We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies.Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other  Alphaherpesvirinae , ATRA , Sodium Valproate, TSA (Trichostatine A) and DMSO (Dimethyl sulfoxyde) were purchased from Sigma Aldrich and used as indicated in the text.Growth factors and cytokines  for cES cells growth were used as described . HMBA [NMonoclonal antibodies (Mabs) to GaHV-2 VP22, gB, ICP4 have been described , 24. Mab5’-TATCGATCGCATCTAGGGAGAACAGAGGGTTGGCGATAGTTGCCTGAATAgtgtaggctggagctgcttcg-3’) and Del37R (5’-TCCAAGACGGGACCGTTGACATAAACGCCCGGATGTGGAGAATAGATTTCcatatgaatatcctcctta-3’) primers, with MDV specific sequences shown in capital letters. BACs were purified by standard anion exchange techniques (Macherey Nagel—France) and characterized by RFLP analyses and southern blotting  BAC20  or the Bblotting .®—Mérial) and serially passaged either on primary cells. Mardivirus inocula consisted in infected cells trypsinized when cytopathogenic effect (CPE) was maximal, cryopreserved in 95% foetal bovine serum (FBS)/5% DMSO and stored in liquid nitrogen.Meleagrid herpesvirus 1 (MeHV-1), strain FC-126, was reconstituted from a lyophilised vaccine preparation (Lyomarex® (Intervet) and passaged on LMH cells as described [GaHV-1 (ILTV) was also reconstituted from the lyophilised vaccine preparation Nobilisescribed . In the aggcctATGTCTGCCGTAACGACCGA) and UL37R (ctgcagTTATGCATTATCACCGTTTG) primers with StuI and PstI sites underlined and MDV UL37 sequence in capital letters. The UL37 ORF sequence was first cloned in pGEM T Easy, sequenced  and then cloned, either in the transfer plasmid pFastBAC for baculovirus expression or in pCDNA3 for expression under the minimal CMV promoter.Plasmid pCDNA3-UL49 has been described . PlasmidPrimary chicken embryo skin cell (CESC) cultures have been described . The cESTelomerase assay , cell-cy5 cell/cm2 into gelatin (Sigma) coated culture dishes in William’s E (WE) medium (Lonza) with 10% FBS. After one day, the concentration of FBS was reduced to 2% and chicken serum (CS) (Gibco—Invitrogen) added to a concentration of 3%, in the same medium. Confluent monolayers were obtained 3 to 4 days after and exposed for 24 to 48 hours to the differentiating drugs in WE with 1% FBS and 1.5% CS. After the differentiating pulse, the cells were infected as described below.cES cells were seeded at 1x104 /cm2 in flasks containing an extracellular matrix from primary CESC, prepared as described [Embryonic Stem Cell Derived Line 1), cultured in DMEM-F12 with 10% FBS, and split twice a week at ratio of 1:3.For the derivation of ESCDL-1, cES cells were seeded at a density of 2 x10escribed . Cells w™ Nucleofector™ technology (Lonza—France) with the Basic Fibroblast kit (ref VPI-1002) and program F024. DNA amounts varied from 4 μg (plasmids) to 8 μg (BAC) for 4x106 cells. After transfection, the cells were seeded in 2 gelatin-coated wells of a 6-well plate for 8 to 16 hours. The medium was then changed to either DMEM-F12 with 1.5% CS and 1% FBS after BAC transfection, or to selection medium when ESCDL-1 were transfected with plasmids coding for viral or reporter genes. Selection medium after transfection of pCDNA3 consisted in DMEM-F12 with 10% FBS and 1 mg/ml G418 (Sigma Aldrich). When needed, plasmid pTK-Hyg (Clonetech), coding for the hygromycine B phosphotransferase (Hph), was co-transfected at a ratio of 1 to 20 with the plasmid of interest and selection was performed with hygromycine B at 80 μg/ml (Sigma Aldrich). Cells at 24 h post transfection with Venus/pCS2 and pTKHYG were either directly exposed to hygromycin or trypsinized and seeded in a 78 cm2 Petri dish to be exposed to hygromycin selection one day later.ESCDL-1 cells were transfected by using the AmaxaTo avoid any contamination of ESCDL-1 or differentiated cES cells by primary chicken cells, initial infection by avian herpesviruses was performed by using either transfection of BACs, FACS-sorted infected CESC  or cell-Infection with sorted infected cells was performed as described . For GaHMardivirus inocula from ESCDL-1 were prepared as described in the upper section.Titration of Mardiviruses was performed as described , except The GaHV-2 copy number in infected cells was quantified in a TaqMan real-time qPCR assay as described , 39 and ® 488 or 594 (Invitrogen—France). Polymerised actin (F-actin) was detected using Alexa Fluor® 594 Phalloidin (Invitrogen- France) and cell nuclei were stained by Hoechst 33342 (Invitrogen—France). Preparations were viewed under a Zeiss Axiovert 200 M microscope, and de-blurred images or optical sections were obtained by using the Zeiss ApoTome structured illumination microscopy (SIM) system. Photographs were obtained with a Zeiss digital camera AxioCam MRm, using Axiovision software. Images were compiled for publication with Adobe Photoshop CS6.Indirect immunofluorescence (IIF) was performed as described . SecondaCells were lysed in sample buffer and proteins were resolved on SDS-polyacrylamide gels , transfe4 PFU per well of vBAC20 virus at passage 2 after transfection on ESCDL-1 or mock-infected. Monolayers were lifted from the dish as specified in the manufacturer’s instructions, immediately fixed and processed as described [ESCDL-1 cells at passage 25 were grown in Nunc UpCell 6-well Multidishes (Nunc UpCell surface—Thermoscientific), infected with 2.9x10https://david.ncifcrf.gov/) and after the probe ID conversion, the GO analysis was performed with a p-value of 0.05 as a limit to define cluster of genes with predicted functions. The R package FactoMineR was used to perform PCA on all 44k probes using the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/) datasets GSE61221 for the CEF-1, GSE38168 for the CEF-2, GSE47191 for the DF1 cells.The cES cells and CESCs were subjected to RNA extraction after exposure to 5mM HMBA and infection by a cell-sorted inoculum of vBAC20UL49GFP, as described . Each ofhttp://www.helixio.com/). One μg of total RNA from ESCDL-1 at passage 31 from the initial cES cells was used to prepare the library using the TruSeq R Stranded mRNA sample preparation kit (Illumina). The average sizes, estimated by Bioanalyser (Agilent) and molarities, estimated by Qubit (Life Technologies) were of 268bp, 277 bp and 267 bp and of 157 nM, 202 nM and 94.5 nM for CEF, cES cells and ESCDL-1 respectively. The paired-end sequencing was performed on the NextSeq 500 sequencer (Illumina) and controlled by the SeqencingAnalysis viewer software (Illumina). The quality control of the ‘Sample-ID.fastq files was performed with the FasQC software (Babraham Institute). The total number of reads was of 85093694, 108943912 and 87951932 and the percentage of aligned reads reached 90.8, 82.9 and 82.2 for CEF, cES cells and ESCDL-1 respectively. The alignment was performed on the chicken genome  using the Hisat, subread and R (DEseq2) package [http://string-db.org/) was used to identify putative networks between proteins in order to better define cellular phenotypes [All the RNAseq analyses were performed by Helixio . Two independent samples were run each in triplicate and the StepOne Plus TM software provides the RQ value for each one with the RSP17 (X07257) as internal control using the ΔΔCt method.Quantitative real time PCR was performed using StepOne Plus TM Real Time PCR system (Applied Biosystems) as described . All oli2 (1.5 mM). After this differentiating pulse, cell monolayers were infected with vBAC20EGFPVP22 infected sorted cells. In all cyto-differentiating agents tested, HMBA, Ascorbic acid-CaCl2, DMSO (3 PFU/ml) compared to the standard CESC which consistently produced titres in the range of 5 x 105 PFU/ml. We exploited this one shot induction approach to identify genes that were up- or down-regulated in response to HMBA exposure.Initial tests performed to evaluate the level of replication of GaHV-2 in pluripotent cES cells showed that they were not susceptible to infection by the highly cell adapted vBAC20EGFPVP22, as concluded from the observation of infected monolayers at day 4 pi (data not shown). Infectivity evaluation assays included tests of media with reduced serum concentration, which induced differentiation in cES cells monolayers leading to cell morphological changes coinciding with an increase in permissiveness . Bearingl2, DMSO  and RA yl2, DMSO . Platingl2, DMSO , but thel2, DMSO . The arel2, DMSO . In thisIn order to identify the genes or pathways that could be implicated in the cell permissiveness to GaHV-2, we performed a comparative microarray analysis on CESCs and cES cells, treated or non treated with HMBA, and either infected and non-infected. A total of 1092 and 548 probe IDs, in CESCs and cES, respectively, were found to be differentially expressed in HMBA treated versus non-treated cells. On virus infected HMBA treated cells, only 425 and 18 probe IDs were differentially expressed in the both type of cells . These IThe Venn diagram  illustraUsing the datasets of primary fibroblasts (CEF) from two different origins, of DF1 fibroblastic cell line and of those CESCs and cES cells, treated and non-treated, a PCA analysis clearly established that the HMBA treatment resulted in a profile for the treated cES cells that resembled neither to CEF nor to DF1 cell line profiles .Interestingly, despite the very different origin of both cell types, when comparing both CESCs and cES treated versus non-treated cells, 96 probe ID (corresponding to 56 unique gene symbol) were found to be differentially expressed in both cell types, with more down-regulated (72) than up-regulated genes (18). Six genes  were found to be up-regulated in treated cES cells, but down-regulated in treated CESCs cells, and Histone genes , genes encoding transmembrane proteins , transferase proteins  and few other ones  were found to be up-regulated in both cell types . No geneOnly 18 probe ID (corresponding to 11 gene ID) genes were up-regulated by the virus in cES cells compared with the HMBA treated cES cells when 425 were found (271 up and 154 down) for the CESCs cells in the same conditions . Among t4 cells per cm2. The initial test for their susceptibility to GaHV-2 was performed on cells at passage 13 by using sorted cells infected with vBAC20 EGFPVP22vBAC20EGFPVP22, and proved to be satisfactory enough to further conduct ESCDL-1 characterization. ESCDL-1 cells displayed morphology of mesodermal cells with numerous actin stress fibers  prepared from primary CESC {Stromal cell-Derived Inducing Activity—SDIA } and to s fibers  and stros fibers . Telomers fibers . Cell cys fibers  and a ras fibers .The complete transcriptomic analysis of ESCDL1 was performed by deep RNA sequencing comparatively to the cES cells and primary embryonic fibroblasts (CEF) . The acq6 of ESCDL-1 at passage 28 , we transfected the BAC20UL17mRFP (5.4 μg) in 4.5 x10ssage 28 . Viral pssage 28 . Viral t5 PFU/ml on primary CESCs vs 2.2±0.22 x 105 PFU/ml on ESCDL-1) while infectious titres from T1P6 virus production on CESC were two-fold lower compared to those obtained for T1P6 production on ESCDL-1 . To evaluate whether the susceptibility of ESCDL-1 could vary over time, we passaged vBAC20UL17mRFP twice on ESCDL-1 at low (<50), intermediate (51<P<90) or high (>95) passages. Initial virus production was performed with T1P8 virus on ESCDL-1 at passages 37, 57 and 97 (MOI of 0.02), yielding T1P9 virus, which in turn was used to produce T1P10 virus on ESCDL-1 at passages 47, 69 and 104 (MOI of 0.01 to 0.02). The resulting viral progenies were titrated and no deleterious effect regarding the viral production on cells at high vs. low passages could be identified, over