses were prone to circulating in a mammalian host and may evolve via adaptive mutation or further reassortment capable of posing a potential threat to human health. For example, our group previously isolated a natural H5N2 virus, A/chicken/Hebei/1102/2010 (HB10), which exhibited high virulence in chickens but was low pathogenic to mice  theoretically generated genome constellations of the two parental H5 and H9 AIVs in the present study, only the combination pattern that containing all the six internal genes from the H9 virus while the two surface genes from H5 virus was investigated systematically to mainly focus on the biological effect especially pathogenicity variation of the internal gene cassette of genotype S H9N2 to reassortant H5 AIVs. Previous studies have shown that the highlighted contribution of a single gene segment to pathogenicity could be influenced by the other seven segments of AIV. For example, Tables 5), although we had previously demonstrated that the single PA or NP gene of genotype S H9N2 virus dramatically increased the virulence of the H5N1 recombinants in mice (Among the 254 ( in mice . In fact in mice . Su W. e in mice .In summary, our results demonstrated the high compatibility between the internal genes of the genotype S H9N2 viruses and the surface genes of the H5 viruses and that H5 reassortant viruses showed attenuated pathogenicity and replication in mice and chickens. In addition, the MDTs of the H5 recombinants were longer than those of their respective parental viruses. The results suggest that the H5 recombinants contained genotype S H9N2 subtypes AIV internal gene cassette might circulate in avian or mammalian host, evolve via reassortment or mutation, which is a potential threat to poultry industry and public health. Hence, our work highlights the necessity for strengthening surveillance to counteract the potential pandemic threat posed by the H9 and H5 reassortant viruses.XH, JH, MG, and XfL conceived and designed this research. XH, JW, and XlL performed experiments. XH and XfL wrote the manuscript. XW, MG, ZH, ZG, DL, JL, XwL, SH, XX, DP, and XJ contributed reagents/materials/analysis tools. XH, JH, MG, and XfL revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the availability of vaccines for Japanese encephalitis virus (JEV), the re-emerging virus remains a clinically important pathogen that causes acute encephalitis and permanent neuropsychiatric sequels. JEV highly targets dopaminergic neuron-rich brain regions including the thalamus and midbrain. The molecular mechanism contributing to the high susceptibility of these particular brain regions remains largely unclear. This study addressed whether this tissue tropism of JEV is associated with signaling of dopaminergic neurons. Three pieces of evidence indicate that JEV exploits dopamine signaling to facilitate its infection: (1) JEV infection modulates dopamine level; (2) a selective dopamine D2 receptor (D2R) agonist enhances JEV infection; and (3) stimulation of D2R activates phospholipase C (PLC) to enhance the surface expression of JEV binding/entry molecules, integrin β3 and vimentin. Overall, JEV may exploit dopamine-mediated neuronal communication to increase the susceptibility of D2R-expressing cells to JEV infection. This study identifies a potential underlying mechanism of viral invasiveness in the dopaminergic brain regions and suggests antiviral strategies against viral infection by targeting D2R-PLC signaling. Japanese encephalitis virus (JEV), transmitted by mosquitoes, is the most clinically important etiological agent of viral encephalitis, with an estimated worldwide annual incidence of 30,000 to 50,000 cases. About 25–30% of these cases are fatal and 50% result in permanent neuropsychiatric sequel including impaired cognition, poliomyelitis-like paralysis and Parkinsonian movement disorders . JEV, a The midbrain is rich in number of dopaminergic neurons as the major source of dopamine in the mammalian CNS . In addi3R) via inositol triphosphate (IP3), the product of phospholipase C (PLC) hydrolysis  pathway. To produce cAMP from adenosine triphosphate (ATP), D1-like receptors activate adenylyl cyclase (AC) by inducing heterotrimeric G proteins Gαs and Gαolf. On the other hand, through Gαi and Gαo, D2-like receptors may inhibit the activation of AC . Alternadrolysis .Dopamine receptors are involved in virus infections. Dopamine can enhance HIV infection by up-regulating the surface HIV entry co-receptor in human macrophages and DR antagonists can inhibit this enhancement . Prochloin vitro and in vivo studies and was propagated in mosquito C6/36 cells as described  or 8 mg QH (D2R agonist)/kg body weight intravenously in the absence  or presence of either 8 mg D1R antagonist/kg body weight  or 8 mg D2R antagonist/kg body weight . The animal survival was monitored daily.Mouse experiments were approved and performed in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Use Committee. Groups of 4-week-old AG129 mice were used to evaluate the effect of D2R agonist and antagonists on animal survival. Mice were challenged intraperitoneally with 5 × 10Cells were adsorbed with JEV  for 2 h. Cells were washed to remove unbound virus and incubated for the indicated times. For antiviral assays, cells were adsorbed with JEV  with the indicated doses of chemical compounds for 2 h, washed thoroughly, then incubated for 24 h with and without chemical compounds.The lentivirus vector pLKO.1, which carries a short hairpin RNA (shRNA) targeting the human