Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation.Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen.  The pathogenic fungus Candida albicans uses a variant of histone H2A and regulated depletion of the centromeric histone CENP-A to control the stability of its chromosomes. C. albicans. This organism is the most common agent of fungal disease in humans but primarily exists as a component of mammalian gut, skin, and genitourinary microbiota  mutant, strains that lack H2A.2, Bub1, or Sgo1 exhibit highly elevated rates of marker loss that correlate with their colony sectoring phenotypes , in which 1 copy of Chromosome 5 can be visualized as a fluorescent dot, produced by the binding of a Tetracycline Repressor Protein-Yellow Fluorescent Protein (TetR-YFP) fusion protein to >100 tandem copies of the tet operator  reduced H2A.2 levels result in chromosome instability in diploid cells , (2) C. ome loss , and (3)ome loss , we posiome loss . Viabiliome loss ; by compome loss . The decome loss . By contome loss . These rectoring  and cellectoring  phenotypARG4/Δ diploid strains to low-dose (1 μg/ml) fluconazole, a drug that promotes aneuploidy in this species  strain that lacks noncanonical H2A.1 maintained a low rate of <0.1% despite exposure to fluconazole (p < 0.01 compared to WT). Quantitative polymerase chain reaction (qPCR) analysis of selected arginine auxotrophs and prototrophs confirmed that auxotrophy usually results from loss of Chromosome 7 , and h2a.2 complemented with either H2A.2WT or H2A.2S121A, and the strains were spotted onto YEPD versus YEPD plus 10 μg/ml fluconazole. As shown in bub1, sgo1, h2a.2, and h2a.2+H2A.2S121A) exhibited enhanced survival compared to strains with lower chromosome instability . Of note, because WT exhibits very low tolerance to fluconazole in this assay, it was not possible to determine whether tolerance is further diminished in the  strain that lacks H2A.1. Overall, these results demonstrate that chromosome instability can provide a selective advantage under certain kinds of stress.Because aneuploidy is frequently associated with resistance to fluconazole , the mosC. albicans; however, it does not explain why chromosomes are destabilized in certain ploidy- and stress-dependent contexts. In other words, why are chromosome gain and loss events more frequent in tetraploid cells compared to diploid cells .Extensive biochemical and genetic data support a close interaction between H2A and Cse4/CENP-A within centromeric nucleosomes in revisiae –30. Giverevisiae , we hypoC. albicans. A newly evolved H2A lacking the Bub1 phosphorylation site promotes chromosome instability under standard conditions by generating nucleosomes that are ineffective at recruiting shugoshin. We hypothesize that the presence of variant H2A.1 in centromeric nucleosomes sensitizes cells to changes in other centromere and kinetochore components. Thus, the graded depletion of Cse4/CENP-A from centromeres in cells with increased ploidy (moderate effect) or in stressful environments (strong effect) produces further increases in chromatin instability and aneuploidy.These data indicate that at least 2 pathways promote chromosome instability in C. albicans cell biology, including reduction of chromosome number after mating and adaptation to drug challenges. Although substantial evidence supports the ability of environmental perturbations to trigger chromosome instability in this organism , , and  diploid strains using plasmid pJD1 , , and  were generated in several steps. First, an allele of the Mating Type-Like Locus .RNA was extracted from log or stationary phase cultures grown in YEPD or prespo medium and extracted using a hot acid phenol method, as previously described by Miller and colleagues . RNA wasARG4 allele (on Chromosome 7) was determined by measuring the rate of loss of the ability of an ARG4/argΔ heterozygous strain to grow on SD(-Arg) dropout medium. The ARG4/argΔ heterozygous strains were created by integration of 1 copy of ARG4 from C. dubliniensis to the argΔ/argΔ locus of WT , , , bub1, and sgo1 strains. C.d.ARG4 gene addback fragments targeted to the argΔ locus were generated by fusion PCR using primers SNO143–147 and pSN69 (C.d.ARG4) template, and correct insertion was verified by colony PCR using primers SNO143 plus SNO263 (5ʹ) and SNO147 plus SNO264 (3ʹ). The resultant Arg+ strains were streaked onto prespo agar and propagated at 37 °C for 4 days. Cells were then resuspended in sterile H2O and plated for single colonies (approximately 200 per plate) on YEPD agar, with incubation at 30 °C for 2 days. Colonies were replica-plated onto SD-Arg agar and monitored for growth; colonies unable to grow on SD-Arg agar were scored as a positive event for chromosome loss. To elicit higher rates of chromosome loss in diploid strains, the ARG4/arg4Δ heterozygous WT, , , and  strains were also propagated in YEPD liquid medium containing 1 μg/mL fluconazole  for 3 days at 37 °C, prior to plating as above. Loss of Chromosome 7 in arginine auxotrophs was confirmed using quantitative PCR with primers specific to the left and right arms of Chromosome 1 and Chromosome 7 , h2a.2 plus H2A.2WT, and h2a.2 plus H2A.2S121A were diluted from overnight cultures and grown to log phase (OD approximately 1.0) in a 30 °C shaker at 200 rpm in YEPD. Cells were then serially diluted onto YEPD, YEPD plus 50 μM nocodazole , or 10 μg/ml fluconazole , and grown for 24 hours at 30 °C. Plates were imaged using a Canon EOS Revel T5i camera, and image manipulations were performed using the ImageJ software (NIH).WT, , , , p-values were generated by performing one-way ANOVA with Dunnett’s multiple corrections. Fisher exact test was used to evaluate the significance of the “two dots” comparison. p < 0.05 was used as a cutoff for significance.Except for ChIP-seq analysis described below, ChIP was conducted as previously described by Lohse and colleagues  with min600 = 0.1 in YEPD, prespo media, or YEPD plus fluconazole (10μg/ml). Samples were propagated to OD600 = 1.0 prior to harvesting.Immunoprecipitations were conducted with 10 μg anti-FLAG M2 antibody , 10 μg anti-histone H3 antibody , or 4 μg of anti-Cse4 (Laura Burrack). For all ChIP-seq experiments, cells were propagated in YEPD overnight and seeded the next day at ODSequencing library preparation was conducted as previously described by Dumesic and colleagues . LibraryC. albicans genome using Bowtie [1] with the following settings: bowtie -p[43] -v2 -M1 --best --un {1}_multi_un.fastq --max {1}_multi.fastq {2} -q [51] --sam {1}_multi.samChIP-seq data from a HiSeq 4000 were aligned to the Conversion to BAM files, indexing, and sorting were performed with SAMTools [2].Bedgraphs were created using BEDTools [3]. Bedgraph files were first expanded to include each position in the genome and then smoothed using a rolling mean of 500 base pairs using the Python package pandas (rolling.mean function). Finally, each position in the bedgraph was normalized to the same position in the smoothed WCE bedgraph file to visualize fold enrichment.C. albicans genome was fragmented into 5 kb tiles, and the number of reads aligning within a tile was determined using pysam (https://github.com/pysam-developers/pysam). The RPKM of each tile was calculated by normalizing to the length of the tile in kb and the total number of aligned reads in each sample (readsT) as follows:Each chromosome in the The enrichment (E) of read density in each tile in the ChIP samples over WCE was calculated by dividing the read density from the ChIP sample by the read density from the WCE for each tile:p-Values were determined by Mann-Whitney U test using the Python package scipy. Raw sequence data and enrichment values for each genome tile are available from GEO (accession number GSE122037).S1 FigS/T121 is denoted by an asterisk. S/T121, serine or threonine at position 121.(TIF)Click here for additional data file.S2 Figh2a.1 and h2b.1, which contain homozygous deletions of H2A.1 and H2B.1, respectively, exhibit slow growth on YEPD, whereas the  strain, which contains extra copies of H2A.2 in place of H2A.1, grows normally. (B) Genomic copy number of H2A and H2B genes in h2A.1, h2B.1, h2A.2, and h2B.2 homozygous deletion mutants. Gene abundance was determined by qPCR and normalized to ACT1 (located on Chromosome 5). (C) Cartoon of the genotype of h2a.1, with the extra copy of Chromosome 3 highlighted in red. qPCR, quantitative PCR; r.u., relative units; YEPD, yeast extract peptone dextrose.(A) Colony morphology. Strains were propagated on solid Spider or YEPD medium for 5 days at 30 °C. Please note that (TIF)Click here for additional data file.S3 FigH2A ORF identity in WT, , and  diploid strains. (B) Verification of the genomic copy number of H2A.1 and H2A.2 ORFs in these strains, measured by qPCR and normalized to ACT1. (C) Verification of H2A.1 and H2A.2 mRNA expression in log-phase strains, measured by RT-qPCR and normalized to ACT1. ORF, open reading frame; qPCR, quantitative PCR; RT-qPCR, reverse-transcription quantitative PCR; WT, wild type.(A) Cartoon of (TIF)Click here for additional data file.S4 FigH2A.2 S121 in DNA damage repair. WT, wild type.These data support an additional role for (TIF)Click here for additional data file.S5 FigC. albicans diploid strains maintain 2 sets of 8 chromosomes , whereas tetraploid strains maintain 4 sets. (B) DNA content of WT diploid and tetraploid strains, as visualized by propidium iodide staining followed by flow cytometry. (C) Colony morphology of WT, WT tetraploid , and tetraploids containing only H2A.1 or H2A.2. Strains were propagated for 2 days on YEPD at 30 °C. We note that the H2A.1-containing tetraploid has a more heterogeneous cell size when examined by flow cytometry, and 100% of colonies are highly sectored, consistent with a highly unstable genome. WT, wild type; YEPD, yeast extract peptone dextrose.