two serial passages  and vBAC20 (T1P2) revealed a minor difference in plaque size, as the mRFP virus showed slightly larger plaques than the parental virus . Kineticpassages .5 PFU/ml and the titre of vUL47-mRFP at the same passage was 1.2±0.23 × 105 PFU/ml.The permissiveness of ESCDL-1 for the replication of GaHV-2 was also assayed by using either vBACRB-1BUL17mRFP, the counterpart of vBAC20UL17mRFP in the RB-1B context, or vUL47-mRFP . After t4 in the inoculum to 1.61 (± 0.14) × 105 for passage 2 on primary CESCs and 9.7 (± 0.62) × 104 in ESCDL-1 to reach 1.73 (± 0.17) × 105 for passage 3 (corresponding to the second passage in ESCDL-1).The replication of HVT (MeHV-1) was also assessed on ESCDL-1 by using a first passage of FC126 virus on CESC as an inoculum, at a split ratio of 1/23. A second passage was performed on ESCDL-1 (at a ratio of 1/7) and titrated. The titres progressed from 3.5 (± 0.4) × 10The susceptibility of ESCDL-1 cells for GaHV-1 (ILTV) was also tested, in comparison with the LMH cells, and ESCDL-1 did not support ILTV replication (data not shown).A study of the morphogenesis on ESCDL-1 of cell-adapted GaHV-2 was performed by using vB20 at passage 2 after transfection of the corresponding BAC on ESCDL-1. Infected cell monolayers were harvested without enzymatic dissociation to preserve the integrity of cell connections and surface and collect cells at all stages of infection. Compared to the mock-infected cells , infecte5 PFU/ ml) although approximately two-fold lower for the virus produced in EV7  as a transgene . From thd in EV7 . In the d in EV7 . Interesd in EV7 .Having established the proof of concept on the use of transgenic ESCDL-1 as a cell substrate for GaHV-2, we addressed the question of generating trans-complementing ESCDL-1 clones. We examined the trans-complementation of 2 GaHV-2 mutants in which the essential genes UL49 and UL37 had been invalidated . Trans-c). We further explored the possibility of serially passaging the deleted virus on the complementing cells, in comparison with the parental virus. The development of viral plaques was monitored at passage 2 and 3  were prepared as described  from 12 S1 FigBlnI digestion (B.a) and Southern blotting (B.b) of BACRB-1BΔUL37 clones 1 & 2  compared to BACRB-1BΔUL47 clones  and pRB-1B BAC clone . BlnI digestion of BACRB-1BΔ37 generates an additional fragment (A 1 and 2—arrowhead). As a control BACRB1BDel47 clones (lanes 3 to 5) and BACRB1B (lanes 6 and 7) were submitted to the same digestion. The KanR gene in BACRB1B-Del37 is associated with a 5560 bp fragment (B.b lanes 1 and 2) corresponding to BlnI fragment 85095–90660. In BACRB1B-Del47, a 31,5 kbp fragment contained the KanR cassette. The “SmartLadder” from Eurogentec was used as a size standard (M).A) Monoclonal antibody AA8 stains Baculovirus-UL37 infected Sf9 cells; Nuclei were counterstained with Hoechst 33342 and an AlexaFluor488 goat anti-mouse conjugate was used to reveal the bound antibodies (bar represents 50μm). B) RFLP analysis of mutated BACRB-1B clones after (TIF)Click here for additional data file.S2 FigTelomerase activity (A), cell-cycle (B) and growth curve. A) Telomerase activity of CESC (1), DF1 (2), LMH (3), ESCDL-1 (4) and CLEC 213 (5) cells. B) Cell cycle analysis of ESCDL-1 and primary CESC at 24 and 48 h post plating. C) Growth curve of ESCDL-1 over 25 days.(TIF)Click here for additional data file.S3 FigThe networks were obtained by using the STRING software with the first 150 most differentially expressed genes in ESCDL-1 compared with the initial cES cells as listed on (TIF)Click here for additional data file.S4 FigThe expression of different genes were validated by real time RT-PCR, including ECM2, CPM, DPT and FABP4 found down-regulated in CESC after HMBA treatment (A), OLFM3, IGFBP2, ID2 and THBS2, found up-regulated in CESC after HMBA treatment (B), NANOG, SCNN1A, LCN8 and ATP12A, found down-regulated in cES cells after HMBA treatment (C), KRT15, SCD5, WISP1 and THBS4 found up-regulated in cES cells after HMBA treatment (D), NAPEPLD, LGALS1, IRF1 and AK1 found down-regulated in both CESC and cES cells after HMBA treatment (E), MGAT3, HS6ST3 and TERG1L found up regulated in both CESC and cES cells after HMBA treatment . Expression was also analysed in CEF and DF1 fibroblasts, in LMH, and in ESCDL1 cells. Expression was taken to be 1 in cES as a reference, and two independent samples were run, each in triplicate. Error bars indicate SD.(TIF)Click here for additional data file.S5 FigA and A’) Overview of a non-infected cell. Due to the elongated shape of the cells only the nucleus region is presented to illustrate the characteristic morphology of these cells. A’ inlet shows a magnification of the boxed zone in A. B) Overview of a highly infected cell undergoing vacuolization. Intranuclear A/B/C capsids are present and a primary enveloped particle can be seen in the nuclear membrane lamina (magnification of the boxed area). Intracytoplasmic capsids are encircled in white in the boxed area. Numerous A and C capsids can be seen in the vacuole  in which electron dense material reminiscent of viral tegument particle (L particles in HSV-1) is also present (black circle).(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The legends for the Supporting Information files S2 FigThe black and gray bars indicate individuals with cerebellar ataxia and neurologically healthy controls, respectively, recruited in this study. The average estimated copy number for each probes was around 2, similar to those of the healthy controls.(TIFF)Click here for additional data file.S3 FigThe sequences between the human and rat IP3R1 are 98.5% identical. The crystallographic structure of rat IP3R1 (PDB: 3JAV) [39] was visualized with UCSF Chimera software [40]. Each single subunit of IP3R1 was colored with yellow, green, pink and blue, respectively. The residue V2574 was labeled with black. V2574 residue locating near the pore-forming region was visualized by the side-view of a single subunit of IP3R1 (A), the side-view of a tetrameric IP3R1 structure (B), the top-view of a tetrameric IP3R1 structure (C), and a close-up top-view focusing on the V2574 residue of each subunit (D).(TIFF)Click here for additional data file.


Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. E. coli species are currently divided into 183 O-groups (lipopolysaccharide) and 53 H-types (flagellar antigen)  and wzy (O-antigen polymerase) or wzm (O-antigen ABC transporter permease gene) and wzt (ABC transporter ATP-binding gene), appear highly specific for each serogroup. These can serve as targets for molecular determination of the serogroup by PCR, qPCR or nucleotide sequencing  frequently associated with extra-intestinal diseases in humans and poultry  constitute an emerging public health problem  strains, associated with colibacillosis, a serious illness in poultry worldwide, are the cause of significant economic losses in poultry farms. Poultry strains share some ExPEC virulence traits with human O1:K1:H7 or O2:K1:H7 isolates and with other ExPEC strains frequently isolated from human patients  such as O1:H20, O1:H7, O1:H2, and O1:H1 were sporadically isolated from feces of human patients with diarrhea and STEC O2:K1:H7 have caused sporadic cases of HUS in humans  O2 strains such as O2:H40, O2:H49, or O2:H8  were described  at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany, the associated French National Reference Center for E. coli at the pediatric Hospital Robert-Debré in Paris (France), and from the French Agency for Food, Environmental and Occupational Health and Safety (Anses) in Maisons-Alfort, France.E. coli strains used to test for the specificity of the qPCR assays included in particular the E. coli reference strains belonging to serogroups O1-O186 and H-types H1-H56. E. coli strains carrying the capsular antigen K1 , the O50 reference strain U18-41 and two EPEC O2 strains CB11127 (O2:H49) and CB15123 (O2:H40) were investigated for the gene sequences of their O-antigen locus  and CB14293 (O1:H20), the All strains were grown overnight at 37°C in Luria broth, and DNA was extracted according to the manufacturer's instructions using InstaGene matrix .E. coli O1 and O2 antisera following previously published protocols  and H (flagellar) antigens of the strains received at the BfR was performed with O-specific and H-specific rabbit antisera prepared according to standard methods  and two qPCR-negative O2 strains (CB11127 and CB15123) were determined (see below). A qPCR for the variant wzx gene present in O1 strains CB13533 and CB14293 was developed and used for further typing of the collection of E. coli O1 strains. In a similar way, two qPCRs for the variant wzx genes of CB11127 and CB15123 were developed and used for molecular typing of E. coli O2 strains  in 25-μl reaction volumes, a Light Cycler 1536  in 1.5-μl reaction volumes or a BioMark  in nanofluidic format according to the recommendations of the suppliers. Primers and TaqMan probes were used at 300 nM final concentrations. The following two-steps thermal profile was applied to all instruments: enzyme activation at 95°C for 1–10 min (as recommended depending on the enzyme used), followed by 40 cycles of denaturation at 95°C and annealing at 60°C.galF and gnd genes, using the Expand Long Range PCR system (Roche) and either the 1523/1524 primers , 412/482 primers (CB15123), or 412/482b primers (CB11070) as described previously , producing a single contig for each sample. The sequences were annotated using PROKKA  medium using the DNeasy blood and tissue kit (Qiagen). The O-antigen cluster of the strains was amplified by long range PCR between the Seemann,  on a GalSeemann,  and BlasThe annotated O-antigen cluster sequences of these strains were deposited in DDBJ/ENA/GenBank under the accession numbers listed in Table E. coli wzyO1 gene derived from the sequence of the E. coli O1 strain G1632 (U5-41) . Besides the rmlBDAC genes, which are fairly common genes among various serogroups  of strains CB11070, CB13533, and CB14293 are approximately 10 kb in length; they all contain 10 ORFs between the d Figure . The ovegalF and gnd in the same transcriptional order from galF to gnd  and a glycosyl transferase. However, the second half of the cluster harbors a different set of glycosyl transferase genes. The nucleotide sequence of the wzx gene of strain A47 is somewhat related to that of strains CB11070, CB13533, and CB14293 as they share 89% identity (89.1–89.2%). As expected, it is distantly related to that of G1632 (54.4% identity) and no significant similarity was found in the nucleotide database of Genbank (Supplementary Table mnaA gene and glycosyl transferase (PROKKA_00007) following wzx do not reveal any significant similarity in the nucleotide database of Genbank (Supplementary Table wzy gene however is very different from that of G1632 (44.7% identity) and to those of the 3 other strains (48% identity). A blastn search indicates that it is 89% identical to the wzy gene of O10:K5:H4 strain Bi8337-41. Interestingly, a blast analysis of the glycosyl transferase genes surrounding wzy  shows 90–94% identity to the O10 strain Bi8337-41 O-AGC  are significantly similar to a single sequence in Genbank nucleotide collection corresponding to strain FHI98 (strain of unknown serotype isolated from human feces in 2011)  in Genbank nucleotide collection , while the wzxO1non-A qPCR assay reacted only with strains of serotype O1:H12 (n = 1), O1:H20 (n = 3), O1:H21 (n = 1), and O1:H33 (n = 1). Thus, the qPCR detection method was found to be far more specific for identification