D2R  or LacZ , obtained from the Taiwan National RNAi Core Facility, was cotransfected with pMD.G and pCMVΔR8.91 into HEK293T cells by use of Lipofectamine 2000 (Invitrogen). The lentiviruses were harvested from culture supernatants and concentrated by ultracentrifugation at 35,000 rpm in a Beckman SW41 rotor for 3.5 h at 4°C. The viral pellets were re-suspended and used to transduce BE(2)C cells. The D2R-deficient BE(2)C cells (shD2R-BE(2)C) and LacZ-control cells (shLacZ-BE(2)C) were selected with puromycin (10 μg/ml).Quantitative assay of dopamine was performed according to the manufacturer’s protocol . Briefly, 250 μl of cell culture medium was used for enzymatic conversion. Sample absorbance was determined by use of an ELISA reader (Molecular Devices) at 450 nm.1 was performed according to the manufacturer’s protocol (IP-One Tb Cisbio Bioassays). Briefly, cells were plated in a 96-well black clear-bottom plate  overnight. The level of IP1 in cell lysate was measured by use of SpectraMax M5 . Changes in intracellular cytosolic calcium level were evaluated by using calcium-sensitive dye Fluo-4AM  with a fluorescence microplate reader . Cells were loaded with Fluo-4AM dye in HEPES-buffered Modified Hanks’ Balanced Salt Solution without phenol red and calcium. Cells were incubated for 10 min at 37°C. Quinpirole hydrochloride (QH) at the indicated doses was added to cells and fluorescence intensity was recorded at 5 min intervals over 30 min at room temperature.cAMP level in cell lysate (10 μg) was measured by chemiluminescent enzyme-linked immunosorbent assay kit (Cell Biolabs). Quantitative assay of IPJapanese encephalitis virus was purified and labeled with Lightning-Link Atto-488 (Innova Biosciences) as described . BrieflyCells were adsorbed with JEV for 2 h with rocking on a linear shaker at room temperature for virus binding/entry assay. The cells were washed three times with cold PBS. Virus binding/entry was evaluated by confocal microscopy with fluorescently labeled  and plaque-forming assay with unlabeled JEV . For virus binding assay, cells were adsorbed with JEV for 1 h at 4°C with rocking on linear shaker. For the virus entry assay, cells were adsorbed with JEV for 1 h at 4°C. After a gentle wash with cold medium, cells were shifted to 37°C incubator for 1 h to allow virus entry. Cells were then treated with acid glycine  to inactivate non-internalized virus for 5 min and washed with cold HBSS. Cells were harvested with use of cell scrapers, and the adsorbed viruses were released by passing the cells through a 27-G needle 3 times. Cell lysates were centrifuged at 12,000 rpm for 1 min, and the supernatant was used for plaque-forming assay.Cells were plated in a 6-well plate for overnight. After treatments, cells were immunostained with anti-integrin β3  or anti-vimentin  without cell permeabilization for 1 h, then washed thoroughly and incubated with Alexa Fluor-488-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. After washing, cells were harvested with use of cell scrapers and passed through cell strainer. FACSCanto flow cytometry was used to measure signal intensity of respective proteins.post hoc test was used. Two categorical data were compared by independent Student t-test. The statistical tests were two-tailed and significance was set at P < 0.05 and < 0.01. For immunoblotting, the band density was quantified by use of ImageJ . A survival curve was descriptively analyzed by use of SigmaPlot v10.0 (Systat Software). Determination of the median survival time (T50) and P-values, by the log-rank test, involved used of Prism v5.0 (GraphPad Software).Data are presented as mean ± standard deviation (SD). For comparison of three or more categorical data, ANOVA-Bonferroni Figure 1A), probably due to cytopathic effect caused by JEV. Consistently, JEV also modulated the rate-limiting enzyme of dopamine biosynthesis, with an increase of phospho-TH levels at 3 and 6 hpi and decrease at later times, when JEV-NS3 was abundant . Notably, UV-inactivated JEV (UV-JEV) also significantly increased dopamine and phospho-TH levels at 3 and 6 hpi but different from that of JEV, the levels were not decreased by UV-JEV at 24 and 36 hpi . These data suggest that JEV increases dopamine levels at the early stage of infection probably through viral binding/entry.To investigate whether JEV affects dopamine production, we used dopaminergic human neuroblastoma BE(2)C cells, which show a high level of TH activity for dopamine synthesis . JEV-infFigures 1C,D). SCH23390, a D1R antagonist, also inhibited JEV infection, although it required a relatively high dose . Thus, our data indicate the major role of D2R in JEV infection as compared with D1R.The biological effect of dopamine is mediated by DRs and to elucidate their role in JEV infection, we used selective antagonists targeting dopamine D1- or D2-like receptors . SimilarFigures C). To verify the requirement of D2R in QH-enhanced JEV infection, we knocked down D2R expression in BE(2)C cells by transduction with a lentivirus expressing shRNA targeting human D2R (shD2R). The knockdown of D2R did not affect D1R expression and cellular localization . The enhancement effect of QH on JEV-NS3 expression and viral progeny production was significantly lower in shD2R-BE(2)C cells than parental BE(2)C cells and knockdown control shLacZ-BE(2)C cells . To further confirm the biological significance of D2R activation on JEV infection, we performed animal challenge experiment. QH treatment significantly accelerated animal