(A) Schematic of WT diploid and WT tetraploid genome content. (TIF)Click here for additional data file.S6 FigShown are plots of bedgraphs of normalized read densities Click here for additional data file.S7 FigC. albicans genome for replicate experiments performed for the indicated genotypes  and the indicated conditions. ChIP-seq, Chromatin-immunprecipitation sequencing; Fluc, fluconazole treatment; PreSpo, presporulation medium treatment; WCE, whole-cell extract.Shown are the normalized enrichments (log2) relative to the WCE sample for 5-kb tiles spanning the (TIF)Click here for additional data file.S1 TableC. albicans chromosome were used to quantify the chromosome abundance in the indicated diploid strains. qPCR, quantitative PCR; WT, wild type.qPCR and primers specific to each (XLSX)Click here for additional data file.S2 TableC. albicans Chromosomes 1 and 7 were used to quantify chromosome abundance in the indicated diploid strains. Loss of Chromosome 7 was inferred from depletion relative to the Chromosome 1 reference. qPCR, quantitative PCR.qPCR and primers specific to (XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Objective: To investigate Livin-mediated regulation of H2A.XY142 phosphorylation via a novel kinase activity and its effect on autophagy in colon cancer cells.Methods: The interaction between Livin and H2A.X was tested by immunoprecipitation. H2A.X–/– HCT116 cells were transfected with human influenza hemagglutinin (HA)-tagged WT or Y142F phospho-dead mutantH2A.X plasmids. GST-tagged recombinant Livin protein was used to perform in vitro pull-down experiment and kinase assay. H2A.X–/–Livin+/+ SW480 cells were co-transfected with H2A.XWT/H2A.XY142F plasmid and LC3 EGFP-tagged plasmid to explore whether H2A.XY142F was involved in Livin-mediated autophagy induced by starvation in colon cancer cells.Results: Co-immunoprecipitation studies confirmed that Livin interacted with H2A.X and that it was phosphorylation dependent. In vitro kinase assay confirmed that Livin could phosphorylate H2A.X. Knockdown of Livin (Livin–/–) in SW480 cells or HCT116 cells canceled the starvation-induced autophagy in colon cancer cells; H2A.X–/–Livin+/+ SW480 cells transfected with H2A.XWT activated autophagy induced by starvation while cells transfected with H2A.XY142F had no significant difference; Livin-H2A.XY142F axis activated autophagy in colon cancer cells through transcriptionally regulating ATG5 and ATG7.Conclusion: Livin promotes autophagy in colon cancer cells via regulating the phosphorylation of H2A.XY142. The nucleosome is a structure composed of a about 200 bp DNA molecule coiling outside and a histone octamer in the core, which is a basic structural unit of the chromosome. The histone octamer core consists of four histones identified as H2A, H2B, H3, and H49 . MultiplInhibition of apoptosis is considered as one of the cardinal requirements of cancer cells to survive and propagate . TherefoY142 phosphorylation.Inhibition of Livin promoted the sensitivity of colon cancer cells to 5-fluorouracil (5-FU) via modulating the crosstalk between cellular apoptosis and autophagy . One of HT-29 cells, SW480 cells and HCT116 cells were purchased from ATCC . Cells were cultured in McCoy's 5A Medium . Transfection of expression plasmids or siRNAs were performed when cells were 40–60% confluent.Cells were harvested and 1 mL Trizol  was added to extract cellular RNA. Concentration of RNAs were measured by the optical density (OD) value using spectrophotometer . Reverse transcription of RNA (system: 10 μL) was performed to synthetize cDNA , which was used to template the real-time quantitative polymerase chain reaction (RT-qPCR) using the following settings: 95°C 2 min;  for 40 rounds.One hundred microliters RIPA lysis buffer  containing 1 μL PMSF  was used to extract the cellular proteins. Concentrations of proteins were measured by BCA kit . Antibodies against Livin , H2A.X , H2A.XY142F , GAPDH , ATG5 , ATG13 , ATG14 , LC3-I/II  and β-actin  were used at 1:1000 dilution.Y142F/ Livin-H2A.XY142F for 8 h at 37°C.The dual-luciferase reporter gene assay kit  was used according to the instruction manual. Colon cancer cells were cultured in 24-well plate and then co-transfected with pGL3-ATG5/pGL3-ATG7 plasmid and Livin-H2A.XCells were harvested 48 h after transfection and lysed as above. Part of lysate was kept as input. One microgram of the corresponding antibodies were added to the rest of cell lysate, and incubate at 4°C overnight. 15 μL 2 × SDS loading buffer was added to immunoprecipitated beads, boiled for 5 min, and then resolved by SDS-PAGE and processed for western blotting analysis.3H]-tyrosine  was cultured for 48 h, then the cells are washed with 2 mL of ice-cold Hank's balanced salt solution  twice, followed by culturing the cells for 2 h in DMEM  to degrade short-lived proteins. Cells were washed twice with 2 mL of ice-cold Hank's balanced salt solution  and measure the culture medium and intracellular L-- tyrosine using a liquid scintillation counter. Long-life protein degradation rate = L--tyrosine content in culture medium/.The whole culture medium containing 1.0 μCi/mL L-ATP. Samples were boiled and then resolved by SDS-PAGE and visualized by autoradiography using Phosphor-Image Screens .Recombinant wild-type or Y142F mutant His-tagged H2A.X proteins (400 ng) were incubated with 100 ng of GST-Livin at 30°C for 30 min in 1× kinase buffer containing 10 μmol/L of unlabeled ATP or 10 μCi[γ-Post-autoradiography blots were coomassie stained to confirm equivalent amounts of protein loading across lanes.P < 0.05 was considered as statistically significant.Statistical analysis was performed by GraphPad Prism 6.0 . Data was represented as mean ± SD. Two-way ANOVA was used to test differences between groups. Y142 (H2A.XY142ph) in three different types of colon cancer cells. We found that the levels of Livin in HT-29 cells and SW480 cells were obviously higher compared to HCT116 cells   . In vitrth H2A.X .Y142. In vitro kinase assay showed that recombinant Livin could phosphorylate wild type H2AX but not Y142F phospho-dead mutant H2AX  in SW480 cells or HCT116 cells canceled all these changes induced by starvation  was performed to observe the number of cytoplasmic autophagosomes. The autophagy was induced by starvation when culturing in Earle's balanced salt solution (EBSS). Comparing to the cells culturing in complete medium, the starved group showed a significant increase of autophagosomes in both SW480 cells and HCT116 cells . The proarvation . These rLivin−/− HCT116 cells  after starvation. We found a markedly increase in H2A.XY142ph protein keeping pace with the activated autophagy level, as assessed by increased number of EGFP-LC3 puncta (Y142ph expression downregulated through expressing H2A.XY142F and the autophagy activity decreased (Y142 phosphorylation.Then we tested the kinase activity of Livin for H2A.X and 1 h . This da3 puncta . Howeverecreased . Taking Y142ph axis on autophagy in colon cancer cells was through transcriptionally regulating ATGs. Firstly, the mRNA levels of ATG5 and ATG7 were markedly up-regulated in WT SW480 cells after starvation (Livin−/− SW480 cells after starvation. When the Livin−/− SW480 cells were transfected with transfected with the phospho-dead H2A.XY142F or H2A.XY142A mutants, the mRNA levels of ATG5 and ATG7 were markedly up-regulated after starvation (Y142ph axis could significantly increase both mRNA and protein expressions of ATGs under starved stimulation.Further investigation was done to test whether the activated effect of Livin-H2A.Xarvation , which darvation . The proarvation . This reY142ph axis could directly target ATG5 and ATG7 in colon cancer cells under starvation, while Livin-H2A.XY142F axis had no combination with ATG5/ATG7 in colon cancer cells under starvation (Y142ph axis, while Livin-H2A.XY142F axis canceled the changes (Y142ph-induced transcriptional activation of ATGs.Then the pGL3-ATG5 and pGL3-ATG7 plasmid were constructed for the luciferase assay. The result showed that the Livin-H2A.Xarvation . Moreove changes . These dIn vitro experiments confirmed that after knocking out the Livin gene of lung cancer SPC-A1 cell line, caspase-3 protein level was down-regulated, but there was no significant change in mRNA level (The anti-apoptotic mechanism of the AlPs family was mainly caused by inhibition of caspase protease and its cascade activation reaction. Livin also acted mainly through this pathway, and it also had other anti-apoptotic pathways . A studyNA level . At the Many studies from Livin discovered to date have shown that this gene is expressed in a variety of malignancies. Livin expression was found in both renal cell carcinoma and normal kidney tissues, but its expression in cancer tissues was significantly higher, which was statistically significant . In addi142ph protein keeping pace with the activated autophagy level. However, H2A.XY142ph expression down-regulated through expressing H2A.XY142F and the autophagy activity decreased (Y142ph axis activated autophagy in colon cancer cells through transcriptionally regulating ATGs. These results indicated that Livin had kinase activity for H2A.XY142 phosphorylation. Whether Livin could influence DNA repair in colon cancer cells through H2A.XY142 phosphorylation should be further investigated. It has been shown that H2A.X can be phosphorylated at other tyrosine residues too. Whether Livin is the kinase that phosphorylates at the other tyrosine residues and their function in autophagy need to be investigated in future studies.Among the four core histones, H2A had the most variants, of which H2A.X accounted for <10%, but there have been many reports of H2A.X . Fragmenecreased . The LivY142 phosphorylation which promoted autophagy in colon cancer cells. This finding provided a new sight into the pathogenesis of colorectal cancer, and may be a new target for medical therapy.In general, this study clarified the regulation of Livin in H2A.XAll datasets for this study are included in the article/supplementary material.YG and SL designed experiments. BL carried out experiments. JC analyzed experimental results. YG wrote the manuscript. SL approved the 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.