of E. coli O1 strains than conventional O-serotyping. Moreover, the qPCR assays used here discerned clearly between E. coli O1A and O1non-A type strains, respectively.Major differences between O1A and O1non-A strains were found in their corresponding ed Table . We haveed Table , includiwzx sequences of strains U9-41 (O2:K1:H4), CB11127 (O2:H49), and CB15123 (O2:H40) allowed us to design three qPCR assays, targeting the O2/O50 and two O2-like serogroups respectively (Table n = 2), O2:K5:H1 (n = 1), O2:K1:H1 (n = 2), O2:K5:H4 (n = 2), O2:K1:H4 (n = 3), O2:K1:H5 (n = 3), O2:K1:H6 (n = 4), O2:K1:H7 (n = 1), O2:H25 (n = 4), O2:H27 (n = 3), O2:H32 (n = 1) and O2:K1:H48 (n = 1). The wzxO2-2 qPCR assay reacted with EPEC and STEC strains of serotype O2:H34 (n = 1), O2:H49 (n = 3), and O2:H29 (n = 2). The wzxO2-3 qPCR reacted only with EPEC strains of serotype O2:H8 (n = 1) and O2:H40 (n = 4).The divergence between the neuB qPCR for indicating strains producing the K1 antigen was tested on the serotype reference strains O1-O186 which have been analyzed for their capsular antigens  we developed a qPCR assay for specific detection of O1 strains. While this assay appeared highly specific, as it did not cross react with any other serogroups, several strains identified as O1 with the seroagglutination failed to react. In the same way, a highly specific qPCR assay developed based on the wzx gene of the O2 reference strain G1674 failed to react with several strains identified as O2 with the seroagglutination. The qPCR assay developed for specific detection of the neuB gene of K1 strains proved 100% sensitive and 100% specific.In an effort to build a complete molecular serotyping scheme for L-rhamnose residues, an N-acetylglucosamine residue and an N-Acetyl-D-mannosamine residue. The O1B/O1C variants contain two L-rhamnose residues, an N-acetylglucosamine residue, an N-Acetyl-D-mannosamine residue and a D-Galactose residue  derived from GDP-D-Mannose. Presence of rmlA, rmlB, fdtA, fdtB, and fdtC suggest that the N-acetyl-D-galactosamine residue is conserved. Strain CB15123 conserved both the ability to synthesize L-rhamnose residues and N-acetyl-D-galactosamine residues and also acquired the GDP-D-Mannose pathway. Once again, presence of other residues whose biosynthesis genes are located outside of the O-AGC cannot be ruled out.The O-AGC found in O2 strains CB11127 and CB15123 constitute two new O2-like serogroups. The genetic structure of the O-AGCs in strains CB11127 and CB15123 are different from that of the O2 reference strain G1674 suggesting different LPS structures. The O2 polysaccharide contains three L-rhamnose residues, an Based on the nucleotide sequence of the new O1-variant and the two O2-like serogroups, specific qPCR assays were developed, which proved to be 100% sensitive and 100% specific, and able to clearly distinguish the different genetic variants identified within each serogroup.E. coli strains of serogroups O1 and O2. They provide a significantly improved alternative to classical O-serotyping by their high specificity and sensitivity to detect virtually all types of O1 and O2 strains and their respective associated variants. They are therefore potentially useful for direct identification of clinically important O1 and O2 strains in food and clinical specimens. These different qPCR assays can thus serve as tools for epidemiological investigations and source attribution of human infections. Molecular typing by use of specific qPCRs provides a more specific detection of clinically important O1 and O2 ExPEC strains. Ambiguous results obtained by serotyping such as cross-reactions with other E. coli O-groups and agglutination reactions with O-rough strains are avoided. Moreover, the qPCR approach described in this study provides a better discrimination between O1 and O2 ExPEC and non-ExPEC strains. In combination with the detection of the neuB gene (K1 capsule), life-threatening O1 and O2 ExPEC infections in risk groups such as newborn infants can be more rapidly identified. Asymptomatic carriers, animals and food contaminated with O1 and O2 ExPEC strains can be more easily identified and the spread of pathogens to human risk groups  can thus be prevented. The qPCR detection of ExPEC O1 and O2 strains is a reliable and easy approach to elucidate the significance of zoonotic and food-borne infection of humans with ExPEC O1 and O2 strains in epidemiological investigations.Overall, the various qPCR assays designed in this study can be used for detection and rapid identification of the different types of Conceived and designed the experiments: LB, SD, and PF. Performed the experiments: SD, LB, PM, AF, and SB. Analyzed the data: SD, LB, PM, AF, SB, and PF. Contributed reagents/materials/analysis tools: SD, LB, PM, SB, and PF. Wrote the paper: SD, LB, and PF. Critical revision of the paper for important intellectual content: SD, LB, PM, AF, SB, and PF.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.


Toxoplasma gondii is one of the most successful human pathogens, infecting an estimated 2.5 billion people across the globe. Pathogenesis seen during acute or reactivated toxoplasmosis has been closely tied to the parasite’s intracellular lytic life cycle, which culminates in an event called egress that results in the release of freshly replicated parasites from the infected host cell. Despite the highly destructive, cytolytic nature of this event and its downstream consequences, very little is known about how the parasite accomplishes this step. Previous work has suggested a role for a secreted phospholipase, LCAT, in Toxoplasma egress and roles in cell traversal and egress in the Plasmodium species orthologue. We confirm here that LCAT-deficient tachyzoites are unable to efficiently egress from infected monolayers, and we provide evidence that LCAT catalytic activity is required for its role in egress. Toxoplasma gondii intracellular lytic cycle. This is a process that drives inflammation and is strongly associated with the pathogenesis observed during toxoplasmosis. Despite the link between this process and virulence, little is known about egress on a mechanistic or descriptive level. Previously published work has suggested that a phospholipase, lecithin-cholesterol acyltransferase (LCAT), secreted from the parasite’s dense granules contributes to parasite growth, virulence, and egress. Here we present evidence from several independent mutant parasite lines confirming a role for LCAT in efficient egress, although no defects in growth or virulence were apparent. We also show via genetic complementation that the catalytic activity of LCAT is required for its role in parasite egress. This work solidifies the contribution of LCAT to egress of T. gondii tachyzoites.Egress is a crucial phase of the IMPORTANCEToxoplasma gondii is one of the most successful human pathogens, infecting an estimated 2.5 billion people across the globe. Pathogenesis seen during acute or reactivated toxoplasmosis has been closely tied to the parasite’s intracellular lytic life cycle, which culminates in an event called egress that results in the release of freshly replicated parasites from the infected host cell. Despite the highly destructive, cytolytic nature of this event and its downstream consequences, very little is known about how the parasite accomplishes this step. Previous work has suggested a role for a secreted phospholipase, LCAT, in Toxoplasma egress and roles in cell traversal and egress in the Plasmodium species orthologue. We confirm here that LCAT-deficient tachyzoites are unable to efficiently egress from infected monolayers, and we provide evidence that LCAT catalytic activity is required for its role in egress. Toxoplasma gondii is an obligate intracellular parasite that infects a wide range of host animals, including humans. This parasite is estimated to infect up to one in three people worldwide  formed from the host plasma membrane during invasion  was originally described in nfection . More reatocytes . Since tl strain .Toxoplasma gondii utilizes during egress, we have focused our attention on T. gondii LCAT (TgLCAT), hereafter referred to simply as “LCAT.” During the course of this study, we consistently observed that parasites lacking LCAT were unable to complete the egress event as efficiently as their wild-type counterparts. However, unlike the previously described LCAT mutant  selectable marker. After transfection of a linear knockout construct -tagged wild type (WT) gene (ΔlcatLCAT-HA) and a mutant allele (ΔlcatLCAT*-HA), which harbors an S332A point mutation predicted to destroy catalytic activity from the active site serine. These constructs were integrated into the “empty” Δku80 locus by double homologous recombination  were infected, and the parasites were allowed to replicate undisturbed for 7 days before fixation and staining of the monolayer . Quantifre found  and D.lcat strain in the acute model of murine infection. Due to the highly virulent nature of the type I strain background, we chose to use a low dose of 10 tachyzoites administered subcutaneously to BALB/c mice. By 10 days postinfection, infected mice were displaying signs of morbidity, and the mice were euthanized by 10 to 15 days postinfection. No statistically significant differences in survival were seen, as mice infected with parental, knockout, or complemented strains succumbed to the infection with similar kinetics. To investigate a potential role for LCAT in vivo more thoroughly, we recreated the knockout in the wild-type RH background. LCAT-deficient parasites were generated using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system  to allow for easy identification and differentiation of RH versus RHΔlcat parasites. Both parental and knockout parasites were inoculated into T25 flasks of confluent HFFs, and after each lysis, parasites were collected for quantification via fluorescence microscopy. The results of the in vitro competition assay mirror what was seen in previous growth assays, as there were no significant differences in relative abundance after serial coculture  or knockout (RHΔlcat or RHΔku80Δlcat) parasites. After 30 h of replication, the infected monolayers were then treated with zaprinast to induce egress pharmacologically. Zaprinast is a phosphodiesterase inhibitor that induces egress by activation of the parasite protein kinase G (PKG)  . The culectively  and B. Tectively . These fToxoplasma (PLP1)  , 20, 21,a (PLP1)  that coua (PLP1) , 24. Thend DPAP3 –27 in add DPAP3 ––30.Plasmodium berghei phospholipase (PbPL), was described in P. berghei, which facilitates sporozoite cell traversal  releasing unknown egress factors from sequestration in the parasitophorous vacuole or (ii) allowing the influx of host/environmental factors that stimulate egress. For example, disruption of the HPM and PVM would allow influx of serum components, including serum albumin, which enhances parasite calcium signaling, microneme secretion, and gliding motility via activation of PKG .2+ (ARC) entry channels, encoded by Orai2 and Orai3 proteins . In mammalian cells, AA activates plasma membrane AA-regulated Caproteins . Downstrproteins . Additioproteins , as compAnother interesting aspect of this study is that an egress effector secreted constitutively from the dense granules must somehow be functionally regulated, so as not to induce premature egress of the tachyzoites. Two potential methods of regulation are proteolytic processing and pH. Recently, a subset of dense granule proteins were shown to be processed at PEXEL motifs by the Golgi resident aspartyl protease ASP5 . LCAT