mortality as compared with PBS treatment in JEV-challenged AG129 mice (T50: 4 days vs. 8 days) . Notably, this acceleration of animal mortality was abolished in the presence of D2R antagonist  but not D1R antagonist  . These data demonstrate that stimulation of D2R positively regulates JEV infection.To address the role of D2R activation in JEV infection, we used a D2R-specific agonist with putative dopaminergic properties, QH . TreatmeFigure 3A). Through Gαi/αo, D2R may inhibit AC to negatively regulate cAMP level (2) to inositol 1,4,5-triphosphate (IP3). IP3 then stimulates the release of calcium from intracellular stores before it is rapidly degraded to IP2 and then IP1 . However, the levels of IP1 were significantly increased by JEV infection . Furthermore, treatments with PLC signaling inhibitors at non-cytotoxic doses and in the range of their IC50 . Thus, PLC signaling pathway is involved in JEV infection in dopaminergic BE(2)C cells.To determine the D2R signaling event involved in JEV life cycle, we evaluated the major pathways of AC and PLC downstream of D2R stimulation . This QH-induced IP1 production was associated with D2R because IP1 level was lower in shD2R-BE(2)C than BE(2)C cells and knockdown control shLacZ-BE(2)C cells. Similarly, JEV infection induced IP1 production in a D2R-associated mechanism, since lower IP1 level was noted in cells with D2R knockdown . In addition, QH dose-dependently increased cytosolic calcium level as measured by Fluo-4 AM calcium indicator assay . Notably, cytosolic calcium level was lower in shD2R-BE(2)C than control cells with QH treatment, so QH-mediated calcium flux was associated with D2R. This D2R-dependent effect on cytosolic calcium level was also observed in JEV-infected cells .Next, we verified the activation of PLC pathway by the D2R agonist QH. Treatment with QH dose-dependently increased IPFigure 3G). Together, these data demonstrate that D2R stimulation and JEV infection induces a PLC signaling cascade in BE(2)C cells.Calcium flux may activate calcium-dependent enzymes such as CaMKII, the most abundant neuronal protein kinase in the brain . CalciumFigures 4A,B). Furthermore, this enhancement in QH-treated cells was concomitantly reduced with PLC signaling cascade inhibitors KN93, 2APB, and U73122 . To further address the effect on viral binding and/or viral entry, we performed virus binding and virus entry assays as described in the section “Materials and Methods.” Both JEV binding and entry were significantly increased by QH treatment and this enhancement could be repressed by PLC signaling inhibitors and with D2R knockdown in shD2R-BE(2)C cells . However, D2R stimulation had no effect on post-entry step of JEV infection because treatments with QH after virus adsorption did not significantly change JEV-NS3 expression, virus progeny production, and intracellular JEV RNA level . Therefore, D2R-mediated PLC signaling plays a role in enhancing JEV binding/entry.To dissect the viral life cycle affected by D2R-PLC activation, we performed viral binding/entry study in BE(2)C cells with or without QH pretreatment for 5 h. The amount of JEV binding/entry was significantly increased with QH treatment  dependent on PLC because the PLC inhibitor U73122 reversed this effect . The increased surface expression of integrin β3 and vimentin was not likely due to the effect on protein expression because their total protein levels were not changed by QH .Phospholipase C activation increases cytosolic calcium level, which in turn activates calcium-dependent kinases and/or directly enhances actin assembly to facilitate the formation of focal adhesion on the cell surface . One of Figures 5D,E). These data suggest that D2R-PLC-enhanced JEV binding/entry is associated with the increased surface expression of JEV binding/entry molecules including integrin β3 and vimentin.To support the role of integrin β3 and vimentin in D2R-PLC-enhanced JEV binding/entry, we performed antibody-blockage experiments. BE(2)C cells-treated with QH for 5 h underwent viral binding/entry assay with and without antibodies against integrin β3 and vimentin. The enhanced JEV binding/entry with QH was significantly repressed by anti-integrin β3 and anti-vimentin antibodies as compared with mouse IgG control (2) to inositol 1,4,5-triphosphate (IP3) .Japanese encephalitis virus infection of the brain mostly targets dopaminergic neuron-rich areas, the thalamus and midbrain . Here, wte (IP3) . IP3 thehe brain . CaMKII he brain . Furtherhe brain . Thus, DOur data indicate that JEV can induce hyperdopaminergia during the early stage of infection. Presumably, elevated dopamine level above the basal level may confer favorable conditions for viral life cycle, because hyperdopaminergia may alter cellular responses such as protein kinase C-mediated ERK and Akt expression . InvolveEmerging and clinically important flaviviruses have ability to enter the CNS and infect neuronal cells . For insOverall, our results revealed elevated dopamine level at the early stage of JEV infection to enhance viral entry in D2R-expressing cells via a PLC-mediated signaling pathway. Our study proposes a possible explanation for the high susceptibility of dopaminergic brain regions to viral pathogenesis and suggests a therapeutic strategy against viral infection by targeting D2R-PLC signal transduction.YS and Y-LLi designed research, analyzed data and wrote the paper. YS performed research. YS and J-JL performed animal study. Y-LLe provided critical