The subscripts in the abbreviations indicate the degree of polymerization (DP). Furthermore, AMPS and APTAC were polymerized using a P(SA)91 macro-chain transfer agent to prepare an anionic diblock copolymer (P(SA)91S67) and a cationic diblock copolymer (P(SA)91A88), respectively. The DP was estimated from quantitative 13C NMR measurements. A stoichiometrically charge neutralized mixture of the aqueous P(SA)91S67 and P(SA)91A88 formed water-soluble polyion complex (PIC) micelles comprising PIC cores and amphoteric random copolymer shells. The PIC micelles were in a dynamic equilibrium state between PIC micelles and charge neutralized small aggregates composed of a P(SA)91S67/P(SA)91A88 pair. Interactions between PIC micelles and fetal bovine serum (FBS) in phosphate buffered saline (PBS) were evaluated by changing the hydrodynamic radius (Rh) and light scattering intensity (LSI). Increases in Rh and LSI were not observed for the mixture of PIC micelles and FBS in PBS for one day. This observation suggests that there is no interaction between PIC micelles and proteins, because the PIC micelle surfaces were covered with amphoteric random copolymer shells. However, with increasing time, the diblock copolymer chains that were dissociated from PIC micelles interacted with proteins.An amphoteric random copolymer (P(SA) A mixture of oppositely charged polyelectrolytes in water forms a water-insoluble polyion complex (PIC) due to attractive electrostatic interactions between the polymer chains . Many reProtein fouling on the surfaces of medical devices due to hydrophobic, electrostatic, and hydrogen bonding interactions causes deterioration of the functionality. Therefore, much attention has been given to the surface modification of medical devices using polymer coatings with antifouling properties. In general, protein antifouling polymers can bind water molecules strongly and are electrically neutral. When an antifouling polymer is coated on a substrate, proteins have minimal contact with the polymer on the substrate due to the presence of water molecules between the proteins and the polymers . In part91) macro chain transfer agent (CTA) composed of the same amounts of anionic sodium 2-acrylamido-2-methylpropanesulfonate  and cationic 3-acrylamidopropyl trimethylammonium chloride  via reversible addition-fragmentation chain transfer (RAFT) radical polymerization /([AMPS] + [APTAC])). The PIC micelles were prepared at f+ = 0.5 unless otherwise noted, which represents complete charge neutralization.P(SA)1H NMR and inverse-gated decoupling 13C NMR spectra were obtained in D2O using a Bruker  DRX-500 spectrometer. GPC measurements were performed using a Jasco UV-2075 detector equipped with a Shodex  OHpak SB-804 HQ column working at 40 °C with a flow rate of 0.6 mL/min. An acetic acid (0.5 M) solution containing sodium sulfate (0.3 M) was used as the eluent. Sample solutions were filtered with a 0.2 µm pore size membrane filter. Mn and Mw/Mn for the polymers were calibrated using standard poly(2-vinylpyridine) (P2VP) samples. Dynamic light scattering (DLS) measurements were obtained using a Malvern  Zetasizer Nano ZS with a He-Ne laser (4 mW at 633 nm) at 25 °C. The hydrodynamic radius (Rh) was calculated using the Stokes-Einstein equation, Rh = kBT/(6πηD), where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. The DLS data was analyzed using Malvern Zetasizer software version 7.11. The angular dependence of DLS and static light scattering (SLS) was measured using an Otsuka Electronics Photal  DLS-7000HL light scattering spectrometer equipped with a multi-τ digital time correlator (ALV-5000E), at 25 °C. A He-Ne laser (10 mW at 633 nm) was used as the light source 0) and APTAC ([MAPTAC]0), DP(theory) and Mn(theory) were calculated from the following formulas:0 is the initial concentration of CTA, MWMAV is the average molecular weight of AMPS and APTAC, and MWCTA is the molecular weight of CTA. When [MAMPS]0 = [MAPTAC]0, the total monomer concentration becomes 2[MAMPS]0. The values of DP(theory) and Mn(theory) are listed in The monomer conversions  values for the PAMPS and PAPTAC blocks in P(SA)91S67 and P(SA)91A88 were found to be 67 and 88, respectively, using quantitative 13C NMR measurements (Mn(NMR) values for P(SA)91S67 and P(SA)91A88 were close to the Mn(theory) values. However, the Mn(GPC) values for P(SA)91S67 and P(SA)91A88 deviated from the theoretical values, which indicates that there are interactions between the sample polymers and that the GPC column or the GPC standard sample (P2VP) was unsuitable for determining Mn(GPC) 91S67 and P(SA)91A88 were relatively small (1.04–1.15), which suggests that the obtained polymers have well-controlled structures.The composition of P(SA)erlapped . To deted in D2O a. The courements b,c. The  Mn(GPC) . The Mw/Rh values for P(SA)91S67 and P(SA)91A88 in 0.1 M aqueous NaCl were 5.7 and 6.0 nm, respectively (91S67 and P(SA)91A88 were 0.147–0.169. The Rh for the stoichiometrically charge neutralized mixture of P(SA)91S67 and P(SA)91A88 increased to 29.0 nm, which suggests the formation of PIC micelles. Assuming that the polymer main chain has a planar zig-zag structure, the expanded chain lengths of P(SA)91S67 and P(SA)91A88 are 39.5 and 44.8 nm, respectively. These are longer than the Rh of the PIC micelles (= 29.0 nm). This suggests that a PIC micelle has a simple spherical core-shell structure composed of a core formed from the anionic PAMPS and cationic PAPTAC blocks and an amphoteric P(SA)91 shell. The PDI for PIC micelles was narrower than for P(SA)91S67 and P(SA)91A88, which indicates that the PIC micelles have a uniform size.The ectively . These hq) was calculated using q = (4πn/λ)sin(θ/2), where n is the refractive index of the solvent and λ is the wavelength of the light source (= 632.8 nm). The Γ-q2 plot was a straight line passing through the origin. Therefore, Rh was determined at θ = 90°, because the diffusion coefficient (D) was independent of θ.The relationship between the relaxation rate (Γ) and light scattering angle (θ) was measured for PIC micelles in 0.1 M aqueous NaCl . The scaRh, LSI, and zeta potential for the mixture of P(SA)91S67 and P(SA)91A88 at various f+ were measured in 0.1 M aqueous NaCl 91S67, and the zeta potential was –11.4 mV due to the negative charge of the PAMPS block. At f+ = 1, the zeta potential for P(SA)91A88 was 8.40 mV due to the positive charge of the PAPTAC block. The zeta potential of the stoichiometrically charge neutralized mixture of P(SA)91S67 and P(SA)91A88 at f+ = 0.5 was close to 0 mV. At f+ = 0.5, the Rh and LSI had the highest values for PIC micelles. We will discuss the structure of PIC micelles in more detail, together with the results of the DLS, SLS, and TEM measurements, later in this paper. The ous NaCl . The totRh and LSI of PC micelles were plotted against Cp , which is the number of polymer chains that form one PIC micelle, may be constant and independent of Cp in this region (0.02–1.0 g/L). At Cp ≤ 0.02 g/L, the LSI for PIC micelles was too low to obtain Rh. We studied the time dependence on Rh and LSI for PIC micelles. The Rh and LSI values remained constant until at least 41 h 91A88, and PIC micelles were performed in 0.1 M aqueous NaCl 91S67 and P(SA)91A88. The Mw values of the unimer states of the block copolymers estimated from SLS were close to the weight-average molecular weight calculated from Mn(NMR) and Mw/Mn 91S67 and P(SA)91A88 were above 1, which suggests that the block copolymer chains were relatively expanded due to electrostatic repulsions in the polyelectrolyte blocks in 0.1 M aqueous NaCl. In general, A2 relates to the interactions between a polymer chain and solvent. A large value of A2 indicates a good solvent; however, a small or negative value of A2 indicates a poor solvent ) 91S67 and P(SA)91A88 were 5.7–6.0 nm  . At [NaC7–6.0 nm . The Rh Rh distributions were unimodal, and the Rh values for PIC micelles and BSA were 29.0 and 4.9 nm, respectively. A mixed solution of PIC micelles and BSA was prepared in PBS, and then DLS was performed on the solution within 1 h. After mixing, the distribution became bimodal, with Rh = 5.1 and 31.5 nm. The Rh value of the large size distribution was close to the Rh (= 29.0 nm) of the PIC micelles. Before mixing, the LSI values for the PIC micelles and BSA were 1.9 and 2.0 Mcps, respectively. The LSI for the mixture of PIC micelles and BSA was 2.1 Mcps, which was close to the values before mixing. This indicates that the PIC micelles do not interact considerably with BSA.We evaluated the interactions between PIC micelles (0.1 g/L) and BSA (5.0 g/L) in PBS using DLS . Before Rh and LSI for the mixture of PIC micelles and BSA were determined 91S67/P(SA)91A88 dissociated from the PIC micelles. After four days, the area of the third distribution peak increased, and Rh increased to 281 nm. The average Rh and LSI for the mixture were almost constant for three days; however, these increased after four days. These observations suggest that the interaction between PIC micelles and proteins increased with time. The block copolymer chains can dissociate from the PIC micelles because these are in a dynamic equilibrium state. The unit PICs of P(SA)91S67/P(SA)91A88 detached from the PIC micelles may interact with proteins due to electrostatic attractions. The total charges in the unit PIC are compensated. However, dangling charge loops may be formed in the complex of cationic PAMPTAC and anionic PAMSP blocks in the unit PIC  and FBS (2.1 Mcps) before mixing. This indicates that there is no interaction between PIC micelles and FBS. The time dependences of Rh distributions for the mixture of PIC micelles and FBS were measured  and FBS (40 g/L) in PBS were evaluated by DLS . While Bmeasured . After t91S67 and cationic P(SA)91A88 were prepared via RAFT using P(SA)91 macro-CTA. Water-soluble PIC micelles were formed from a stoichiometrically charge neutralized mixture of 0.1 M aqueous NaCl solutions of P(SA)91S67 and P(SA)91A88. The maximum values of Rh and LSI were observed when f+ was 0.5, and the zeta potential was close to 0 mV. The PIC micelles were in a dynamic equilibrium state with PIC micelles and charge neutralized small aggregates composed of a P(SA)91S67/P(SA)91A88 pair. The particle sizes in the mixture of PIC micelles and proteins in PBS remained almost constant for at least one day, which suggests that there is no interaction between PIC micelles and proteins. The PIC micelles were covered with amphoteric P(SA)91 shells that suppressed their interaction with proteins. However, the interactions of the diblock copolymer chains dissociated from PIC micelles and proteins increased with time. If the lengths of the PAMPS block in P(SA)91S67 and the PAPTSC block in P(SA)91A88 increased, the dynamic equilibrium may shift to form PIC micelles. Therefore, it is expected that the interaction of PIC micelles with long polyelectrolyte chains and proteins can be suppressed for a long time.Oppositely charged diblock copolymers of anionic P(SA)