coPlasmodium spp., with stage-specific expression of different PFTs and stage-specific activity of PbPL (sporozoite transmigration versus merozoite egress). While in these systems a direct role has not been established for cooperation between these two molecules, there are established examples of PFTs directly enhancing phospholipase activity, as is the case in bee venom . All efforts were made to minimize pain and suffering of the mice.Toxoplasma tachyzoites were maintained by serial passaging and growth in human foreskin fibroblast (HFF) cells. Cell cultures were grown in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum , 2 mM glutamine, and 10 mM HEPES and grown in 5% CO2 at 37°C. Parasites were liberated by scraping the infected HFF monolayer and passage through a 27-gauge needle. The liberated parasites were then filtered through a 3-µm size filter (Millipore), counted on a hemocytometer, and added to HFFs at the appropriate density. For routine culture of the parasites, 5 drops of naturally egressed parasites were passed into fresh host cells in a T25 flask.dhfr selectable marker was amplified from pYFP.LIC.DHFR (Addgene) with primer pair ajsP20 plus ajsP21. These three products were used as the template and fused via a final PCR to primers ajsP2 and ajsP5. The linear vector was transfected into RHΔku80 parasites, and stable clones were isolated based on pyrimethamine resistance. The complement construct (pLCAT.Ku80.HXG) was created via Gibson cloning . The vector pM2AP.Ku80.HXG was prepared by double digestion with AscI plus SpeI and purified by gel extraction (Qiagen). The 5′ and 3′ lcat flanking sequences were amplified from RH gDNA with primer pair ajsP62 plus ajsP52 and primer pair ajsP63 plus ajsP59, respectively. The lcat cDNA was amplified from a cDNA library with primer pair ajsP53 plus ajsP58. The catalytically dead construct, pLCATs332a.Ku80.HXG, was generated using site-directed mutagenesis  and primers ajsP64 plus ajsP65. The constructs were linearized prior to transfection by double digestion with KpnI plus ApaLI and transfected into RHΔku80Δlcat parasites, and stable clones were isolated based on resistance to mycophenolic acid (MPA) plus xanthine (Xan). For creation of RHΔlcat lines using CRISPR-Cas9, 20 bp of lcat-specific guide sequence was inserted into pCRISPR-Cas9-Ble, using site-directed mutagenesis and the primer pair ajsP200 plus ajsP201. PCR was used for genetic confirmation shown in ku80 locus (indicated by the red B in ku80 locus (indicated by the red C in The knockout construct was constructed via fusion PCR. 5′ and 3′ homologous flanks were amplified from RH genomic DNA (gDNA) using primer pair ajsP1 plus ajsP22 and primer pair ajsP6 plus ajsP23, respectively. The The following primers were used in this study: ajsP1, CTGCATGGGACACAAACAGT; ajsP2, TCGTCACAGCCATCGAAATC; ajsP5, TCGGTCACTGAGCAGCTAG; ajsP6, CAGTTACTTTCAGATCCAACC; ajsP15, ATGGACTTCCTCTCTGGAGG; ajsP19, AGGCTCGAGGAGGATGTTTA; ajsP20, GAGGTCGACGGTATCGATAA; ajsP21, TAGAACTAGTGGATCCCCCT; ajsP22, TTATCGATACCGTCGACCTCTCTGGAAGGAGCGGAACAC; ajsP23, AGGGGGATCCACTAGTTCTATAGAGGGAACTGAGTCGGGA; ajsP52, CCTCCAGAGAGGAAGTCCATTTCTGGAAGGAGCGGAACACT; ajsP53, AGTGTTCCGCTCCTTCCAGAAATGGACTTCCTCTCTGGAGG; ajsP58, CGTAGTCCGGGACGTCGTACGGGTACGTGCTGTCTGCCATAATCG; ajsP59, GTACGACGTCCCGGACTACGCGTAATAGGAGGGAACTGAGTCGGGA; ajsP62, CTTGGTCGTAAGAGAAGAGGAGCGCCCTGGAACAACATAACACA; ajsP63, ATTCGCACCCTCCAAACTAGTGGTCCGAGCGTTTGTTGCAA; ajsP64, AAAGTTGACCTGATCGCCCACGCCTTGGGCAGCATTATTCTGTGT; ajsP65, ACACAGAATAATGCTGCCCAAGGCGTGGGCGATCAGGTCAACTTT; ajsP138, CGTAGGTGGCGTTCATGTAGTAGTCTAGGT; ajsP142, AGGCGACGACAAAGCCGGATCCTCGAGCATAT; ajsP143, GAGGATCCGGCTTTGTCGTCGCCTTTCTCC; ajsP144, GACTACGCGTAAGCTAACAAAGCCCGAAAG; ajsP145, TTTGTTAGCTTACGCGTAGTCCGGGACGTC; ajsP200, GGCACTGGCAAACCAAAGAAGTTTTAGAGCTAGAAATAGC; ajsP201, TTCTTTGGTTTGCCAGTGCCAACTTGACATCCCCATTTACC; XhoI_ku80_5\’Flank.f, GACTCGAGTGGAGATCCAAGCGAGGACTTA; and XhoI_ku80_5\’Flank.r, GACTCGAGGCTTCGAGTCGTCTGTTCTGG.Infected monolayers in a T25 flask were washed three times with 37°C phosphate-buffered saline (PBS) prior to scraping and host lysis as described above. Parasite suspensions were made via serial 10-fold dilutions to reach a concentration between 75 and 150 parasites/150 µl PBS. Monolayers of HFFs in individual six-well plates were immediately inoculated with 150 µl of the parasite solution. The infected monolayers were then incubated, undisturbed, for 7 days prior to fixation with 2% crystal violet. Plaques were quantified using ImageJ on wells that had been digitally scanned with a ruler included for scale.Infected HFFs were fixed with 4% paraformaldehyde at either 16 or 32 h postinfection. The fixed monolayers were permeabilized with 0.01% Triton X-100 and labeled with rabbit anti-SAG1 and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). At least 250 vacuoles were quantified per strain per experiment.lcat coding sequence was amplified with primers ajsP143 plus ajsP145 from a Toxoplasma cDNA library and was subcloned into pET15b via Gibson assembly.The bacterial expression construct pET15b was linearized with inverse PCR (iPCR) with primer pair ajsP142 plus ajsP144. The T. gondii tachyzoites subcutaneously in 150 µl of PBS. Delivery of an accurate dose of infectious parasites was confirmed by performing plaque assays in parallel (described above). In the event of mice surviving the infection, seropositivity was tested by enzyme-linked immunosorbent assay (ELISA).Groups of five female BALB/c or Swiss Webster (Jackson) female mice, aged 6 weeks, were infected with either 10 or 50 lcat parasites were transfected with a glycosylphosphatidylinositol (GPI)-anchored green fluorescent protein (GFP) variant, and stable clones were selected following treatment with pyrimethamine. Extracellular parasites were counted on a hemocytometer, and roughly equal numbers (5 × 105) of both RH and RHΔlcatGFP parasites were coinoculated into a T25 flask of HFFs. Following lysis of the host cell monolayer, extracellular parasites were collected, filtered, and fixed on poly-l-lysine-coated glass slides. Parasites were immunolabeled with rabbit anti-SAG1 and enumerated via fluorescence microscopy. This process was repeated for a total of three passages.RHΔ5/ml, and 100 µl was used per well (5 × 104 tachyzoites). Prior to egress assays, infected wells were washed three times with 37°C Ringer’s buffer. After the final wash, the cells were treated with either 57 µM zaprinast diluted in Ringer’s buffer or Ringer’s solution with an equal volume of dimethyl sulfoxide (DMSO). The treated plates were allowed to incubate at 5% CO2 and 37°C for 20 min before removal and immediate placement on ice. Fifty microliters of the supernatant was removed and placed into individual wells of a 96-well round-bottom plate. The round-bottom plates were centrifuged at 4°C at 500 × g for 5 min. Thirty microliters of solution was removed, stored at 4°C, and used within 1 h as source assay material for lactate dehydrogenase (LDH) content (BioVision).Thirty hours prior to assay, infected monolayers in a T25 flask were washed three times with 37°C PBS prior to scraping and host lysis as described above. Following centrifugation, parasites were resuspended to a density of 5 × 10


To elucidate the adaptive strategies of influenza A viruses (IAVs) to human, we proposed a computational approach to identify human-adaptive mutations in seasonal IAVs, which have not been analyzed comprehensively. We compared representative PB2 sequences of 1425 avian IAVs and 2176 human IAVs and identified a total of 42 human-adaptive markers, including 28 and 31 markers in PB2 proteins of seasonal viruses H1N1 and H3N2, respectively. Notably, this comprehensive list encompasses almost all the markers identified in prior computational studies and 21 novel markers including an experimentally verified mutation K526R, suggesting the predictive power of our method. The strength of our analysis derives from the enormous amount of recently available sequences as well as the recognition that human-adaptive mutations are not necessarily conserved across subtypes. We also utilized mutual information to profile the inter-residue coevolution in PB2 protein. A total of 35 and 46 coevolving site pairs are identified in H1N1 and H3N2, respectively. Interestingly, 13 out of the 28 (46.4%) identified markers in H1N1 and 16 out of the 31 (51.6%) in H3N2 are embraced in the coevolving pairs. Many of them are paired with well-characterized human-adaptive mutations, indicating potential epistatic effect of these coevolving residues in human adaptation. Additionally, we reconstructed the PB2 evolutionary history of seasonal IAVs and demonstrated the distinct adaptive pathway of PB2 segment after reassortment from H1 to H3 lineage. Our study may provide clues for further experimental validation of human-adaptive mutations and shed light on the human adaptation process of seasonal IAVs. The transcription and replication of influenza viruses are catalyzed by the viral polymerase complex composed of three subunits: polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acidic protein (PA). The PB2 subunit binds the 5′ 7-methylguanosine cap of host pre-mRNAs, which are subsequently cleaved off 10–15 nucleotides downstream by PA3. PB1 protein, a viral RNA-dependent RNA polymerase, catalyzes the addition of nucleotides to the resulting capped short RNA primer and initiates viral transcription. The cooperation between the polymerase complex subunits is essential for viral replication and transcription4.Influenza A virus (IAV) genome consists of 8 negative-sense RNA segments encoding 11–12 proteins5. The host spectrum of influenza virus is mainly dictated by hemagglutinin (HA) glycoprotein and the viral polymerase complex4. Specific mutations in the receptor-binding domain of HA alter the specificity and affinity for the receptor and affect host tropism, while the mutations of viral polymerase proteins influence viral replication efficiency in new hosts4. Particularly, a single PB2 mutation E627K can cause highly increased replication efficiency and enhanced pathogenicity8, enabling the replication of avian-origin IAVs in human cells. In addition, the PB2 mutations D701N10, G590S/Q591R12, and K526R13 significantly increase replication efficiency in mammalian hosts.Migratory waterfowl is the natural reservoir of avian IAVs, from which IAVs are transmitted into other hosts, such as humans, domestic poultry, swine, and other species14. Amino acids L473V and L598P of PB1 protein from an avian-origin IAV contributed to higher polymerase activity, especially in mammalian cells15. Non-structural protein 1 mutations F103L and M106I led to increased viral growth of a human H5N1 isolate in vitro (mouse and canine cells) and enhanced virulence in mice16. Furthermore, epistatic effects of combinatorial mutations have been observed in IAV studies. Epistasis describes non-additive interactions among genetic sites, namely, the consequence of a mutation at one site depends on the presence of mutations at other sites. Epistasis commonly exists and plays an important role in immune escape and drug resistance in various pathogens17. In terms of the epistasis in IAVs, three PB2 mutations I147T, K399T, and A588T showed marginal effect when individually introduced into H5N1 but highly increased the polymerase activity when introduced in combination18.Studies of human adaptation have been extended to other viral proteins. For example, T85I, G186S, and L336M in PA protein were identified to increase the polymerase activity of 2009 pandemic H1N1 (pH1N1) virusTo date, most