reagents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Breast cancer (BC) is a heterogeneous disease characterised by variant biology and patient outcome. The amino acid transporter, SLC7A5, plays a role in BC although its impact on patient outcome in different BC subtypes remains to be validated. This study aimed to determine whether the clinicopathological and prognostic value of SLC7A5 is different within the molecular classes of BC.n = 1980), and proteomic level, using immunohistochemical analysis and tissue microarray (TMA)  in well-characterised primary BC cohorts. SLC7A5 expression correlated with clinicopathological and biological parameters, molecular subtypes and patient outcome.SLC7A5 was assessed at the genomic level, using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data (p = 0.001). High expression of SLC7A5 mRNA and protein was associated with poor patient outcome (p < 0.001) but only in the highly proliferative oestrogen receptor (ER)+/ luminal B (p = 0.007) and HER2+ classes of BC (p = 0.03). In multivariate analysis, SLC7A5 protein was an independent risk factor for shorter breast-cancer-specific survival only in ER+ high-proliferation tumours (p = 0.02).SLC7A5 mRNA and protein expression were strongly correlated with larger tumour size and higher grade. High expression was observed in triple negative (TN), human epidermal growth factor receptor 2 (HER2)+, and luminal B subtypes. SLC7A5 mRNA and protein expression was significantly associated with the expression of the key regulator of tumour cell metabolism, c-MYC, specifically in luminal B tumours only  contains supplementary material, which is available to authorized users. MYC [Rb) [Altered metabolic pathways have been readily accepted as part of the revised hallmarks of cancer where cancer cells are able to regulate their metabolism to provide energy and cellular building blocks required for their unremitting proliferation . Many caMYC , 5 and tMYC [Rb) .SLC7A5 is a sodium-independent transporter and acts as an amino acid exchanger by transporting large neutral amino acids such as leucine, phenylalanine and tryptophan by exchange with intracellular glutamine –10. For It has been reported that SLC7A5 is highly expressed in a variety of cancers including oesophageal carcinoma , oral caSLC7A5 gene copy number aberrations (CNA) and gene expression. DNA/RNA was isolated from fresh frozen samples and genomic and transcriptional profiling was obtained using the Affymetrix SNP 6.0 and Illumina HT-12v3 platforms respectively. CNA were considered at the gene level by segments and the Šidák correction [http://bcgenex.centregauducheau.fr) and breast cancer data from The Cancer Genome Atlas (TCGA) [SLC7A5 copy number/or mRNA expression.A cohort of 1980 BC tumours in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC)  was usedrrection  was applrrection . In thiss (TCGA)  were usen = 1110) presented at Nottingham City Hospital between 1989 and 1998, while the validation set (n = 1554) includes patients who were presented between 1998 and 2006. Patient management was uniform and based on tumour characteristics by Nottingham Prognostic Index (NPI) and hormone receptor status. Patients within the NPI excellent prognostic group (score ≤3.4) received no adjuvant therapy, but those patients with NPI >3.4 received tamoxifen if ER-positive (± goserelin (Zoladex) in case the patients were premenopausal). Conversely, classical cyclophosphamide, methotrexate and 5-flurouracil (CMF) were used if the patients were ER-negative and fit enough to receive chemotherapy. None of the patients in this study received neoadjuvant therapy. Clinical history, tumour characteristics and information on therapy and outcomes are prospectively maintained. Outcome data included development and time to distant metastasis (DM) and breast-cancer-specific survival (BCSS). There was no difference in the distribution of clinicopathological parameters between the discovery and validation cohorts or the METABRIC series of patients   was validated using western blotting in human embryonic kidney (HEK) 293 T over expression lysate  and MDA-MB-175 , T47D and MCF7   breast cn = 1110) were arrayed as previously described using a single 0.6-mm core sampled from the periphery of each invasive tumour [n = 1554) were similarly arrayed using a tissue microarray (TMA) GrandMaster (3D Histech). Immunohistochemical (IHC) staining was performed on 4-μm TMA sections using the Novolink polymer detection system . Briefly, tissue slides were deparaffinised with xylene and rehydrated through three changes of alcohol. Heat-induced antigen epitope retrieval was performed in citrate buffer (pH 6.0) for 20 min using a microwave oven (Whirpool JT359 Jet Chef 1000 W). Endogenous peroxidase activity was blocked by peroxidase block for 5 min. Slides were washed with Tris-buffered saline , followed by application of protein block for 5 min. Following another TBS wash, sections were incubated with the primary SLC7A5 antibody diluted at 1:50 in Leica antibody diluent  overnight at 4 °C. Slides were washed with TBS followed by incubation with post primary block for 30 min followed by a TBS wash. Novolink polymer was applied for 30 min: 3,3′-diaminobenzidine (DAB) chromogen was applied for 5 min. Slides were counterstained with Novolink haematoxylin for 6 min, dehydrated and coverslipped.The discovery set , at × 20 magnification. Evaluation of staining for SLC7A5 was based on a semi-quantitative assessment of digital images of the cores using a modified histochemical score (H-score) which includes an assessment of both the intensity and the percentage of stained cells . StaininImmunhistochemical staining and dichotomisation of the other biomarkers included in this study were as per previous publications , 22–30. t test  or Mann-Whitney test . Survival curves were analysed by the Kaplan-Meier and log rank test. Cox’s proportional hazard method was performed for multivariate analysis to identify the independent prognostic/predictive factors. The p values were adjusted for multiple testing using the Bonferroni correction. A p value ˂0.05 was considered significant. The study endpoints were 10-year BCSS or distant metastasis-free survival (DMFS).Statistical analysis was performed using SPSS 22.0 statistical software . Spearman’s correlation coefficient was calcualted to examine the association between continuous variables. The chi-square test was performed to analyse relationships between categorical variables. For the continuous variables, differences between three or more groups were assessed using one-way analysis of variance (ANOVA) with the post-hoc Tukey multiple comparison test  or Kruskal-Wallis test . Differences between two groups were assessed using the SLC7A5 was amplified in 0.3% and 0.6% of BC cases in the METABRIC and TCGA datasets, respectively, while deletion  was detected in 56% and 68% of cases in the same cohorts respectively. Point mutations in SLC7A5 were extremely rare, where TCGA data reported just one case with a missense mutation ; all the annotated genes, which were located on 16q [SLC7A5 and assess whether these aberrations were locus-specific or involved large chromosomal segments. There was significant positive correlation between SLC7A5 deletion and the deletion of all genes  showed significant association with the amplified SLC7A5  in the afore-mentioned datasets together.d on 16q  were selhttps://medicine.yale.edu/lab/rimm/research/software.aspx; Yale University). Positive SLC7A5 expression (>15 H-score) was observed in 191/1110 (17%) and 268/1554 (17%) of cases in the discovery and validation sets, respectively, while high SLC7A5 mRNA expression (log2 intensity >8) was observed in 1019/1923 (53%) of the METABRIC breast cancer cases. A total of 49/1980 (2.4%) of cases had a copy number (CN) gain of SLC7A5 and 530/1980 (26.7%) a CN loss. Significant association was observed between SLC7A5 copy number variation (CNV) and SLC7A5 mRNA expression . Regarding BC metastatic sites, high SLC7A5 protein levels were associated with the development of distant metastases (DM) in the brain (p < 0.001) and lung (p = 0.04), while there was no association with development of DM in the bone or liver.Table p < 0.001), higher grade , positive nodal metastasis . Both SLC7A5 mRNA and SLC7A5 protein were associated with medullary-like tumours. Where data were available, these associations were confirmed using the Breast Cancer Gene-Expression Miner v4.0 . There was positive association between SLC7A5 copy number gain and MYC gain . Similarly, within the METABRIC integrative clusters, high SLC7A5 mRNA expression was associated with clusters 5 (ERBB2 amplified), 9  and 10  ) subtypes , high exSLC7A5  and the positive relationship between MYC, HIF2A and SLC7A5 was only observed in luminal B tumours .Correlation between SLC7A5 mRNA and associated genes was investigated in the METABRIC dataset (Table p < 0.02).High SLC7A5 mRNA expression was specifically associated with the enzymes involved with conversion of glutamine (Gln) to proline, where PYCR1 and ALDH18A1 showed a positive relationship with SLC7A5 in almost all subtypes (p < 0.02).The majority of glutamine transporters were significantly associated with SLC7A5 expression primarily in triple negative tumours and to a lesser extent luminal and HER2+ subtypes. SLC7A5 was significantly correlated with SLC1A5 in all subtypes .p < 0.001, Table p < 0.001), while the low expression of SLC7A5 was associated with high levels of p-mTORC1 (p < 0.001)   expression were associated with poor BCSS in the discovery and validation sets  Fig.  and protets Fig. . This asp = 0.007, Fig. p = 0.03, Fig. p = 0.006, Additional file p = 0.02, Table While SLC7A5 mRNA expression was not predictive of BCSS in any specific molecular class Fig. , high exp < 0.001; Additional file p = 0.03, Additional file . The relationship between high SLC7A5 mRNA expression and poor patient outcome in ER+ disease, but not ER- disease, was confirmed using Breast Cancer Gene-Expression Miner  , which was located in 16q22.1, was also implicated. It is widely known that most lobular tumours harbour loss of heterozygosity (LOH) at chromosome 16 and are missing the wild type CDH1 allele [SLC7A5 loss were associated with invasive lobular histology. In addition, SLC7A5 protein expression in lobular carcinoma has a relatively lower mean rank value compared to the other histological subtypes, confirming that deletions involve large segments of q16, which can reflect the BC phenotype.1 allele . In thisSLC7A5 is widely expressed in many human cancers and various cancer cell lines . The curWith respect to the breast cancer ER+ subtypes, SLC7A5 expression was lower in ER+ tumours that have low proliferation  compared with the highly proliferative ER+  malignancies, and it was primarily associated with poor patient outcome and shorter DMFS in the latter class only. This is most likely due to their heavier energy and nutrient requirements for cell survival, proliferation and metastasis. This was anticipated, as it has been