Specifically, enzymatic removal of heparan sulfate from primary human hepatocytes, CRISPR/Cas9 manipulation of heparan sulfate biosynthesis in human hepatoma cells, or pharmacological manipulation of heparan sulfate–protein interactions using sodium chlorate or surfen dramatically reduced baseline and BMP6/SMAD1/5/8-dependent hepcidin expression. Moreover inactivation of the heparan sulfate biosynthetic gene N-deacetylase and N-sulfotransferase 1 (Ndst1) in murine hepatocytes (Ndst1f/fAlbCre+) reduced hepatic hepcidin expression and caused a redistribution of systemic iron, leading to iron accumulation in the liver and serum of mice. Manipulation of heparan sulfate had a similar effect on IL6-dependent hepcidin expression in vitro and suppressed IL6-mediated iron redistribution induced by lipopolysaccharide in vivo. These results provide compelling evidence that hepatocyte heparan sulfate plays a key role in regulating hepcidin expression and iron homeostasis in mice and in human hepatocytes.Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis. Its expression is regulated by the bone morphogenetic protein 6 (BMP6)/SMAD1/5/8 pathway and by the proinflammatory cytokine interleukin 6 (IL6). Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear. Here, we show that modulation of hepatic heparan sulfate significantly alters hepcidin expression and iron metabolism both  Hepcidin is the key peptide hormone regulating systemic iron homeostasis, and it is expressed primarily in the liver. Hepcidin is produced as a prepropeptide and processed by the convertase furin into the mature and active 25-amino acid peptide before being secreted into the circulation. Circulating hepcidin binds ferroportin, the only known cellular iron exporter , resultiA variety of stimuli trigger hepcidin expression, including high tissue and plasma iron levels, inflammation, hypoxia, and erythropoiesis . Iron-deBMP6 signaling involves activation and phosphorylation of the BMP receptors, followed by phosphorylation of SMAD1/5/8, the recruitment of SMAD4, and the translocation of the complex into the nucleus where it binds a response element in the hepcidin promoter . InflammA). Plasma membrane and extracellular matrix HSPGs can act as receptors for cytokines and growth factors, or as co-receptors for signaling complexes on the cell surface (in vivo in mice. The results show that hepatocyte HS is a master regulator of hepcidin expression and iron homeostasis.All cells express heparan sulfate proteoglycans (HSPGs), glycoproteins that contain one or more heparan sulfate (HS) chains A. Plasma surface . In the HAMP mRNA, compared with saline-treated cells (B). Partial degradation of cell surface and extracellular matrix HS with bacterial heparin lyases decreased HAMP expression significantly, indicating that HS in primary human hepatocytes facilitates BMP6-dependent induction of hepcidin expression (B). Similar results were obtained using hepatocarcinoma cells, a model for human hepatocytes .To examine if hepcidin expression depends on HS in primary human hepatocytes, we obtained cells through the Liver Tissue Cell Distribution System . BMP6 (50 ng/ml) stimulated 6-fold hepcidin expression, as measured by qPCR of ed cells B. Partiapression B. SimilaEXT1) and EXT2 in human hepatocarcinoma cells with siRNAs. EXT1 and EXT2 encode components of the co-polymerase that alternately transfer glucuronic acid and GlcNAc units during HS chain elongation (A). Transfection of cells with siRNAs reduced mRNA levels of EXT1 and EXT2 by ∼80–90% (C) and expression of EXT1 and EXT2 proteins . Simultaneous silencing of EXT1 and EXT2 caused a dramatic reduction of HAMP mRNA expression in naive cells and after stimulation with BMP6 (D). Silencing of EXT1 and EXT2 also reduced pSMAD5 in unstimulated cells and after stimulation with BMP6 (E and F). Expression of ID1 (inhibitor of DNA binding protein 1), another marker of the BMP6/SMAD pathway, also was diminished, although not to the same extent (G). Control experiments using siRNA to GFP showed no impact on expression of EXT1, EXT2, HAMP, and ID1, or phosphorylation of SMAD5 (C–G). Together, these studies demonstrate that HS strongly regulates hepcidin mRNA expression in human hepatocytes.The partial reduction of hepcidin mRNA expression induced by heparin lyase treatment could reflect incomplete removal of HS. To obtain additional evidence that HS regulates hepcidin expression, we silenced EXOSTOSIN 1 . Downstream processing of the chains by 6-O-sulfation of glucosamine residues and epimerization and 2-O-sulfation of uronic acids depends on prior NDST action. Thus, diminishing NDST1 expression depresses overall sulfation of the chains. A clonal line (NDST1−/−) was isolated bearing two independent frameshift alleles in exon 1 of NDST1 that caused premature stop codons in both alleles downstream from the frameshift (A). We noted markedly reduced NDST1 mRNA expression (82 ± 3% reduction in NDST1−/− compared with WT)  and binding of fibroblast growth factor 2 (FGF2) to the mutant, which reflects the loss of cell surface HS, and the extent of reduction occurred similarly in WT cells treated with heparin lyases (B). Isolation of HS from the mutant and depolymerization of the chains into disaccharides showed that NDST1−/− cells exhibited a decrease of disaccharides bearing N-sulfoglucosamine residues  and accumulation of nonsulfated disaccharides (D0A0). Overall N-sulfation of glucosamine residues was reduced by 73%, leading to ∼40% reduction in 6-O-sulfation of glucosamine residues and ∼90% reduction of 2-O-sulfation of uronic acids because of coupling of these downstream reactions to N-sulfation (C). The reduction of sulfation was incomplete because of expression of NDST2 in the cell line, as observed in other cell types (–NDST1 expression reduced HAMP expression by 70% in the absence of BMP6 and by 90% after stimulation (D).To study how the structure of HS influences hepcidin expression, we inactivated residues A. Downstameshift A. We notn lyases B. Isolatulfation C. The rell types –21. Nevemulation D.HAMP expression on sulfation of HS. Sodium chlorate is an inhibitor of the universal sulfate donor 3′-phosphoadenyl-5′-phosphosulfate (PAPS) (m sodium chlorate caused a significant suppression of both unstimulated and BMP6-stimulated HAMP expression (A). The addition of 25–50 mm NaCl had no effect on HAMP expression , indicating that the inhibitory effect of sodium chlorate was not because of changes in osmolarity of the medium.Pharmacological studies also demonstrated the dependence of e (PAPS)  and blocpression A. The adHAMP expression in HepG2 cells (B). The effect was greatly magnified in cells treated with the more potent analog oxalylsurfen (>90% inhibition) (C), whereas treatment with the inactive analog hemisurfen had no effect on hepcidin expression . Cells treated with these different pharmacological agents showed reduced SMAD5 phosphorylation after BMP6 challenge (D and E). Together these findings show that interference with HS sulfation or HS-protein interactions results in diminished HAMP expression under basal conditions and after BMP6 stimulation.Recently, we reported that surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) and oxalylsurfen  block HS-dependent processes by interference with protein-HS interactions , 24. SurG2 cells B. The efibition) C, whereahallenge , D and ENdst1f/fAlbCre−) and mice in which Ndst1 was inactivated specifically in hepatocytes (Ndst1f/fAlbCre+)  (A) and absence of NDST1 protein (B). Inactivation of Ndst1 diminished basal and BMP6-stimulated Hamp expression (C) and SMAD5 phosphorylation (D). Sulfation of HS is not completely abolished in Ndst1f/fAlbCre+ hepatocytes .As a segue to studying the impact of altering HS on hepcidin expression and iron homeostasis in mice, we obtained primary hepatocytes from control mice (AlbCre+) . Ndst1f/in (Tbp) A and abs protein B. Inactipression C and SMArylation D. Sulfatatocytes ; thus thma cells D.in vivo, we measured Hamp mRNA, serum hepcidin, and iron levels in mutant and WT mice fed an iron-balanced diet (0.2 g/kg of carbonyl-iron) and after 1 and 3 weeks on an iron-rich diet (8.3 g/kg of carbonyl-iron) (A). Ndst1f/fAlbCre+ mice fed an iron-balanced diet exhibited 4-fold lower hepatic Hamp mRNA expression than control mice . Serum hepcidin did not differ significantly (119 ± 41 versus 192 ± 85 ng/ml) . Plasma iron levels were elevated in Ndst1f/fAlbCre+ mice  , as was liver iron content   and ferritin-iron . No difference in spleen iron content was observed .To study the impact of altering hepatic HS on iron metabolism yl-iron) A. Ndst1fin vivo system to study the impact of altering HS in hepatocytes on hepcidin expression. Iron-loading for 1 week greatly enhanced both Hamp mRNA and serum hepcidin in Ndst1f/fAlbCre− control mice, whereas the effect was significantly less in Ndst1f/fAlbCre+ mice (3.7 ± 0.8 versus 2.2 ± 0.5) . Serum hepcidin showed a similar trend (850 ± 180 in the mutant versus 460 ± 160 ng/ml in the control mice) (C). Serum iron levels increased as expected after 1 week of iron-loading, and their levels were consistently higher in the Ndst1f/fAlbCre+ mice (290 ± 15 μg/dl versus 250 ± 13 μg/dl) (D). Liver iron content was also elevated in the mutant compared with the WT after 1 week  (E).Iron overloading increases BMP6 expression , 9, thus2 ± 0.5) B, 1 