of the human-adaptive mutations have been identified from epidemic or pandemic influenza virus isolates. However, those in seasonal human IAVs such as H1N1 and H3N2 subtypes remain poorly investigated. To fill the knowledge gap, we conducted a large-scale sequence analysis to identify the potential human-adaptive mutations in seasonal IAV PB2 protein and infer the adaptive evolutionary history of seasonal IAVs.1, we aim to compare the PB2 segments of seasonal IAVs to avian IAVs for the discovery of potential adaptive markers in seasonal human IAVs. We surveyed 3457 and 6690 PB2 sequences of avian and human IAVs, respectively, with collecting years spanning from 1918 to 2016, in order to obtain a large pool of viral sequences. The distributions of collecting date and subtype were calculated. Avian IAVs are more diversified with 86 subtypes compared to 9 subtypes of human IAVs. The three major subtypes of avian viruses are H5N1 (16.7%), H3N8 (7.7%), and H6N2 (7.3%), while H1N1 (62.1%) and H3N2 (35.5%) are the dominant subtypes in human IAVs. We evaluated and eliminated sampling biases in both avian and human viruses, such as the oversampled H5N1 subtype in avian IAVs and the 2009 pandemic H1N1 in human IAVs. As a result, a total of 1425 avian and 2176 (1086 H1N1 and 1090 H3N2) human IAV sequences were retained for downstream analyses.On the basis that PB2 segments of seasonal H1N1 and H3N2 are both derivatives of 1918 pandemic H1N1 virus20. Additionally, we identified 12 markers, A44S, M64T, T81M, T105V, I292T, R368K, L475M, D567N, T569A, V613T, A674T, and G682S, which have been proposed to be human-adaptive markers in similar computational studies22. Notably, the other 11 markers, including T106A, V109I, V114I, I354L, R355T, A395V, I399V, Q447L, S490N, T491A, and V547I, in H1N1 and 10 markers, including I67V, N82S, E120D, Q194R, V227I, I382V, P453H, N456S, I463V, and T676I, in H3N2 have never been documented. A661T was excluded from both H1N1 and H3N2, since it was recently reported to have negligible effect on polymerase activity23. More specifically, among the 11 novel markers in H1N1, T106A, V109I, and V114I are located in Nter domain; I354L, R355T, A395V, I399V, and Q447L in cap-binding domain; S490N and T491A in cap-627 linker domain; and V547I in 627 domain. Comparatively, among the 10 novel markers in H3N2, I67V, N82S, E120D, and V227I are located in Nter domain; Q194R in Lid domain; I382V, P453H, N456S, and I463V in cap-binding domain; and T676I in 627 domain 25. Since identification of coevolution with MI requires sufficiently large alignment of homologous sequences24, we combined avian, swine, and seasonal human IAV PB2 sequences. However, MI values can be misleading when homologous sequences are not collected properly or the sequence alignment is not built correctly26. In order to take different homologous sequences into account more equivalently and to make the MI values more comparable between H1N1 and H3N2, we performed resampling to construct balanced samples with equal number of avian, swine, and seasonal human IAVs. Using this approach, we identified 35  (Supplementary Text 30) was reconstructed with 115 sequences, with roughly 1.26 sequences per year. In contrast, the evolutionary history of H3N2 from 1968 to 2016 is covered by 3.54 sequences per year. Accordingly, the mutational path of H3N2  tree and the corresponding mutational path were summarized from the Bayesian Markov chain Monte Carlo (MCMC) simulations. The MCC trees illustrating the evolution of the PB2 gene of seasonal H1N1 and H3N2 have a “cactus-like” shape with a strong temporal structure Figs.  and 6a. 3N2 Fig.  is more 3N2 Fig. .Fig. 5MC1. It acquired human adaptation mutations, such as K526R and A684S, which are absent in seasonal H1N1 viruses.We also estimated the emerging dates of the identified markers Table . Among tAdaptive mutations in the PB2 polymerase play a critical role for avian influenza virus replication in mammalian cells and enable some avian IAVs to establish infection in humans. In this study, we conducted a large-scale sequence comparison between avian and human IAVs, whereby a comprehensive list of novel human-adaptive markers in PB2 protein of H1N1 and H3N2 viruses were identified. To our knowledge, this is basically an exhaustive list, which encompasses most well-characterized human-adaptive markers in PB2 protein identified in prior studies as well as the novel markers obtained from this study. The identification of a large pool of adaptive markers allows us to uncover the coevolution pattern among these markers, which implicates their correlated function in host adaptation. Additionally, we demonstrated the distinct evolutionary pathways of seasonal H1N1 and H3N2 viruses.19. Intriguingly, we noted that two well-characterized human-adaptive mutations K526R and A684S are specific in the H3N2 subtype, suggesting the necessary concerns for subtype-specific mutations. Therefore, we relaxed the assumption and compared seasonal H1N1 and H3N2 to avian viruses separately. With our approach, a number of novel markers that have not been reported in similar computational studies were identified in both H1N1 and H3N2 subtypes. Notably, an experimentally verified mutation K526R, which has not been identified in previous computational studies31, was revealed in our study. The outperformance of our approach may originate from two improvements. First, we relaxed the assumption in previous approaches that human-adaptive mutations should be conserved across subtypes as aforementioned. Second, the dramatically expanding databases provide large-scale thoroughly sampled sequence data. With the availability of comprehensive sequence data of seasonal IAVs, we can significantly improve the predictive power.Previous similar computational studies tend to focus on human-adaptive mutations that are conserved in all subtypes33. The functional effect of a given substitution frequently depends on the presence or absence of other substitution(s)17. Thus epistatic sites usually present covariation or coevolution and exhibit particular patterns in multiple sequence alignment (MSA) of homologous proteins. The algorithms for detecting coevolution can be divided into two general classes: algorithms such as MI and statistical coupling analysis that score covariation between all pairs of columns in a sequence alignment; while global probabilistic models such as direct coupling analysis that assess the likelihood of covariation between sites24. The former algorithm has been utilized in our study to quantify the inter-residue covariation, since it is conceptually straightforward, technically simpler to implement, and often sufficiently powerful to provide useful insights of coevolution.There has been a growing recognition that epistasis plays a key role in functional evolution of proteins by constraining accessible evolutionary pathways and increasing the role of contingency in adaptation24. Indeed, a number of the identified coevolving pairs in this study showed significant epistasis in previous wet-lab studies. For instance, combination of coevolving pair A199S and E627K imposed a strong synergistic effect on replication efficiency28. R368K, a coevolving partner of E627K, showed limited effect when introduced alone into a H5N1 virus strain but significantly increased replication efficiency and pathogenicity when combined with 627K28. Moreover, the adaptive effects of a number of mutations in PA and NP proteins also showed dependency on E627K. For instance, three NP mutations R100V/I and L283P28 can cause a failure of virus rescue but showed highly enhanced replication efficiency with 627K28. Integrating the prior findings and discoveries obtained from our study, we believe that the strong human-adaptive effect may not solely result from the verified mutations as demonstrated previously6. Instead, the effects of verified mutations may depend on the intricate interplays with other mutations. Currently, we only focused on the inter-residue coevolution within PB2 protein due to the lack of adequate whole-genome sequences. Upon the availability of sufficient whole-genome sequences in the future, inter-protein coevolution analysis could uncover important residues that are involved in protein–protein interactions.Analyses of amino acid coevolution within protein family can serve as a valuable guide for identifying residues that are functionally coupled35, which may override the true distinctions between avian and seasonal human IAVs. Therefore, we carefully eliminated pH1N1 prior to comparison. Despite all the efforts, our comparative method does have limitations. We are unable to identify newly fixed adaptive mutations, since they are unlikely to achieve predominance to fulfill the first criterion. For instance, two verified mutations M535L and E249G, which were acquired by seasonal H1N1 . Subtype distributions of both avian and human IAVs were estimated. For avian IAV viruses, replicate sequences within the same subtype due to oversampling were removed. For human IAV viruses, non-seasonal subtypes such as 2009 pandemic H1N1 (3083 sequences), H5N1 (367 sequences), and H7N9 were excluded. The remaining IAVs were subdivided into two subsets, H1N1 and H3N2. Replicate sequences within each subset collected in the same year were excluded to eliminate oversampling in certain years. Nonsense characters of each sequence were trimmed while those with partial length (<759 aa) were removed. MSA of PB2 was constructed using the Muscle v3.736 software with the fastest parameters (-maxiters 2 without refinement), due to the large amount of sequences.The PB2 sequences of avian and human IAVs were retrieved from the OpenFluDB database (F) of amino acids in each aligned position were calculated. The predominant amino acid of each site is defined as that with the largest F. Pearson’s chi-square test was used to test the statistical significance of amino acid distribution difference in the corresponding position between avian and human IAVs. Cramer’s V test was utilized to normalize the chi-square statistic to control for dataset size and quantify the effect size. Accordingly, two criteria were employed to define an amino acid as a human-adaptive marker in a given site: (i) the frequency of the amino acid is predominant (F > 0.5) in human IAVs and minor (F < 0.5) in avian IAVs; (ii) the Cramer’s V value is >0.8. The marker is indicated as “A + site + B” where A and B are the predominant amino acids of avian and human IAVs, respectively, in that site and the substitution from A to B is assumed to be responsible for human adaptation. Only the counts of amino acid A and B in each aligned position of avian and human IAVs were taken into tests to ensure the same degree. We used 1% of the total count as a pseudo value in tests when the observed count is <5. The sequence comparison was implemented using Python and Biopython framework37. Statistical tests were performed using R language38. Sites with host-specific amino acids were mapped onto the polymerase complex (PDB code: 4WSB) by using Pymol. The source code is available upon request.Site-wise amino acid compositions in PB2 of H1N1 and H3N2 subsets were compared to those of avian IAVs, respectively. Frequencies (H) is a measure of uncertainty or randomness39. The entropy of a column c in a MSA is calculated with the following equation:p(xi) is the observed frequency of amino acid i occurring at a site. All values were calculated using a log20 so that the range of position entropy scores is 0–1. The covariation between two sites is quantified using MI, which quantifies