shown that over expression of SLC7A5 is actively involved in the proliferation of vascular smooth muscle cells  and it iSLC7A5 mRNA and protein was also highly expressed in TNBC and HER2+ BC, in concordance with Furuya et al. . HoweverPrevious studies have shown regulation of SLC7A5 by other proteins including the tumour oncogene Myc, which induces SLC7A5 , 5. In tThis study further investigated the association of SLC7A5 expression with other glutamine transporters, in which some variability in the expression of amino acid transporters across molecular subtypes was observed. For example, the TN subtype was the main class associated with the transporters required for glutamine influx, perhaps because it depends on delivery of glutamine instead of synthesis. In contrast, positive correlation between SLC7A5 and the glutamine synthase enzyme GLUL was observed in luminal A tumours, suggesting that this subtype might rely on glutamine neosynthesis rather than uptake. SLC1A5 functionally couples with SLC7A5 to allow the cellular influx and efflux of glutamine, as SLC1A5 mediates uptake of glutamine, while SLC7A5 uses intracellular glutamine concentrations to adjust the essential amino acid cytoplasmic pool for metabolic demands and signalling to mTORC1 . Here wePrevious studies have raised awareness and revealed the importance of the proline-glutamine (Pro-Gln) regulatory axis in BC. SLC7A5 appears to have a pivotal role in this regulatory axis, as its expression was highly associated with the enzymes that mediate glutamate degradation to form the amino acid proline, which has been shown to play a role in assisting tumour growth by different mechanisms ..Blocking of SLC7A5 using its inhibitor, 2-aminobicyclo--heptane-2-carboxylic acid (BCH), efficiently decreased colony formation of MDA-MB-231 TNBC cells . Even thThis study revealed and confirmed that the glutamine transporter SLC7A5 is associated with poor prognostic characteristics and poor survival outcome. Over expression of SLC7A5 appears to play a role in the proliferation and progression of the aggressive ER+ subtype of breast cancer, thus it could act as a potential therapeutic target. Functional assessment is necessary to reveal the specific role played by this amino acid transporter in the highly proliferative subclass and HER2+ BC.Additional file 1:Table S1. Clinicopathological parameters of the METABRIC and Nottingham discovery and validation series. (DOCX 16 kb)Additional file 2:Figure S4. SLC7A5 mRNA expression, in the TCGA data, and its association with copy number alteration (A), staging system (B), ER status (C), PR status (D) and HER2 (E) status. (PPTX 176 kb)Additional file 3:Table S2. List of genes with copy number loss that was significantly associated with SLC7A5 deletion in the METABRIC and TCGA data. (DOCX 16 kb)Additional file 4:Figure S1. SLC7A5 gene expression and its association, using Breast Cancer Gene-Expression Miner v4.0, with: tumour grade (A), NPI (B), ER status (C), PR status (D), HER2 status (E), Triple Negative status (F)and PAM50 subtypes (G). (PPTX 117 kb)Additional file 5:Table S3. SLC7A5 mRNA and patient outcome. (DOCX 14 kb)Additional file 6:Figure S2. SLC7A5 vs DMFS in all cases in the discovery set (A), all cases in the validation set (B), ER + low proliferation tumours in the combined discovery and validation set cases (C), ER + high proliferation tumours in the combined discovery and validation set cases (D), triple negative tumours in the combined discovery and validation set cases (E) and HER2+ tumours in the combined discovery and validation set (F). (PPTX 407 kb)Additional file 7:Figure S3. SLC7A5 mRNA and breast cancer patient outcome using Breast Cancer Gene-Expression Miner in unselected cases (K), ER+ disease (L) and ER- disease (M). (PPTX 80 kb)


Cip1/CDKN1A (p21) is an important and universal Cdk-interacting protein. Recently, we have reported that p21 is involved in the regulation of the mitotic kinase Cdk1/cyclin B1 and critical for successful mitosis and cytokinesis. In the present work we show that S130 of p21 is phosphorylated by Cdk1/cyclin B1 during mitosis, which reduces p21′s stability and binding affinity to Cdk1/cyclin B1. Interfering with this phosphorylation results in extended mitotic duration and defective chromosome segregation, indicating that this regulation ensures proper mitotic progression. Given that p53, the major transcriptional activator of p21, is the most frequently mutated gene in human cancer and that deregulated Cdk1 associates with the development of different types of cancer, this work provides new insight into the understanding of how deregulated p21 contributes to chromosomal instability and oncogenesis.The multifunctional protein p21 Cip1/CDKN1A (p21) is an intrinsically unstructured protein [in vitro by non-receptor tyrosine kinases, which influences its Cdk/cyclin binding and stability [Cyclin-dependent kinases (Cdk), a family of proline-directed serine/threonine kinases, regulate the cell cycle . Cdk1 is protein , which b protein –7. It is protein , 8. p21  protein , 8. The  protein . It has  protein , whereas protein . Phospho protein , 12. In  protein . It is p protein , Cdk2/cy protein  and Cdk6 protein . Moreovetability .We have recently shown that p21 is involved in the regulation of the mitotic progression, whereby its loss prolongs mitotic duration resulting in defects in chromosome segregation and cytokinesis . Moreovest panel, lanes 2, 4, 6, 8 and 10), p21 is clearly present accompanied by an appearance of