weekol mice) C. Serum 3 μg/dl) D. Liver ctively) E.Hamp mRNA, serum hepcidin, and liver iron remained elevated, but the values for the mutant were similar to the control . Serum iron levels remained elevated at 3 weeks in the mutant compared with the WT (D). Ferritin-iron complexes also were significantly elevated in liver samples in the mutant compared with the control (F). Spleen iron content rose dramatically with iron loading but showed no difference between control and mutant mice (G).After 3 weeks on the iron-rich diet, the values for h the WT D. Ferrit control F. Spleenant mice G.H, 0 time point). Under these conditions, ferritin L (FtL) was significantly elevated in the mutant. Liver pSMAD5 was reduced significantly in the mutant, which tracked with reduced Hamp mRNA expression  and a trend toward reduced serum hepcidin . Iron loading for 1–3 weeks did not alter expression of FPN or TfR1, but FtL remained elevated in the mutant . pSMAD5 remained depressed in the mutant , consistent with the reduction in Hamp mRNA expression . As expected, Id1 expression increased with iron-loading in WT mice, whereas mutant mice exhibited no response, suggesting that BMP6 signaling was impaired in the mutant . Bmp6 expression in both control and mutant mice trended toward higher levels with iron-loading .Analysis of several proteins related to iron storage and metabolism showed that liver ferroportin (FPN) and transferrin receptor (TfR1) did not differ in mutant and WT animals fed an iron-balanced diet . Both unstimulated and IL6-induced Hamp expression was reduced in the mutant (A). To confirm these findings, we treated hepatoma cells with surfen, which reduced IL6-induced stimulation of HAMP expression (B). Similarly, siRNA-mediated silencing of EXT1 and EXT2 diminished both baseline and IL6-stimulated HAMP expression, whereas an siRNA directed against GFP had no effect (C). Surprisingly, silencing of EXT1 and EXT2 had no effect on STAT3 phosphorylation (D) or suppressor of cytokine signaling 3 (SOCS3) expression, which is downstream of STAT3 (E and F). Thus, the IL6 induction of hepcidin expression depends on HS, but it occurs independently of STAT3 phosphorylation and SOCS3 expression.Inflammation also affects hepcidin expression and iron metabolism, mediated primarily through IL6/JAK/STAT3 signaling . To evale mutant A. To conpression B. Similao effect C. Surprirylation D or suppof STAT3 , E and Fin vivo, we injected Ndst1f/fAlbCre− and Ndst1f/fAlbCre+ mice intraperitoneally with lipopolysaccharide (LPS). Previous studies have shown that LPS induces a robust increase in hepatic Socs3 and Hamp mRNA expression as well as a reduction of serum iron levels in mice (Socs3 mRNA expression in both Ndst1f/fAlbCre+ and Ndst1f/fAlbCre− mice (A). In contrast, Hamp mRNA expression was reduced nearly 3-fold in Ndst1f/fAlbCre+ mice compared with control mice (B). This trend was mirrored by reduced serum hepcidin in the mutant (C). As expected, the reduced hepcidin level in the mutant after LPS injection was associated with higher serum and liver iron compared with control mice (D and E). Although there was a trend toward reduced spleen iron in the mutant, the difference was not significant (F), and LPS induced strong STAT3 phosphorylation in both mice strains (G). These findings suggest that hepatic HS also plays a key role in the regulation of IL6-induced hepcidin expression in mice.To examine the impact of an inflammatory challenge  in mice . LPS indre− mice A. In conrol mice B. This te mutant C. As exprol mice , D and Enificant F, and LP strains G. These Hamp expression and serum hepcidin. Evaluation of pSMAD5 in LPS-stimulated WT mice showed a significant increase, suggesting activation of the pathway. In the mutant, baseline of pSMAD5 was reduced as shown in Fig. 5H. Importantly, LPS did not stimulate SMAD5 phosphorylation above baseline in the mutant (G). To analyze the effect of heparan sulfate on IL6/STAT3 pathway, we transfected WT and NDST1-deficient Hep3B cells with a HAMP-luciferase reporter construct and measured luciferase activity upon stimulation with IL6 with and without siRNA silencing of STAT3. Silencing of STAT3 eliminated the heparan sulfate–dependent regulation of IL6-induced HAMP expression as measured by luciferase activity (H). We performed a similar experiment to evaluate the impact of NDST deficiency on BMP6/SMAD5 pathway by siRNA silencing of SMAD5. Silencing of SMAD5 did not diminish BMP6-dependent HAMP expression (I). SMAD5 siRNA and STAT3 siRNA reduced SMAD5 and STAT3 mRNA levels, respectively, compared with scrambled siRNA in both WT and NDST1−/− cells .The lack of effect of HS on IL6-induced STAT3 signaling suggested that an alternative pathway might be responsible for reduced e mutant G. To anaactivity H. We perpression I. SMAD5 in vivo based on the accumulation of serum and liver iron in mice bearing a hepatocyte specific alteration of HS biosynthesis. Regulation of the system depends not only on the amount of HS produced, but also on its degree of sulfation and its capacity to interact with heparan sulfate–binding proteins.In this paper, we provide genetic and pharmacological evidence for cell autonomous control over hepcidin expression by HS in human and murine hepatocytes. Moreover, the modulatory effects of HS, demonstrable in cell culture, translate to altered iron homeostasis The studies reported here provide the first direct evidence that hepatic HS alters iron metabolism. HS affects baseline, BMP6-stimulated, and IL6-stimulated hepcidin expression, suggesting a common underlying mechanism. However, siRNA-mediated reduction of STAT3 expression eliminated HS-dependent regulation of IL6-induced HAMP-luciferase expression, whereas siRNA-mediated reduction of SMAD5 did not eliminate the HS dependence of BMP6 signaling.Ndst1 inactivation depressed hepcidin expression suggests that HS may act as a “ligand” for one or more factors central to iron regulation. Previous studies showed that exogenous heparin, a highly sulfated form of HS, inhibits hepcidin expression in hepatoma cells and in mice , and the extracellular matrix proteoglycans perlecan, collagen 18, and agrin . Furtherml-glutamine (Gibco) at 37 °C under an atmosphere of 5% CO2/95% air. Mouse hepatocytes were isolated from 10-week-old animals as described previously .Primary human hepatocytes were obtained from a public repository managed by the University of Minnesota under contract from the NIDDK at the National Institutes of Health (Liver Tissue Cell Distribution System). Cells were derived from resected liver sample from a 63-year-old female with a bleeding liver cyst (no chemotherapy history) and seeded in 6-well plates. The human hepatoma cell lines, HepG2 and Hep3B (American Type Culture Collection), were cultured in minimum essential medium  containing 10% endotoxin-free fetal bovine serum (FBS) (Sigma-Aldrich), 0.04 mg/ml gentamicin (Gibco), and 2 meviously . The cel5 cells/well) in the presence or absence of different concentrations of sodium chlorate or NaCl (25–50 mm) for 72 h or surfen, oxalylsurfen, or hemisurfen for 24 h (Hepatocytes were seeded in 12-well plates (1.5 × 105 cells/well) in MEM with 10% FBS. After 24 h the cells were transfected with siRNA for Ext1 and 2 or GFP siRNA  with RNAiMax (Life Technologies) according to the manufacturer's instructions.HepG2 was seeded in 12-well plates (1 × 10N-deacetylase N-sulfotransferase-1 (NDST1−/−) were generated using NDST1 guide RNAs designed according to Broad Institute published resources (m) were annealed using T4 polynucleotide kinase (New England Biolabs) and integrated into vector pSp-Cas9(BB)-2A-Puro (a gift from Dr. Feng Zhang) using T7 ligase (New England Biolabs). The vectors were sequenced by Sanger sequencing to confirm their correct construction and transfected into Hep3B cells using Lipofectamine-2000 according to the manufacturer's instructions. Transfected Hep3B cells were selected with puromycin (3 μg/ml) and single cell clones were isolated by limiting dilution. Mutations in the targeted region were determined by PCR amplification and Sanger sequencing.Mutant cell lines lacking GlcNAc esources  and wereNdst1f/fAlbCre+ mice were generated and genotyped as described previously  were maintained on an iron-balanced diet  for 1 week and then switched to an iron-rich diet (8.3 g/kg carbonyl-iron) for 1 or 3 weeks (4–5 mice per group). To mimic an inflammatory stimulus, mice were treated intraperitoneally with 1 mg/kg lipopolysaccharide (Sigma-Aldrich) and euthanized after 6 h. Blood, liver, and spleen were collected for further analysis.Control −ΔΔCt). The primers are indicated in Table S1.Total RNA was isolated from tissues or cells using TRIzol Reagent (Ambion), according to the manufacturer's instructions. cDNA was generated by reverse transcription, using 1 μg RNA and SuperScript III Reverse Transcriptase kit (Invitrogen). Samples were analyzed by quantitative RT-PCR (qRT-PCR) using PowerUp SYBR Green Master Mix (Life Technologies) according to the manufacturer's instructions. All data are normalized to the expression of the proper housekeeping gene depending on the type of sample; specifically HPRT1 for HepG2, GAPDH for Hep3B, and primary human hepatocytes and Tbp (for murine-derived samples) and expressed by relative quantification method ) or using a lysis buffer . Protein was quantified by BCA assay (Thermo Scientific-Pierce). Samples (40–50 μg of protein) were separated by 10–14% SDS-PAGE and transferred to Hybond-P Membrane . The primary antibodies used for immunoblotting were anti-l-ferritin ; anti-GAPDH ; anti-phospho-SMAD5 ; anti-phospho-STAT3, anti-SMAD5, anti-STAT3 ; anti-actin ; anti-TfR1 ; anti-ferroportin ; and anti-EXT1, anti-EXT2, and anti-NDST1 . After incubation with horseradish peroxidase–conjugated secondary antibodies, membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific-Pierce) and visualized by imaging (Lycor Odyssey). Densitometric analysis was performed using ImageJ software. Mouse serum hepcidin1 was quantified using Hepcidin Murine-Compete ELISA Kit (Intrinsic LifeSciences) according to the manufacturer's instructions.Mouse tissues or cell extracts were prepared using RIPA