the mutual dependence between two variables. The MI between two positions in a MSA is given asHc and Hd are entropies of column c and d, respectively, and Hcd is the joint entropy of column c and d calculated by the same method using the frequencies of occurrence of each combination of residues in column c and d. The MI scores range from 0 to the minimum of Hc or Hd. The raw MI values are normalized by dividing by the joint entropy of the positions, Hcd, to reduce the influence of entropy on MI. Normalized MI values range from 0 to 125.Shannon’s entropy  was utilized to infer the parsimony tree with JTT model. The tree was then visually analyzed using TempEst41  to identify potential violations that substantially deviated from the linear regression of root-to-tip genetic distance against divergence time. We removed the outliers and then repeated these two steps to achieve a high consistence between molecular clock and stamped dates. The remaining sequences were used in downstream analyses.Preprocessed human seasonal IAV sequences were used for the construction of MCC tree and evolutionary history. A maximum of 10 sequences of H1N1 and H3N2 per year were retained to avoid the trees being too large. Next, RAxMLhttp://beast.bio.ed.ac.uk). BEAGLE (http://beast.bio.ed.ac.uk/BEAGLE) was utilized to boost the core computation. The BEAST XML input file was generated using the combination of BEAUTI (inside BEAST package) and hand-annotation. This XML file that specifies date-stamped sequences, a strict molecular clock and a JTT model of substitution, was used in multiple runs of MCMC simulation. The MCMC chain was set as 100 million iterations, with subsampling every 10,000 iterations. Tracer  was used to track the log file of combined runs with the initial 10% of the chain as burn-in to ensure good MCMC convergence. The MCC tree was summarized using TreeAnnotator  on the basis of merged simulations. FigTree  was used to visualize the tree file, manually recolor the branches, and export to tree images. The mutational path was extracted from the merged trees using Mutpath python package33 (available at http://github.com/jbloom/mutpath), with manually created input file.To infer the evolutionary history and the MRCA for the PB2 sequences, a Bayesian MCMC method was applied as implemented in the BEAST package (version 1.8.3; The datasets used and/or analyzed during the current study are available from the corresponding author on request.Figure S1Table S1Table S2Table S3Table S4Text S1Text S2Text S3Supplementary Figure an Table legend


The 3D modelling results identified four unique amino acid polymorphisms, Ala73, Trp156, Arg219 and Met304, that may affect the function of the HLA-C*12:02:02 allele. KIR2DL5  and the KIR Bx haplotype (p = 0.0000275) were protective against Taiwanese AS, while KIR 2DS4/1D  appeared to be a risk factor for it. KIR2DL5 combined with the HLA-C1/C2 heterozygous genotype showed a protective effect ; in contrast, KIR 2DS4/1D combined with the HLA-C1C1 homozygous genotype  represented a risk factor for AS development. Our data suggested that interactions between KIRs and their cognate HLA-C ligands may contribute to the pathogenesis of AS.Human leukocyte antigen (HLA) class I ligands and Killer immunoglobulin-like receptors (KIRs) regulate the cytolytic activity of natural killer (NK) cells and certain T cells. We examined their genetic predisposition to disease susceptibility and clinical phenotypes in Taiwanese ankylosing spondylitis (AS) patients. KIR genotyping and Human Leucocyte Antigen C (HLA-C) sequencing were performed in 653 Taiwanese AS patients and 952 healthy controls. KIR genotype distributions and HLA-C allele frequencies were compared in patients and controls and among patients with and without HLA-B27 positivity, early age onset and spinal syndesmophytes. HLA-C alleles were functionally characterized using 3D structural modelling with peptide simulation. This study discovered that the HLA-C*12:02:02 allele (43.42% vs. 3.31%;  Ankylosing spondylitis (AS), a prototypical chronic immune-mediated inflammatory arthritis disorder of spondyloarthritides (SpA), is a highly familial, heritable disease as shown by strong evidence from genetic association twin and family studies ,2,3. MulNatural killer (NK) cells dynamically interact via inhibitory surface receptors with human leukocyte antigen (HLA) ligands to acquire a “licensing” process, and express functional competence during development . LicenseKIRs (killer immunoglobulin-like receptors) regulate the cytolytic activity of NK cells and certain T cells through binding to HLA class I ligands . KIRs coKIR gene clusters show extensive genetic diversity, as do the HLA Class I loci, which encode ligands for KIR molecules. The activating KIR genes and their corresponding HLA ligand groups show strong negative correlations across populations with distinct evidence for coevolution [KIR and HLA genotypes are associated with certain human disease outcomes, although the functional explanation for these associations is poorly understood. The interaction of HLA-C with KIR plays the dominant role to control human NK cell response [The volution ,14. The response . In addiresponse ,17,18. HThis study recruited 653 Taiwanese AS patients  with a mean age at disease onset of 25.18 ± 9.27 years old, as shown in p < 0.00001 Odds Ratio (OR), 16.88; 95% Confidence Interval (CI): 11.27–25.28). In contrast, other alleles, including HLA-C 01:02:01 , HLA-C 03:02:01  and HLA-C 03:04:01 , revealed protective effects in Taiwanese AS patients , as shown in The current study successfully performed HLA-C allele discrimination in 653 AS patients and 952 normal controls using sequencing-based genotyping. We identified 39 HLA-C alleles in the Taiwanese population. The HLA-C*12:02:02 allele frequency  was significantly higher in AS patients than in normal controls . In contrast, the KIR 2DS4/1D genotype was identified as a risk factor for AS, although this association was insignificant after multivariate analysis . We next investigated whether KIR gene variations had different genetic background effects in AS phenotypes. As shown in KIR genes and AS clinical characteristics. Thus, our data indicate that ethnic differences in KIR distribution have no significant associations with Taiwanese AS clinical phenotypes.KIRs and their ligand pathways play an important immune regulatory role during antigen presentation. We applied an allele-specific genotyping method for nine KIR genes in Taiwanese AS patients and healthy controls were performed, with two broadly classified KIR groups (AA and Bx). We then investigated whether the KIR haplotypes affected AS. The KIR AA haplotype showed a significant risk association with Taiwanese AS development . In contrast, the KIR Bx haplotype showed a protective role in Taiwanese AS subjects . These findings suggest that KIR haplotypes with predominant inhibitory functional receptors play regulatory roles in AS pathogenesis that contributes to disease development. Again, we did not observe any significant associations between KIR haplotypes (AA and Bx) and AS clinical characteristics. Thus, the inhibitory dominant KIR haplotypes do not contribute to the development of other clinical phenotypes and complex disease characteristics of AS.Haplotype construction and the analysis of KIR genes and their HLA-C ligands may be implicated in the outcome of AS. As shown in KIR2DL5 combined HLA-C1/C2 heterozygous genotype was significantly lower than that in normal controls . In contrast, the frequency of AS in the KIR2DS4 combined HLA-C1C1 homozygous genotype was significantly higher than that in normal controls . These results suggest that interactions between KIRs and their cognate HLA class I ligands play a crucial role in the pathogenesis of AS.Genetic variation in KIRs is responsible for the complex gene effects impacting NK cell function, while HLA class I molecules tightly regulate cytotoxic T lymphocyte responses. We speculated that the specific combinatorial effect of Similar to the HLA-A and HLA-B groups, the heterodimer proteins of HLA-C alleles are composed of an α chain (heavy chain) and a β chain (β-2 microglobulin). The α chain consists of α1 and α2 domains, forming a peptide-binding groove between the two domains, followed by the α3 domain and a transmembrane helix in the carboxyl end of the HLA protein . We compKIRs, HLA Class I genes and their combinations under natural selection result in the diverse characteristics of individuals and populations, and contribute to AS pathogenesis in the Taiwanese population [KIR AA haplotype, HLA-C1202 allele and HLA-C1 epitope confer risk for AS development, while KIR2DL5, KIR Bx and HLA-C2 have a protective effect.Genetic differences in MHC alleles, especially HLA-B27, confer the greatest risk to AS but are not the only significant genetic factor driving the disease development process. The highly polymorphic KIRs modulate NK and T cell actions against target cells through their interactions with HLA-C ligands. The current study shows that specific pulation ,20. As aKIR3DL1/KIR3DS1, 2DL5 and KIR2DS1 are risk factors in the pathogenesis of AS [KIR gene content of particular KIR haplotypes nor KIR3DL2 polymorphisms contribute to AS in Caucasian (UK) patients [KIR3DS1 segregates as an allele with a short cytoplasmic tail and characterizes activation receptors that are expressed by a higher percentage of NK cells in KIR3DS1 homozygous donors than in heterozygous donors [KIR3DS1 shows an increased frequency association in combination with HLA-B alleles carrying Bw4-I80 in the trans position in Spanish and Azoreans populations with AS, compared with B27 controls, whereas KIR3DL1 was decreased in AS patients [KIR genotypes is responsible for AS susceptibility. With the genetic background of HLA-B27, variation at the KIRs and their corresponding specific HLA-C ligands may contribute to the pathogenesis of AS. Both HLA-C and HLA-B are located at the MHC cluster on Chromosome 6. We found HLA-C1202 allele had strong linkage with HLA-B27 positivity. However, their influence on the ability of NK cells and T cells to recognize and lyse targets in the immune response and the functional effects of KIR polymorphisms remain largely unknown [KIRs are involved in the activation/inhibition of NK cells through their interaction with MHC class I, particularly on target cells. KIRs and MHC class I ligands, the HLA-B group (particularly HLA-B27) and possibly other HLA molecules have been associated with differential NK cell activity and function, adding to the growing evidence for the involvement of KIRs in AS disease development . Previouis of AS ,22. A popatients ,24. KIR3s donors . KIR3DS1patients ,27. KIR3patients . The actpatients ,29. As a unknown ,29,30.KIR gene variations with activating effects might associate with susceptibility to AS by influencing NK cell activity once HLA-C2 epitope ligands are present [ present ,17,18,21 present . Howeverpfishier = 0.004833). KIR2DL5 belongs to the KIR B haplotypes and shows associations with activating receptors related to high viral loads in primary human cytomegalovirus (HCMV) infection following HCMV-positive renal transplant, severe pandemic influenza A (H1N1) and dengue virus infections [KIR2DL5 was shown to decrease the risk of systemic lupus erythaematosus (SLE), but increased the overall risk for viral infections in Japanese subjects [KIR2DL5A is the predominant genotype in Asian populations, which is consistent with the results from our 282 commercially genotyped individuals [KIR B haplotypes also showed a