several additional slowly migrating bands indicative of being post-modified . To address if these slowly migrating bands are induced by phosphorylation events, we treated cells additionally with λ-phosphatase (λ-PPase) and the band/bands disappeared , suggesting that the slowly migrating band/bands of p21 are indeed induced by phosphorylation events. Moreover, when cells were treated with the proteasome inhibitor MG132, p21 was stabilized in prometaphase, both fast and slowly migrating bands . Cytoplasmic-nuclear separation of HeLa and MCF7 cells revealed that phosphorylated p21 is more abundant in the cytoplasm than in the nucleus of prometaphase cells . To follow the p21 phosphorylation status over the cell cycle, thymidine release kinetics was performed. When the cyclin B1 level peaked at 8 h post release, p21 was phosphorylated, which disappeared with the degradation of cyclin B1 , phosphorylated p21 almost disappeared in cells deficient of cyclin B1 , whereas phosphorylated p21 receded only slightly in cells treated with siRNA targeting Cdk2 , indicating that Cdk1 could be the main kinase for phosphorylating mitotic p21. The signals of phospho-histone H3 (p-HH3) were slightly reduced yet still abundant in cells knocked down of cyclin B1 or Cdk2, indicating that most of these cells with left cyclin B1 or Cdk2 were still able to enter mitosis . This was further supported by cell cycle analysis showing a reduction of 6-7% in the G2/M population in cyclin B1 or Cdk2 downregulated cells relative to control siRNA treated cells , further underscoring that phosphorylated p21 is associated with the activity of Cdk1. Moreover, the phosphorylation signal of p21 was abolished in mitotic HeLa cells released into medium containing RO-3306, a specific and efficient Cdk1 inhibitor [rd panel, lane 4). To halt RO-3306 treated cells in mitosis, mitotic cells were treated additionally with the proteasome inhibitor MG132. In this case, the phosphorylation signal of p21 was definitely decreased , whereas the levels of the mitotic markers Plk1 and cyclin B1 were hardly changed , suggesting that the reduction of the phosphorylation signal is ascribed to the reduced Cdk1 kinase activity. Of note, RO-3306 treated mitotic cells showed hardly the p-HH3 (S10) signal , which is in line with the previous observation [To define the mitotic kinase responsible for this phosphorylation, HeLa cells were treated with siRNA targeting cyclin B1, the regulatory subunit of Cdk1, or siRNA against Cdk2, and synchronized to prometaphase for Western blot analysis. Compared to control siRNA treated cells , the levels of precipitated wild type FLAG-p21 and its mutant FLAG-p21S130A  [in vitro [To reveal if the mutants affect the kinase activity of Cdk1, an n (MCAK) , in the in vitro , 25, a drd panel, lane 4), its protein stability was also affected by the pan-caspase inhibitor Z-VAD , whereas calpain cleavage inhibition impacted scarcely its protein turnover . Of note, p53 is increased after the inhibition of the caspase and the proteasome pathway, indicating that the accumulated p21 could result from both the stabilization of the protein and increased transcription via the p53 signaling. These data support the common observation that the degradation of p21 is majorly dependent on the proteasome pathway.The protein degradation and stability of p21 is well studied and it is known that the degradation is often triggered by its site-specific phosphorylation and different binding partners , 26, 27.nd panel). Interestingly, compared to HCT116 cells with p21, cyclin B1 is more stable in HCT116 cells without p21 , which was also observed in HeLa cells treated with siRNA against p21 , suggesting that the p21 status may influence directly the stability of cyclin B1 or indirectly via interfering with mitotic progression. Intriguingly, while non-phosphorylated p21 retained till 90 min in cells released from nocodazole treatment and incubated with fresh medium containing CHX , the phosphorylated form was reduced at 45 min  and undetectable at 75 min , when cyclin B1, the regulatory subunit of Cdk1, started to be degraded . The data indicate that phosphorylation of p21 alters its turnover during mitosis.To study precisely the turnover of p21 during mitosis, HCT116 cells with and without p21 were synchronized to prometaphase and released into medium containing cycloheximide (CHX), a protein synthesis inhibitor, and harvested for Western blot analysis. p21 is a short-lived protein during mitosis , non-phosphorylatable FLAG-p21S130A was more stable , whereas phosphomimetic FLAG-p21S130D was less stable . Moreover, FLAG-p21S130D degraded faster than wild type FLAG-p21 and FLAG-p21S130A during the release course and it was almost disappeared at 2 h in HeLa cells . Further quantification analysis clearly indicates that FLAG-p21S130D is the most unstable form at 4 h  compared to nocodazole treated cells . To underscore these results, we transfected HCT116 p21−/− cells with wild type FLAG-p21 or its mutants and the CHX kinetics was carried out. In line with the observations from HeLa cells, FLAG-p21S130D was less stable compared to wild type FLAG-p21 and its non-phosphorylatable mutant FLAG-p21S130A . Notably, relative to wild type FLAG-p21 , both mutants were hardly affected by the treatment with the proteasome inhibitor MG132 , suggesting strongly that S130 is involved in the proteasome dependent degradation of mitotic p21. Taken together, the data underline the notion that phosphorylation of S130 by Cdk1 