buffer (150 m6 cells were digested with 0.4 μg/ml Pronase (Sigma-Aldrich) at 37 °C for 16 h and crude GAGs were isolated by anion exchange chromatography using DEAE-Sephacel . The columns were washed with 0.25 m NaCl and the glycosaminoglycans were eluted with 2 m NaCl. Heparan sulfate chains were depolymerized with heparin lyase I, II, and III (2 milliunits/ml of each) and unsaturated disaccharides were analyzed using glycan reductive isotope labeling liquid chromatography/mass spectrometry techniques as described elsewhere  of clarified acid extract were added to 240 μl of working chromogen reagent in a 96-well plate . The solutions were then incubated for 30 min at room temperature and the absorbance measured at 535 nm in a plate reader. A standard curve was prepared with a precalibrated solution of FeCl3 (Sigma-Aldrich). Blood was collected and serum iron was determined with a commercial kit , according to the manufacturer's instructions.Spleen and liver iron content was determined spectrophotometrically as described previously with minor modifications . Briefly2O2 in 20 mm Tris HCl, pH 7.4, for 15–30 min. The reaction was blocked by washing with tap water.Liver homogenates were pretreated at 70 °C for 10 min to enrich the thermostable ferritins. Samples  were loaded on 8% nondenaturing PAGE and electrophoresed for 3 h at 160 V. The gels were washed with water and incubated in 2% ferrocyanide and 2% HCl for 1 h. To enhance the signal, the gels were incubated in 0.025% 3,3′-diaminobenzidine (Sigma-Aldrich) and 0.05% HNDST1−/− cells were transfected with scrambled, SMAD5, or STAT3 siRNAs, along with pGL2Hamp-luciferase and pGL2TK-Renilla plasmids (Promega). After 24 h, cells were treated with BMP6 (50 ng/ml) or IL6 (50 ng/ml) and incubated for 6 h. Luciferase activities were measured as described . Differences were defined as significant for p values < 0.05 or < 0.001, respectively, and values are shown in the figures.Data are shown as mean ± S.D. or mean ± S.E. as indicated in the figures. Generally, mRNA expression levels were scaled to control values and exhibited as -fold change or percentage. Comparison of values between untreated or treated cells and between M. P. and J. D. E. conceptualization; M. P., F. A.-E.-K., and J. D. E. data curation; M. P., P. L. S. M. G., and J. D. E. formal analysis; M. P., F. A.-E.-K., M. A., P. R., M. G., and A. D. investigation; M. P., F. A.-E.-K., P. L. S. M. G., and J. D. E. writing-original draft; M. P., P. L. S. M. G., P. A., and J. D. E. writing-review and editing; P. L. S. M. G., P. A., and J. D. E. supervision.


Notably, the T cell-mediated control was shown to be perforin-independent but IFNγ-dependent. In total, this study systematically identifies crucial antiviral factors present in lung NIFs for early containment of a local MCMV infection at the single cell level.Human cytomegalovirus (CMV) and mouse cytomegalovirus (MCMV) infection share many characteristics. Therefore infection of mice with MCMV is an important tool to understand immune responses and to design vaccines and therapies for patients at the risk of severe CMV disease. In this study, we investigated the immune response in the lungs following acute infection with MCMV. We used multi-color fluorescence microscopy to visualize single infected and immune cells in nodular inflammatory foci (NIFs) that formed around infected cells in the lungs. These NIFs consisted mainly of myeloid cells, T cells, and some NK cells. We found that the formation of NIFs was essential to reduce the number of infected cells in the lung tissue, showing that NIFs were sites of infection as well as sites of immune response. Comparing mice deficient for several leukocyte subsets, we identified T cells to be of prime importance for restricting MCMV infection in the lung. Moreover, T cells had to be present in NIFs in high numbers, and CD4 as well as CD8 T cells supported each other to efficiently control virus spread. Additionally, we investigated the effects of perforin and interferon-gamma (IFNγ) on the virus infection and found important roles for both mechanisms. NK cells and T cells were the major source for IFNγ in the lung and in  Cytomegalovirus (CMV) is worldwide a highly prevalent β-herpesvirus. While the primary infection in healthy individuals does not cause disease, infection of immunocompromised patients can lead to multiple organ disease and can sometimes be lethal. CMV becomes latent and uses a series of mechanisms to circumvent its elimination by the immune system. Therefore, the establishment of therapies or vaccination strategies is a difficult endeavor. Murine CMV (MCMV) is a well-established model to study CMV infection in mice, since it targets many immune defense mechanisms in a similar manner as human CMV. Thus, studying immune responses of mice to MCMV can help to develop new therapeutic strategies for patients. In this study we focused on MCMV infection in adult mouse lungs and found that, in the immunocompetent host, immune cells infiltrate the lung tissue as nodular inflammatory foci that help to control the acute infection in one week. We identified specific lymphocyte subsets that are pivotal for efficient containment of the infection and showed that this process requires cooperation between CD4 and CD8 T cells. Furthermore, these cells need to secrete the multipotent cytokine IFNγ to successfully clear the lungs from infectious viruses. The immune response against CMV infection in humans and murine cytomegalovirus (MCMV) in mice has been studied for decades . UnderstThe immune response against CMV infection seems to be affected by the site of infection, since various cell populations and effector mechanisms differentially contribute to virus control in different organs –16: Whilin vivo . Th. ThRag2-We next studied the dynamics of the number of lymphocytes present in NIFs of MCMV-infected wild-type mice. Histological analysis at 1 dpi revealed hardly any lymphocytes residing in close proximity to infected cells, whereas at 3 dpi and 5 dpi a substantial number of T cells is present in NIFs. The number of T cells per NIF is even further increased at 8 dpi, when most of the infected cells already disappeared. In contrast, the number of NK cells present in NIFs peaked at 3 dpi and declined until 5 dpi . These dRag2-/- recipients , IFNγ(Ifng-/-), or IFNγ-receptor-1 (Ifngr1-/-). At 8 dpi, we quantified the number of infected cells per NIF but did not find any significant differences between WT and the three knock-out strains tested  were sufficient to reduce the viral titer or the amount of infected cells in several organs of infected immunocompromised recipients [7) led to T cell densities in NIFs that contributed to sufficient local virus control to a similar degree as observed in WT mice 8 dpi. This high number of polyclonal naïve T cells is essential to provide sufficient numbers of precursors for MCMV-specific T cells that can be expanded and subsequently control the infection.Using various experimental approaches, we show in lungs that T cells have a major effect on NIF dynamics and virus control in NIFs. Effector CD8 T cells are known to essentially contribute to the control of MCMV infection in mice –44 and hcipients –44. In cThe role of CD4 T cells in controlling cytomegalovirus infection has been discussed controversially. Several adoptive transfer experiments showed that CD4 T cells on their own cannot control MCMV infection ,45,46. NRag2-/- mice, we showed that CD4 T cells support CD8 T cells in controlling virus infection in the lungs. This effect might be mediated by a type of T helper-1 function supporting CD8 T cell differentiation as suggested elsewhere [Applying adoptive transfers into lsewhere  but it slsewhere ,55. Intrin vitro assay we found that IFNγ affected plaque size of lung stromal cells after MCMV infection, indicating that IFNγ stimulates intrinsic cell mechanisms to interfere with virus replication [Furthermore, we showed that the secretion of IFNγ by T cells was essential to reduce the number of infected cells while the absence of perforin did not affect the T cell mediated control. IFNγ has been reported to have multiple effects on different cell types –58. For lication ,61. Intelication . It is klication . Previoulication . In addiBased on the data presented in this manuscript we envisage the following model how T cells contribute to the local control of MCMV in lung NIFs: T cells are primed in lymphoid organs and within NIFs to secrete IFNγ directly at the site of infection. This secretion of IFNγ not only slows down the viral cell-to-cell spread but might also interfere with MHC I molecule downregulation on the surface of infected cells allowing for better recognition of viral antigen by MCMV-specific CD8 T cells. Furthermore, the IFNγ secretion by NK, as well as both CD4 and CD8 T cells, can stimulate macrophages to efficiently phagocytose infected cell remnants and clear the tissue from infectious virus particles. In parallel, NK cells contribute to MCMV control in a perforin-dependent manner. Thus, a combination of virus-specific CD8 and CD4 T cells, with the addition of NK cells, might be an optimal recipe for anti-CMV adoptive immunotherapy.2 anesthesia by cervical dislocation.All animal experiments were performed according to the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations and Society of Laboratory Animals and approved by the institutional review board and the Niedersächsische Landesamt für Verbraucherschutz und Lebensmittelsicherheit . All experiments were performed according to the Tierschutzgesetz and the Tierschutz-Versuchstier-Verordnung. Anesthesia of mice was performed with Ketamin and Xylazin injection. Mice were sacrificed following COIghmtm1Cgn [Cd3etm1Mal [Rag2tm1 [Rag2tm1Il2rgtm1[Ifngtm1Ts [Ifngr1tm1Agt [Prf1tm1Sdz [Ncr1gfp/wt [Rag2-/-Prf1-/- and Rag2-/-Ifng-/- mice were generated by intercrossing Rag2-/- and Prf1-/- or Rag2-/- and Ifng-/- mice, respectively. 