protective role for AS in our study, although the combinatory effects of activating KIRs are difficult to explain. Our study suggests that KIR2DL5 may serve as an independent KIR protective marker for Taiwanese AS, although the ligand is unknown [KIR2DL5 exhibits distinct inhibitory capacities through recruiting both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than that of KIR3DL1 . Notablyfections ,33,34,35subjects . Howeversubjects ,38. KIR2ividuals . KIR B h unknown .2DS4 presents as either a fully functional (KIR2DS4-f) or deleted non-functional (KIR2DS4/1D) variant with a 22-bp deletion in exon 5, due to a frame shift mutation and premature stop codon yielding a truncated KIR2DS4 protein with loss of the transmembrane and cytoplasmic domains [KIR2DS4 deletion carriers with low KIR2DS4 functional expression were at risk for AS. Additionally, our study showed that KIR A haplotypes with multiple KIR inhibitory genes represented risk factors, suggesting that this inhibitory regulatory receptor is critical for AS susceptibility.KIR2DS4 was identified as a risk factor for AS based on a previous meta-analysis . KIR2DS4 domains . Our datKIR gene structure and human evolution by genetic selection over long-term environmental stimulation. The remarkable polymorphism of KIR and HLA genes warrants descriptive gene frequency studies in different populations. The diversity distribution of Asians, especially Taiwanese citizens, demonstrates a similar high frequency (>98.6%) in framework-specific KIR genes  among normal subjects and AS patients, although we did not apply these results to functional analysis. Nevertheless, the present study extends the association and confirms the contribution of the KIR genes to AS susceptibility, with an imbalance between activating and inhibitory KIR genes seeming to influence susceptibility to AS [These discrepant results indicate the complex diversification of ty to AS .KIR genes and their HLA-C ligands may reveal the interaction effects contributing to AS pathogenesis. We observed protective effects for KIR2DL5 with the heterozygous HLA-C1C2 ligand combination genotype in a multivariate analysis of Taiwanese AS development. Our results indicated that HLA-C was beneficial in KIR2DL5 AS carriers possessing at least one C2 allele, while the opposite was not true for homozygous C1 carriers (C1/C1), resulting in risk outcomes. In contrast, the KIR2DS4 deletion and HLA-C1C1 ligand combination was identified as a risk factor for AS pathogenesis. These results suggest that the KIR gene content in combination with the specific ligand may enhance the influence on AS development.Combinatorial analyses of The present study recruited patients who fulfilled the 1984 revised New York diagnostic criteria for AS. Radiographs of the cervical, thoracic and lumbar spine were examined by rheumatology specialists to evaluate syndesmophyte formations according to the modified Stoke’s Ankylosing Spondylitis Spinal Score (mSASSS). To ensure the accuracy of evaluation, two rheumatology specialists independently scored the syndensmophyte formations by blindly reading radiographs of AS patients to establish inter- and intra-reader reliability. The X-ray observations were further classified into three groups: Group 1 patients showed no spine erosion, sclerosis or syndesmophyte formation; Group 2 patients showed <4 fused syndesmophyte formations (mSASSS < 24); and Group 3 patients showed >4 syndesmophyte formations (mSASSS ≥ 24), as described previously . For theTM HLA-B27 kit with fluorescein-conjugated anti-HLA-B27 phycoerythrin-conjugated anti-CD3 antibodies that specifically bind to leucocyte antigen. Samples were then analyzed using a FACSCalibur flow cytometer and HLA-B27 software . In PCR assays, two sets of primers were used to amplify HLA-B gene with genomic DNA. The first set of primers detect human HLA-B27/B40/B55 while the second set of primer specifically target HLA-B27. Amplification of human β-globin gene in the same PCR reaction was included as an internal control.HLA-B27 antigen positivity was determined by flow cytometry analysis and/or polymerase chain reaction (PCR) assays. Briefly, the whole blood samples were stained with BDGenomic DNA was isolated from Ethylenediaminetetraacetic acid (EDTA) anti-coagulated peripheral blood using the Puregene DNA isolation kit .KIR genes (KIR gene profiles) were established using the PCR-SSP KIR genotyping kit (KIR Genotyping SSP Kit). KIR2DL1 (279), 2DL3 (281), 2DL4 (282), 3DL1 (278), 3DL2 (282), and 3DL3 (282) showed positive rates over 98.5%. Thereafter, nine KIR genes  in the remainder of the samples were genotyped using an in-house PCR-SSP primer set, the results of which were consistent with commercial kits applied to the same 282 samples. PCR reactions were prepared at a volume of 10 µL containing 0.08 µL Taq DNA polymerase (5 U/L), 9 µL Ready PCR buffer and 1 µL DNA (40 ng/L). All of the KIR genes were amplified using an ABI 9700 PCR Cycler  under the following conditions: initial denaturation at 96 °C for 2 min, followed by 10 cycles of 15 s at 96 °C and 1 min at 65 °C, and 20 cycles of 15 s at 96 °C, 50 s at 61 °C, and 30 s at 72 °C, with a 4 °C hold. The PCR products were electrophoresed on a 2% agarose gel and then visualized under ultraviolet light.The presence and absence of Genotyping of the HLA-C allele was performed using a commercial sequencing-based typing (SBT) kit . HLA-C genotypes were assigned by the HLA SBT uTYPE 6.0 software .http://hla.alleles.org). The α chain of HLA-C*12:02:02 is a protein of 342 amino acids. Based on a sequence search using Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov), the structural information for HLA-Cw3 (Protein Date Bank codes: 1EFX) [The amino acid sequences, residue numbers and the sequence alignment of HLA-C alleles were searched for on the HLA nomenclature website (s: 1EFX) . This RRs: 1EFX) , with ths: 1EFX)  was useds: 1EFX) . The mols: 1EFX) .KIR genes, KIR haplotypes (group A and group B) and KIR/HLA-C pairs between patients and controls were made using logistic regression or the χ2-test, and Fisher’s exact test was applied when appropriate. Based on the risk allele identified, p-values, odds ratios (ORs), and 95% confidence intervals (CIs) were then calculated. To account for multiple testing corrections, false discovery rate (FDR)-corrected p-values were generated using an FDR correction method in the modified version of QVALUE software . To investigate the genetic association with clinical characteristics, we controlled for each of the clinical characteristics and performed stepwise logistic regression analyses.Frequency comparisons of HLA-C alleles, individual Collectively, our data suggest that the HLA-C*12:02:02 allele and KIR2DL5 have distinctive functional roles in AS development. The present study provides new insight into the effects of KIR, HLA, and KIR-HLA combinations on the immune response of NK and specific T cells, and their contributions in Taiwanese patients with AS.


STAT5 is an essential transcription factor in hematopoiesis, which is activated through tyrosine phosphorylation in response to cytokine stimulation. Constitutive activation of STAT5 is a hallmark of myeloid and lymphoblastic leukemia. Using homology modeling and molecular dynamics simulations, a model of the STAT5 phosphotyrosine-SH2 domain interface was generated providing first structural information on the activated STAT5 dimer including a sequence, for which no structural information is available for any of the STAT proteins. We identified a novel intramolecular interaction mediated through F706, adjacent to the phosphotyrosine motif, and a unique hydrophobic interface on the surface of the SH2 domain. Analysis of corresponding STAT5 mutants revealed that this interaction is dispensable for Epo receptor-mediated phosphorylation of STAT5 but essential for dimer formation and subsequent nuclear accumulation. Moreover, the herein presented model clarifies molecular mechanisms of recently discovered leukemic STAT5 mutants and will help to guide future drug development. D816V-positive systemic mastocytosis (SM)649S710F into mice identified STAT5 as a putative oncogene capable of inducing tumor initiation in many hematopoietic cell types, independent of dysregulated upstream kinasesSTAT5, represented by the highly homologous isoforms STAT5A and STAT5B, is an essential transcription factor for the proliferation, survival and differentiation of myeloid cells24In addition to these functions in physiologic and malignant myelopoiesis, STAT5 represents a master regulator of lymphopoiesis, controlling the proliferation and differentiation of T and B cells through various cytokines including IL-2 and IL-7121415161718The addiction to STAT5-mediated signal transduction processes has been considered to be a potential Achilles heel in leukemic signaling networks, resulting in intense research aiming at the pharmacological inhibition of STAT5. Hence, prevention of STAT5 dimer formation is thought to interfere with tumor cell proliferation and survival2021Oncogenic somatic STAT5 mutations are characterized by stimulation-independent activation and predominantly map to the SH2 domain  of the transcription factor. However, constitutive STAT5 activation is additionally promoted by somatic mutations located in the sequence following phosphotyrosine 694 , a region for which only limited or no structural information is available for any of the STAT dimers .Therefore, the structural determinants of the STAT5A dimer interface were predicted by bioinformatics approaches and molecular dynamics (MD) simulations. The modeled system includes the SH2 domain (residues 589–687) and the C-terminal tail segment , which consists of a short flexible linker , the phosphotyrosine motif  and the N-terminal part of the transactivation domain  . The ini24Next, 2,000 ns-long MD simulations were performed  on the h2324M2) interacts with the SH2 domain of M1 with the same orientation as pY694M1. Salt bridges formed by pY694 and the highly conserved and invariant R618 located in the βB strand of the opposite SH2 domain are key interactions in both intermolecular PTM-SH2 domain interfaces. Indeed, they are fully preserved during the MD simulations with occupancies of 100% (pY694M1) and 97% (pY694M2), respectively. Moreover, the coordination of pY694 in both monomers is supported by the formation of transient hydrogen bond interactions with K600, S620, S622 and T628 in the opposite monomer during the MD simulations  in the CTS of monomer 1 (M1) interacts with the SH2 domain of the opposite monomer (M2) in a direction orthogonal to the strands of the central β-sheet . In a syulations . Notablyulations 2425.Our model further identified two novel residues involved in the recognition of pY694. These are the βD residues N642 (βD5) and K644 (βD7), which stabilize the aromatic ring of the phosphotyrosine by hydrophobic interactions . Finally24M1 and K700M1 with Q701M2, and I699M2 and Q698M2 with E705M1 . In the opposite monomer, F706M2 forms aromatic interactions with F633M2, Y665M2 and W631M2 with occupancies of the hydrophobic contacts of 41.4%, 41.3% and 21.7%, respectively. Such a network of hydrophobic interactions substantially contributes to the recognition of F706 in their respective interfaces /JAK2-mediated phosphorylation of Y694, dimer formation and nuclear accumulation of STAT5A being mutated at residue F706 (F706A) or in the hydrophobic acceptor interface . These mutants were studied in comparison to STAT5A wild-type and the non-functional R618Q mutant. HeLa T-REx cells stably