renders p21 unstable.To explore if the phosphorylation of p21 by Cdk1 impacts its stability, HeLa cells were transfected with wild type FLAG-p21 or its mutants, synchronized and released into CHX-containing medium Figure . Westernh Figure . InteresTo gain insight into the binding affinity of p21 and its mutants to Cdk1/cyclin B1, pulldown assays were performed using different GST-p21 recombinant proteins incubated with cellular extracts from mitotic HCT116 p21−/− cells. Compared to wild type p21 or the phospho-mimetic mutant p21S130D Figure , lane 4.To analyze if phosphorylation changes p21′s subcellular localization, which has been reported for T145, S153 or T57/S130 , HeLa ceTo examine the effect of the phosphorylation at S130 on mitotic progression, time-lapse imaging was performed in living HCT116 p21−/− cells stably expressing fluorescent histones (H2B-tdTomato in red) [nd panel, DNA staining), which were partially alleviated by wild type FLAG-p21 . While p21S130A was able to mitigate these failures in a marginal extent, p21S130D expressing cells exhibited defects comparable to p21-deficient cells , indicative of the importance of subsequent dephosphorylation of p21 during mitosis.We were then interested in the mitotic phenotype of cells overexpressing wild type FLAG-p21 or its mutants. Transfected cells Figure  were staIn the present work we show that S130 of p21 is mainly phosphorylated by Cdk1/cyclin B1 during mitosis, which attenuates its stability and reduces its binding to Cdk1/cyclin B1. This regulation is of importance for a proper mitotic progression evidenced by extended prometaphase and metaphase in cells expressing the non-phosphorylatable mutant FLAG-p21S130A or the phosphomimetic FLAG-p21S130D, respectively. Interfering with this phosphorylation leads further to defective segregation and delayed mitotic exit indicating that a precise control of p21 is important for a successful mitotic progression.in vitro as well as ex vivo. In support of our notion, mass spectrometry analysis revealed that S130 in p21 is indeed phosphorylated in mitotic HeLa cells [Cdk1 becomes activated for the mitotic entry, its activity increases at prophase, reaches its peak shortly after nuclear envelope breakdown, remains almost constant in metaphase and is immediately reduced at the onset of anaphase by degradation of its regulatory subunit cyclin B1 –37. It hLa cells , 40, altLa cells , Cdk6/cyLa cells  and ERK2La cells . These dLa cells . In thisWe showed previously that p21 deficiency causes segregation defects which could be rescued by inhibiting Cdk1 activity with the Cdk1 inhibitor RO-3306 or by adding back wild type p21  and chroTaken together, we show here that p21 is phosphorylated at S130 by Cdk1/cyclin B1 during mitosis influencing its stability/degradation and ensuring a successful mitotic progression. Given that p53, the major transcriptional activator of p21, is the most frequently mutated gene in human cancer  and that® HD in a ratio 1:3 .HCT116 p21+/+, HCT116 p21−/−, HeLa, MDA-MB-231, MCF7 and U2OS cells were cultured as instructed. Stable HeLa 776-6, expressing shRNA targeting cyclin B1, were generated as described , 19. To BamH I/Not I sites of the pGEX-5×3 vector , into the EcoR I/BamH I sites (5′-AATTGAATTCTATGTCAGAACCGGCTGGGGATGTC-3′ and 5′-GATAGGATCCTTAGGGCTTCCTCTTGGAGAAGATCAG-3′) of the 3xFlag-CMV 7.1 vector (Sigma), and into the BamH I/Hind III sites of the pBI-CMV3 vector (Clontech), described in Kreis et. al [Full-length human p21 cDNA (pOTB7-vector) was obtained from ImaGenes GmbH (Berlin). By use of primers (5′-GATAGGATCCAGATGTCAGAACCGGCTGGGGATGTC-3′ and 5′-GACAGCGGCCGCTTAGGGCTTCCTCTTGGAGAAGATCAG-3′), the cDNA was amplified by PCR and subcloned into the s et. al . Point ms et. al . Recombi® M2 . Mouse monoclonal antibody against calnexin was from BD Biosciences and against lamin B1 from MBL . To generate a polyclonal antibody against p21 phosphorylated on S130, rabbits were immunized using the peptide GEQAEGpSPGGPGD (p = phospho), and antibodies were affinity purified . Kinase assay in vitro was performed as described [ex vivo was performed with HeLa cells transfected with FLAG-p21 or FLAG-p21S130A. Cells were synchronized with nocodazole and immunoprecipitated with FLAG® M2 beads (Sigma). The beads were subjected with radioactive ATP  without adding additional kinase and incubated for 30 min at 37°C with or without inhibitors.Cells were lysed in RIPA buffer and Western blot analysis was performed, as described . Cytoplaescribed . Cdk1/cy®, Cambridge, UK), monoclonal mouse antibody against FITC-conjugated α-tubulin (Sigma-Aldrich) and human immune serum against centromere . Cy3 and Cy5-conjugated secondary antibodies were obtained from Jackson Immunoresearch . DNA was stained using DAPI . Slides were examined using an AxioObserver.Z1 microscope with a HCX PL APO CS 63.0×1.4 oil UV objective  and images were taken using a confocal laser scanning microscope .For indirect immunofluorescence staining, cells were seeded on Nunc™ Lab-Tek™ II CC2™ chamber slides from Thermo Fisher Scientific (Schwerte). Briefly, cells were fixed for 15 min with 4% PFA and permeabilized for 5 min with 0.1% Triton™ X-100 at room temperature. The following primary antibodies were used for staining: polyclonal rabbit antibody against pericentrin (abcamt-test (two tailed and paired or homoscedastic) was used to evaluate the significance of difference between the different conditions. Difference was considered as statistically significant when p < 0.05.Student's