6–14 weeks old mice were used for the experiments. All mouse experiments were performed in accordance with local animal welfare regulations.Mice were bred at the central animal facility of Hannover Medical School under specific pathogen free conditions. The different mouse strains  were mam129 ORF resulting in an intact MCK2 protein was used for intranasal infection [6 PFU MCMV-3D or MCMV-3DR. In some experiments C57BL/6 mice were irradiated with a total dose of 6 Gray in one irradiation procedure the day before intranasal infection.The MCMV strain named MCMV-3D has been described previously . Virus snfection . All aniRag2-/- mice were used as recipients of adoptive T cell transfers. T cells were isolated from lymph nodes and spleens of untreated wild-type B6, Ifng-/-, Ifngr1-/-, or Prf1-/- mice using MACS pan T cell, CD4 T cell, or CD8 T cell negative selection kits (Miltenyi Biotech) and adoptively transferred intravenously in parallel to MCMV infection. For in vivo depletion of NK cells in B6 and Rag2-/- mice 300 μg anti-NK1.1 (antibody clone PK136) was applied intraperitoneally four hours before and every second day after MCMV infection.In experiments in situ with cold PBS, explanted and stored in 500μL DMEM containing 10% FCS and 1% P/S. Next, the organs were mechanically disrupted by metal beads and shaking at 25/s over 4 min . Then, 350 μL of the homogenate were separated and stored at -80°C for viral plaque titration, while the rest was centrifuged at maximum speed for 15 min. 20 μL of the substrate (1 μg Coelenterazine / ml in PBS) were added freshly to 180μL of a 1:10 dilution of the organ homogenate and measured for 10s directly with Lumat LB 9507 (Berthold Technologies) in duplicates. The viral plaque titration was performed on MEF monolayers in triplicates.Organs were perfused in situ with PBS solution containing 2% paraformaldehyde and 30% sucrose. Fixed lungs were embedded in OCT compound  before freezing at -20°C.Mice were sacrificed and lungs were perfused with cold PBS via the right heart ventricle and then filled 7 μm thick cryosections were blocked with rat serum in TBST and stained with antibodies and nuclei were stained with DAPI. The following antibodies (clones) were used after additional blocking of Fc receptors with rat anti-CD16 and anti-CD32 (2.4G2): CD11b-bio (MAC1), CD3-Cy5 (CD317A2), CD11c-APC (N418), CD45.2-FITC (104), CD45-APC (30-F11), and CD8a-PE (53–6.7). For staining with anti-mouse NK1.1-APC (PK136) and GFP-binding protein , rehydration was performed in 0.5% Triton-X following blocking with mouse serum and Fc receptor blocking, while slides were stained in 1%Triton-X. Images were taken with an AxioCam MRm camera (Carl Zeiss) attached to an Axiovert 200M fluorescence microscope (Carl Zeiss) with PlanApochromat objectives 10x/0,45 and 20x/0,75  and with AxioScan.Z1 (Carl Zeiss) and processed with AxioVision 4.8.2 software and with ZEN blue 2.3 software.gfp/wt mice and additional staining with a GFP-binding protein in the area of infiltration that was semi-automatically measured based on CD45 staining using ImageJ. Between 10 and 15 NIFs were analyzed per animal.Lung sections were analyzed at defined anatomical positions . Sections were stained with DAPI. For each animal, 4 lung sections were analyzed by an observer blinded for mouse identity. Manual counting of virus-infected cells and NIFs was performed directly at the microscope. Intact cells were distinguished from remnants of infected cells based on their mCherry expression, morphology, and DAPI staining while NIFs were quantified based on localized accumulation of nuclei as well as presence of mCherry signals. The number of independent experiments performed is indicated in each figure. In general, we only show data from at least 4 mice and at least 2 independent experiments. For the analysis of infected cells per NIFs, microscopy pictures were taken with the 20x/0.75 objective. T cells were quantified based on CD3 staining, CD8 T cells were quantified based on CD8a staining, and NK cells were quantified based on GFP expression in Ncr1in vitro in 200 μL RPMI/FCS/HEPES. For re-stimulation 50 ng/mL PMA and 500 ng/mL Ionomycin were added and cells were incubated for 2 hours at 37°C with 5% CO2. To allow detection of intracellular proteins 10 μg/mL Brefeldin A were added and incubated for another 2 hours at 37°C and 5% CO2. Fc-receptors were blocked in PBS with 3% FCS (PBS/FCS), 5% serum, and rat antibodies to mouse CD16 and CD32 (2.4G2). Surface staining was performed for 15 min on ice following the fixation and intracellular staining procedure with the Intracellular Fixation & Permeabilization Buffer Set (eBiosciences) according to the manufacturer protocol. Cells were re-suspended in PBS/FCS for flow cytometry analysis using a LSRII (BD Biosciences). Flow cytometry data was analyzed using FlowJo 7.5 and 10. Gates for IFNγ+ cells were set based on the border of 99% of the isotype control. Cells from in vitro culture experiments were trypsinized at room temperature (for 3 min using PBS containing 0.5 mg/mL Trypsin and 3 mM EDTA), blocked for Fc-receptors and stained with antibodies before FACS analysis. Following antibodies (clones) were used for flow cytometry staining: CD8a-PE (53–6.7), CD8-APC (RmCD8-2), Thy1-Alexa488 (RMT1), CD103-FITC (M290), CD31-FITC (MEC13.3), Ly6G-PE (1A8), CD11b-eF450 (M1/70), CD45.2-APC (104), EpCAM-eF450 (G8.8), F4/80-APC-eF780 (BM8), gp38-bio (eBio8.1.1), Ly6C-PerCP-Cy5.5 (HK1.4), CD11c-PeCy7 (N418), CD19-BV510 (6D5), CD4-PerCP (RM4-5), CD45.2-PerCP-Cy5.5 (104), IFNγ-PE (XMG1.2), NK1.1-PeCy7 (PK136), Pdgfra-PE (APA5), and Pdgfrb-APC (APB5).Leukocytes from lungs were isolated as described before . Briefly+ cells with the use of a MACS CD45 positive selection kit (Miltenyi) or cultured as a mixed population. Cells were cultured in RPMI with FCS, P/S and L-Glutamine, and medium was exchanged every day to remove dead and non-adherent cells. After 6 days of culture, cells were trypsinized, seeded on 48 well plates and cultured for additional 7 days to reach a confluent layer. Cells were then infected with 20 PFU/well for 30 mins. Afterwards, virus solution was discarded and cells were covered with carboxy-methyl-cellulose in addition to different concentrations of mouse IFNγ protein . After 4 and 8 days, mosaic images were taken with Zeiss Axiovert M microscope, and plaque sizes were measured with ImageJ.Animals were sacrificed and the right heart ventricle was perfused with cold PBS to remove blood cells from the lungs. Lungs were explanted, chopped into pieces and digested in RPMI with 0.125 mg DNAse I and 0.269 mg collagenase D at 37°C for 2h. Cell suspensions were treated with erythrocyte lysis buffer and stromal cells either purified by depletion of CD45Rag2-/- mice , P<0.05 (*), P<0.01 (**), P<0.001 (***).Statistical analysis was performed using GraphPad Prism 4. A non-parametric two-tailed t-test (Mann-Whitney test) was applied to compare data from experimental groups to that of wild-type mice  or of Raice Figs  and 7. IS1 Fig6 PFU MCMV-3D i.n. At 5 and 8 dpi salivary glands were homogenized and viral titers (A) or luciferase activities (B) were determined. Dots represent means of (A) triplicates or (B) duplicates per organ. (A+B) Median plus interquartile range are shown in grey; Mann-Whitney test. (C) Correlation of viral titers and luciferase activity in salivary glands. r: Spearman coefficient. (A-C) DL: detection limit; data from 2 independent experiments per time point are shown. PFU: plaque forming units; RLU: relative light units.Animals were either irradiated with 6 Gy or not (control) and infected one day post irradiation with 10(TIF)Click here for additional data file.S2 Fig6 PFU MCMV-3D. Quantification of NIF size at (A) 5 and (B) 8 dpi. Dots represent NIFs in 1–2 independent experiments; median + interquartile range (red); Kruskal-Wallis test with Dunn’s Multiple Comparison to WT-mice.Wild type mice and various mouse strains deficient for different immune cell populations were infected i.n. with 10(TIF)Click here for additional data file.S3 Fig6 PFU MCMV-3D. PFA-fixed lung cryosections were prepared from mice and analyzed at 5 dpi. Infected cells were quantified per (A) NIFs and (B) lung section. Dots represent (A) NIFs or (B) means of 4 lung slices analyzed per animal. Median plus interquartile range shown in red; Mann-Whitney test performed with mean values of individual animals.Animals were depleted or not for NK cells before i.n. infection with 10(TIF)Click here for additional data file.S4 FigRag2-/- mice were infected i.n. with 106 MCMV-3DR  or MCMV-3D  and lungs were analyzed at 8 dpi. Infected cells were quantified per (A) NIF and (B) lung section. Dots represent (A) NIFs and (B) mean of 4 lung sections analyzed per animal; median + interquartile range (grey); Mann-Whitney test performed with mean values of individual animals.; data shown from 2 independent experiments.WT or (TIF)Click here for additional data file.S5 Figgfp/wt mice were i.n. infected with 106 PFU MCMV-3D. (A) Histology of representative NIFs GFP, blue; anti-CD8a, orange; anti-CD45, green; infected cells, red; scale bar 50 μm. (B+C) Quantification of the number of (B) NK cells and (C) CD8 T cells per NIF at 3 and 5 dpi. Dots represent NIFs; median + interquartile range; Mann-Whitney test. (D+E) Quantification of number of (D) NK cells or (E) T cells per NIF area; Dots represent NIFs.Ncr1(TIF)Click here for additional data file.S6 Fig6 PFU MCMV-3D i.n. and lungs were analyzed at 5 and 8 dpi. For FACS analysis cells were stained with a mixture of three different tetramers . (A) Number of tetramer+ cells per lung and (B) frequency of tetramer+ cells of CD8+ T cells. Data points represent animals; median + interquartile range (grey); data from 2 experiments shown.Animals were infected with 10(TIF)Click here for additional data file.S7 FigIfngr1-/- mice were cultured in the (A+B) presence or (C+D) absence of lung-resident CD45+ hematopoietic cells. Confluent cells were infected with 20 PFU MCMV-3D per well. After infection, carboxy-methyl-cellulose and various concentration of recombinant mouse IFNγ was added. Quantification of plaque sizes (A+C) 4 dpi and (B+D) 8 dpi after treatment with IFNγ as indicated. Mean of plaque sizes per well in dependence on the amount of IFNγ added . Data from 3–4 independent experiments are shown.Primary lung stromal cells of (TIF)Click here for additional data file.


Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2–3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection. Genetic analysis has demonstrated that avian H5N1 viruses are highly diverse and prevalent with distinct regional genetic lineages becoming apparent in the past decade [Human infection with avian H5N1 subtype virus was first documented in Hong Kong in 1997, where it caused 18 infections and six deaths . Availabt decade . While ht decade ,7,8. TheIn addition to adopting host-specific receptor binding through changes to viral hemagglutinin, host adaptation mutations in the basic polymerase subunit PB2 are recognized to be a critical step for avian influenza viruses to gain the ability to infect and replicate in mammalian cells ,10,11. VSince PB2-E627K is rarely found among H5N1 human isolates from Indonesia, it seems possible that, for the genetic constellation of Indonesian H5N1 viruses, optimal function of the polymerase complex for virus replication is independent of E627K, and instead requires K526R adaptive substitution. Similarly, a specific PB2 adaptation mechanism has been observed in 2009 pandemic H1N1, which utilizes 590S/591R, rather than 627K, for replication in humans . Becausehttp://gisaid.org), were analyzed for prevalence of the K526R substitution.All influenza viruses used in this study were rescued using the reverse genetics (RG) technique to ensure the genetic homogeneity of the virus. Viruses were propagated in embryonated chicken eggs (9–11 days old) for 24 h (P1) and titered by plaque assay in MDCK Cells . The two strains of Indonesian H5N1 viruses used in this study are a human isolate, A/Indonesia/5/05 (IND5), and an avian isolate, A/Chicken/Indonesia/2A/03 (CK2A) ,28. AddiLuciferase activity-based minigenome reporter assays were performed as described previously . The cod2. Culture supernatants were collected at different time points and viral titers determined by plaque assay in MDCK cells. MDCK and A549 cells were cultured at 37 or 33 °C, while DF-1 cells were cultured at 39 °C. MDCK, A549  and DF-1 cells were used in the virus growth kinetics study. In general, confluent cells were infected with RG virus at the indicated multiplicity of infection (MOI). The viral inoculums were removed after 1 h of adsorption at 37 °C and replaced with infection medium containing 1 µg/mL of TPCK-trypsin. Infected cells were further incubated at the indicated temperatures, supplied with 5% CO50 determination, groups of four or six mice were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine, and then intranasally inoculated with 25 µL of RG virus diluted in PBS, the dose range being 100–106 PFU. Body weight and survival were monitored daily for 14 days after infection. The MLD50 was calculated by the method of Reed and Muench [http://www. aaalac.org/about/guidelines.cfm). The “Guide for the Care and Use of Laboratory Animals, 2011, NRC, USA”  is the main document used by the HKU CULATR. The risk assessment of this study was approved by the safety committee on BSL-3 facility and infectious agents, Li Ka Shing Faculty of Medicine, the University of Hong Kong.BALB/c mice, aged 4–6 weeks, were obtained from the Laboratory Animal Unit, the University of Hong Kong. For MLDd Muench . To asseTo determine the expression levels of vRNA and viral mRNA in infected cells, A549 cells were infected with different RG viruses at an MOI of 1. Total RNA was extracted at the indicated time points and 1 µg of total RNA reverse transcribed using a High Capacity cDNA Reverse Transcription Kit . For detection of vRNA and mRNA, uni-12 and oligo dT primers, respectively, were used in the reverse transcription (RT) reaction. Expression of these two NP RNA species was quantified using SYBR Premix Ex Taq reagent (Takara) in an ABI 7500 Fast Real-Time PCR machine. For the detection of NP, NP-545s (5′-cagtgaaggggatagggaca-3′) and NP-746 as (5′-ccaggatttctgctctctcg-3′) primers were used. β-actin expression in each sample was used for normalization. Expression of NP genes was estimated by relative quantitative RT-PCR and normalized with the β-actin gene, in accordance with a previously described method . The ampMutation at the PB2 526 position was performed using the QuikChange Site-Directed Mutagenesis Kit (Agilent). Growth competition of the H5N1 avian wild type virus A/Chicken/Indonesia/2A/03 (PB2 526K and 627E) against its mutants (PB2 526R or PB2 627K) was carried out according to established procedures ,32. DF-1To study the interaction between NP and PB2 in the RNP complex, pCX plasmids expressing PB1, PA, NP and flag-tagged PB2 were transfected into HEK293T cells together with an RNP reporter plasmid. After 48 h, cells were lysed in cell lysis buffer . Cell debris was cleared by centrifugation. One microliter of laboratory made polyclonal NP antibody (1:3000) was added to the cell lysate and the mixture incubated for 3 h at 4 °C. Thirty microliters of washed Protein A sepharose beads  was added to the lysate, followed by incubation for 1 h at 4 °C. The beads were washed with the lysis buffer and finally boiled in SDS buffer at 95 °C. Proteins were resolved in SDS-PAGE and detected by Western blot using anti-NP and anti-Flag  antibody.Phylogenetic trees were constructed using the neighbor-joining method with the Tajima-Nei model of nucleotide substitution using MEGA X. All available sequences of H5N1 PB2, HA and PB1 from Indonesia were acquired from GenBank and GISAID for analysis. The bootstrap values from 1000 replicates were calculated to evaluate the reliability of the phylogenetic tree. Statistical analysis was carried out using GraphPad Prism V5 software .Apart from human cases infected with Qinghai-like viruses, which harbor 627K PB2 in avian species, and Indonesian viruses the later which rarely contain 627K PB2, about 30% of human isolates from rest of the affected areas mainly from China, Vietnam and Thailand contain 627K, with the E627K substitution presumably being acquired following infection. As reported previously, about 80% of H5N1 human isolates from Indonesia contain K526R PB2 . To undeTo understand if K526R substitution contributes to virus replication, we created two pairs of reverse genetic viruses based on A/Chicken/Indonesia/2A/2003  and A/Indonesia/5/2005 , which originally contained 526K and 526R PB2, respectively . MinigenInteraction between PB2 and nuclear exporting protein (NEP) may regulate the virus–host adaption process during virus replication, although a detailed mechanism remains elusive. Multiple functions of NEP during the influenza virus life cycle have start to emerge in recent years . PreviouTaken together, these results clearly show that K526R substitution in PB2 significantly enhances virus replication in MDCK and A549 cells, but does not attenuate virus replication in avian cells, supporting the contention that, similar to E627K, K526R is a host adaption marker important in the context of viral replicative function in humans.2 to 106 PFU, showed that both 526K and 526R PB2 viruses are virulent in mice at the higher dose inoculums of 104 to 106 PFU, with all infected mice dying between four and nine days of infection [3 PFU of IND5 virus still killed all infected mice, regardless of whether it was carrying WT-526R or back mutated 526K  reduces virus virulence in mice (We previously showed that K526R enhances avian H7N9 virus replication in mice when combined with 627K . Since tnfection . At loweted 526K A. In conecovered B. Measur in mice . WT IND5 in mice C. In sumAcquisition of the E627K substitution in PB2 following infection in humans or other mammalian hosts has been observed for avian H5N1, H7N7 and H7N9 viruses . Our resApart from seasonal H3N2 viruses arising since the 1970s and the Indonesian H5N1 viruses, few other circulating influenza viruses carry the PB2 polymerase K526R substitution. It was suggested that, subsequent to the 1968 reassortment event, which incorporated an avian PB1, acquisition of K526R in the PB2 gene of H3N2 enhanced the function of PB2-627K in the polymerase complex to optimize replication, and perhaps also transmission, of H3N2 in humans . UniquelCross species transmission of avian influenza A virus is restricted by host barriers which may include receptor specificity, replication fitness and host innate immunity. Emerging human infections with avian influenza viruses have increased in incidence over the past 20 years . Major cHistorical pandemics were caused either by reassortant viruses incorporating avian viral segments or whole animal viruses ,41,42. TIndonesia has the second largest total number of H5N1 human cases, with 199 recorded since 2005. However, most human H5N1 isolates from Indonesia do not harbor the E627K or D701N substitutions frequently observed in other human isolates from avian H5N1 or H7N9 virus infections. We previously reported that the K526R substitution enhances the effect of E627K in promoting replication of H3N2 and H7N9 viruses, and that K526R is present in 80% of H5N1 human isolates from Indonesia . Here, wThe functions of K526R-PB2 in H5N1 virus from Indonesia and E627K in H5N1 virus from Egypt to predispose towards the ability to replicate in humans suggests that mammalian host adaptive mutations can be stably acquired by avian influenza viruses and maintained in poultry. These adaptations do not hinder replication fitness of virus in avian species but provide H5N1 viruses with panzootic potential. Laboratory adaptation of H5N1 viruses in ferrets showed that only a handful of additional substitutions in the HA is sufficient to allow H5N1 viruses to transmit between animals, indicating that at least some genetic variants of currently circulating H5N1 viruses are likely able to replicate in humans but are merely restricted by receptor binding specificity ,10. Beca