expressing eYFP-tagged STAT5A constructs and the EpoR revealed a severely impaired tyrosine phosphorylation of all mutants in response to Epo stimulation. Intriguingly, the inhibition of phosphatases by sodium vanadate treatment rescued the Epo-induced phosphorylation of STAT5A mutants F706A, F633A or Y665A demonstrating that the reduced phosphorylation of these mutants in the absence of vanadate is caused by phosphatase activity rather than dysfunctional receptor recruitment . Of noteTo study the impact of these mutations on STAT5A dimerization, blue-native PAGE was performed. Since phosphorylation at Y694 is a prerequisite for the formation of STAT5A dimers, lysates were analyzed after treatment of cells with Epo and vanadate. Phosphatase inhibition resulted in a 2.15-fold increase in dimerization of STAT5A wild-type in response to Epo stimulation, illustrating that the EpoR/JAK2/STAT5A signaling pathway in steady-state is highly counter-regulated by phosphatases. Surprisingly, while in response to vanadate treatment STAT5A proteins carrying the F706A mutation appeared to be phosphorylated markedly above reference levels (Epo stimulation of STAT5A wild-type in the absence of vanadate), their dimerization was severely impaired (−2.37-fold) . LikewisIn contrast, vanadate treatment failed to restore the receptor-mediated phosphorylation of the STAT5A mutants F633A/Y665A, W641A and R618Q to reference levels , suggestIn resting cells, STAT5 is evenly distributed between cytoplasm and nucleus. Formation of phosphorylated dimers is a requirement for nuclear accumulation of STAT5. Confocal imaging of fluorescently labeled STAT5A revealed that vanadate treatment resulted in a forced nuclear enrichment of the wild-type protein in erythropoietin-stimulated cells, while the investigated STAT5A mutants did not accumulate in the nucleus reflecting their impaired capability to form dimers . Taken tIn a defined subset of aggressive lymphoid cancers, the somatic mutations N642H (βD5) and T628S (β-sheet C) have recently been identified, which map to the phosphotyrosine-binding pocket in interface 1 of the STAT5 SH2 domain , and 4b.15171416N642H and STAT5AT628S by the detection of dimers that appeared 2.10-fold and 1.48-fold increased in Epo-stimulated cells, suggesting that both mutations interfere with appropriate deactivation of the JAK-STAT signaling pathway  demonstrating that this mutation confers a stronger constitutive activation to STAT5 proteins compared to T628S . Strikingly, phosphatase inhibition failed to restore STAT5A dimerization back to reference levels confirming that the phosphorylation of STAT5A is necessary yet not sufficient for dimerization . These fN642H, mutations in the PTM resulted only in a weak factor-independent constitutive activation, illustrating that both mutants are more sensitive to dephosphorylation similar to STAT5AT628S. The introduction of the F706A mutation into I699L and Q701L mutants again significantly diminished dimerization in stimulated cells compared to the corresponding single mutants (3.13-fold and 2.54-fold), despite boosted phosphorylation levels caused by vanadate treatment  appears at a lower frequency of 2.0% in lymphomas of NK or γδ T-cell origin (1/94)reatment . Withoutreatment . Notably of F706 . Neverthed cells .Y665F showed a significant 1.82-fold (p = 0.0020) increase in the amount of dimers in stimulated cells compared to reference levels, while vanadate treatment remained again insufficient to rescue pSTAT5AF706A or pSTAT5AF706G dimerization rization .Y665F carrying the F706A (3.90-fold) or F706G (3.50-fold) mutation, suggesting that the elimination of the Y665-hydroxyl group is sufficient to enable appropriate dimerization of STAT5AF706A/G. Nevertheless, these dimers remained highly susceptible for phosphatases. Therefore, it appears evident that besides its role in the dimerization process, binding of F706 to the hydrophobic interface is critical to protect STAT5A dimers from rapid inactivation  and 6.16-fold (p = 0.0007) in stimulated cells compared to the Y665F single mutant . Surpristivation . In accotivation .The crystal structures of the activated STAT1 and STAT3 dimers were used to predict the structural determinants of the fully assembled SH2 domain interface of STAT5A by the combination of homology modeling and MD simulations. The resulting model, validated with experimental data, revealed novel and unique features of the dimerization interface of STAT5A and was used to unravel the impact of disease-related STAT5 mutations on protein structure and function.Not unexpected, the model corroborates reciprocal phosphotyrosine-SH2 domain interactions as a conserved driver of STAT dimerization resulting in a grossly symmetric dimer2428The binding interface selectivity of SH2 domains is mediated by individual binding pockets, additional to the phosphotyrosine-binding site, that primarily recognize residues in the pY+1 and pY+3 positions located C-terminal to the phosphotyrosine residue. In most SH2 domains, the βD5 residue critically contributes to selectivity by recognition of the pY+1 position3124In STAT1 and STAT3, the PTM forms a pair of cross-over connections with the opposite PTM significantly increasing the intermolecular contact area at the dimer interface24In recent years, an increasing number of somatic STAT5 mutations has been identified in leukemia patients. The vast majority of lymphoma-associated STAT5 mutations such as N642H, T628S, I699L, Q701L and Y665F map to the SH2 domain and PTM of the transcription factor and are characterized by an activating phenotype1415161718T628S directly strengthens phosphotyrosine coordination by the SH2 domain. Importantly, the improved phosphotyrosine-binding ability of both SH2 domains did not suffice to rescue dimerization of STAT5A proteins lacking F706, further strengthening the relevance of intramolecular interactions for STAT dimerization (The mutations N642H and T628S reside in the phosphotyrosine-binding interface and peptide precipitations confirmed an increased binding affinity for both of the mutated SH2 domains suggesting that constitutive activity is a consequence of an impaired inactivation by STAT5 phosphatases . MD simu1734rization .I699L/F706A and STAT5AQ701L/F706A exhibited a weaker dimerization defect compared to the F706A mutant providing evidence that L699 (pY+5) and L701 (pY+7) significantly support the dimerization of STAT5A. Hence, constitutive activation of STAT5AI699L and STAT5AQ701L is most likely a consequence of increased binding affinities between the two PTMs . These are bacteria CobB sirtuin  were initially obtained by using homology modeling. The latter was based on the correspondent domains in the crystal structures of activated STAT1 dimer  scoring functionErythropoietin  was used at a concentration of 1 U/ml. Anti-pY694-STAT5  and anti-GFP  were used for immunoblotting. Anti-rabbit and anti-goat antibodies conjugated to horseradish peroxidase (HRP) were ordered from, DAKO .® XL Site-Directed Mutagenesis Kit according to manufacturer’s instructions .The cDNA of murine STAT5A was either cloned into pLentiLox  or pcDNA3.1  expression vectors and fused to the cDNA of the enhanced yellow fluorescent protein (eYFP). The cDNA of the human erythropoietin receptor (EpoR) was cloned into the pcDNA5/FRT/TO expression vector and fused to the cDNA of the hemagglutinin tag (HA). Fluorescent proteins were fused to the C-terminus of the target proteins, whereas the HA-tag was fused to the N-terminus of the human EpoR. Point mutants of STAT5A were generated with PCR based QuikChange®-LT1  according to manufacturer’s instructions. Stable transfection of HeLa T-Rex cells for the inducible expression of HA-EpoR was performed with the Flp-In system  using 250 μg/ml hygromycin B  and 15 μg/ml blasticidin  for selection. The expression of HA-EpoR was induced with 5 ng/ml doxycycline  for 24 h. All media were supplemented with 10% FCS  and 25 U/ml penicillin/streptomycin . Cells were incubated at 37 °C in a water-saturated atmosphere with 5% CO2. Phosphatase inhibition was carried out by sodium vanadate (Na3VO4) treatment (1 mM) for 2 h.HeLa T-Rex  cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/GlutaMAX . Transient transfection of HeLa T-REx cells was performed using Trans ITTo achieve stable and constitutive expression of STAT5A constructs in inducible HeLa T-Rex cells, self-inactivating lentiviral constructs were used according to the guidelines of the RNAi consortium. Briefly, the cDNA of STAT5A-eYFP and the point mutants of STAT5A-eYFP were cloned into the lentiviral pLentiLox expression vector downstream of the CMV promotor . HEK293-T packing cells were seeded on 6-cm dishes on to be 80–90% confluent at the time of transfection. On the second day, HEK293-T cells were transiently co-transfected with the lentiviral vector, psPAX2 (packing plasmid) and pMD2.G (envelope plasmid). Eighteen hours after transfection the culture medium was replaced by high serum growth medium containing 30% FCS. Viruses were harvested 24 hours after medium exchange. For the infection of HeLa T-Rex cells, virus-containing supernatant was supplemented with 8 μg/ml polybrene  prior to infection.2HPO4, 1.5 mM KH2PO4, adjusted to pH 7.4) once and lysed with RIPA lysis buffer  supplemented with phosphatase/protease inhibitors , 5 μg/ml aprotinin, 2.5 μg/ml leupeptin) according to standard procedures. The proteins were separated by SDS-PAGE and transferred to a PVDF membrane with subsequent immunodetection using specific antibodies. Primary and HRP-conjugated secondary antibodies were diluted in TBS-N buffer . Membrane-bound antibody complexes were detected by chemiluminescence .HeLa T-REx cells were washed with PBS  supplemented with 2% digitonin . The lysates were cleared by centrifugation and incubated with Coomassie brilliant blue G-250 and separated over night at 4 °C using NativePAGETM Bis-Tris gel system with a gradient polyacrylamide gel (4–16%) according to the manufacturer’s instructions. Dimerization of STAT5A-eYFP constructs was analyzed by detection of the eYFP fluorescence with a typhoon fluorescence scanner  by excitation with a 488 nm laser line. The emission was detected using a 515–555 nm bandpass filter.Transiently transfected HeLa T-REx parental cells were lysed with storage buffer  supplemented with phosphatase/protease inhibitors. Lysates were cleared by ultracentrifugation . Peptides corresponding to the phosphotyrosine motif of STAT5 (b-tat-AKAVDGpYVKPQIKQVVPEFVNASADAG-a) were synthesized with >95% purity, modified by N-terminal biotinylation (b-) and C-terminal amidation (-a) and contain the HIV tat protein derived GRKKRRQRRRPQ (tat) sequence at the N-terminus. 25 nmol of the peptides were coupled to NeutrAvidin Agarose Resin  for 2 h at 4 °C, washed twice with storage buffer and incubated with 1 mg of the lysate over night at 4 °C. Peptide/protein complexes were washed 3 times with storage buffer for 10 minutes and the precipitates were analyzed by SDS-PAGE and immunoblotting.HeLa T-REx cells expressing fluorescently labeled fusion proteins were grown on glass cover slips. After paraformaldehyde (3.7%) fixation cells were stained with DRAQ5  and Phalloidin-Atto 550  to label nuclei and actin, respectively. Cover slips were mounted with ImmuMount  as has been described previouslyInformation on statistics and sample numbers is given in the corresponding figure legends.How to cite this article: Fahrenkamp, D. et al. Intramolecular hydrophobic interactions are critical mediators of STAT5 dimerization. Sci. Rep.6, 35454; doi: 10.1038/srep35454 (2016).