Non-small cell lung cancer (NSCLC) is a prevalent subtype of lung cancer, whose mortality is high. Long non-coding RNAs (lncRNAs) have caught rising attentions because of their intricate roles in regulating cancerization and cancer progression. Long intergenic non-protein coding RNA 461 (LINC00461) has recently shown oncogenic potential in several cancers, but the function of LINC00461 in NSCLC remains to be investigated. Our study planned to unveil the regulatory role of LINC00461 in NSCLC. It was validated that LINC00461 was highly expressed in NSCLC tissues and cell lines and exhibited prognostic significance. Furthermore, LINC00461 expression in advanced stage was much higher than in early stage. Loss-of-function experiments suggested that LINC00461 knockdown impaired cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT). Subcellular fractionation revealed the predominant location of LINC00461 in cytoplasm. Mechanistically, LINC00461 up-regulated E2F transcription factor 1 (E2F1) expression through sponging miR-4478. Besides, E2F1 bound to the promoter of LINC00461 to induce its transcription. Finally, rescue experiments verified that LINC00461 aggravated proliferation, migration, and EMT through targeting miR-4478/E2F1 axis. In consequence, the present study illustrated that LINC00461/miR-4478/E2F1 feedback loop promoted NSCLC cell proliferation and migration, providing a new prognostic marker for NSCLC. Non-small cell lung cancer (NSCLC), taking up approximately 80–85% of lung cancer cases, is a main histological subtype of lung cancer, and can be further classified into adenocarcinoma and squamous cell carcinoma ,2. AlthoLong non-coding RNAs (lncRNAs) are defined as non-coding transcripts more than 200 nucleotides in length, with none or limited protein-coding potential ,9. Many Mechanistically, lncRNAs could realize their regulatory function through competing endogenous RNA (ceRNA) network in cytoplasm. Through sponging microRNAs (miRNAs), lncRNAs released downstream mRNAs which targeted by miRNAs . miRNAs,E2F transcription factor 1 (E2F1) is reputed as a key transcription factor and regulates cell-cycle progression in cancers . BesidesThe present study was proposed to probe the role and molecular mechanism of LINC00461 in NSCLC, and it was discovered that LINC00461 exhibited a high expression level in NSCLC. Additionally, LINC00461 mediated by E2F1 facilitated NSCLC cell proliferation and migration through targeting miR-4478/E2F1 axis, revealing LINC00461/miR-4478/E2F1 feedback loop in NSCLC.Ninety paired cancer and adjacent para-cancerous tissues were collected from Gansu Provincial Hospital of TCM. All enrolled patients had signed written informed consent. The study was permitted by the Institutional Ethics Committee of Gansu Provincial Hospital of TCM. All tissues were maintained in liquid nitrogen and stored under −80°C. No patients received any preoperative therapies. Following the directions of World Medical Association Declaration of Helsinki, this work has been carried out. The ethical approval ID number is AF/SC-07/02.0.2.The human normal bronchial epithelial cell line 16HBE, human NSCLC cell lines A549, H1299 ; H23 and SPC-A1  were used. For cell culture, Dulbecco’s Modified Eagle’s Medium  were applied for 16HBE, RPMI-1640 Medium (Invitrogen) for H1299, H23, and SPC-A1 cells, and F-12K Medium (Invitrogen) for A549 cells. All above-mentioned mediums were supplemented with 10% fetal bovine serum (Invitrogen) and penicillin–streptomycin  in the moist incubator at 37°C with 5% COsiNC: CGAUGUUACAUAACUUAUUAGsiLINC00461#1: CGAUAAGUUAUGUAACAUUAGsiLINC00461#2: GUUAAUUGUAGUAGACAAUGGshNC: CCGCCGATGTTACATAACTTATTAGCTCGAGCTAATAAGTTATGTAACATCGTTTTTGshLINC00461#1: CCGCCGATAAGTTATGTAACATTAGCTCGAGCTAATGTTACATAACTTATCGTTTTTGshLINC00461#2: CCGCGTTAATTGTAGTAGACAATGGCTCGAGCCATTGTCTACTACAATTAACTTTTTGNC mimic: GUCAGCCUGCUGAGGAGmiR-4478 mimic: GAGGCUGAGCUGAGGAGNC inhibitor: CUCCUCAGCAGGCUGACmiR-4478 inhibitor: CUCCUCAGCUCAGCCUCSmall interfering RNAs (siRNAs) targeting LINC00461 (siLINC00461#1/2) and short hairpin RNAs (shRNAs) targeting LINC00461 (shLINC00461#1/2) were used to knock down LINC00461. The pcDNA3.1/LINC00461 or pcDNA3.1/E2F1 was used to overexpress LINC00461 or E2F1. siNC, shNC, and pcDNA3.1 vectors were controls. The microRNA 4478 (miR-4478) mimic and miR-4478 inhibitor were used for miR-4478 overexpression and knockdown, NC mimic and NC inhibitor as controls. Plasmids sequences used in the present study were listed as below:All vectors were produced by GenePhamar . The introduction of plasmids was accomplished by Lipofectamine 2000 (Invitrogen) as demanded, and cells were harvested 2 days after transfection.To obtain RNA extracts, TRIzol reagent (Invitrogen) was used. The complementary DNA (cDNA) was produced from extracted RNAs utilizing PrimeScript RT reagent Kit and First-Strand cDNA Synthesis Kit  with the genomic DNA (gDNA) Eraser kit . The PCR reactions were accomplished utilizing SYBR Premix Ex Taq II (Takara) on a StepOne Plus Real Time PCR System (Life Technologies). Small nuclear RNA U6 (for miRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for lncRNA and mRNA) were internal controls. Primers were as follows:Cell counting kit-8 (CCK-8) cell counting kit  was applied for cell viability detection. Transfected cells were inoculated in 96-well plates (1000 cells/well). 10 μl CCK-8 solution was added at incubation time points at day 1, 2, 3, and 4. Following further incubation for another hour at 37°C, the optical density (OD) value (450 nm) was estimated under a microplate reader .5/well) was added to the upper insert, whereas 20% serum-contained medium was added into the lower insert. After 20 h incubation at 37°C, cells remained in upper chambers were scrubbed off by the cotton swab, and migrated cells on the bottom surface were fixed utilizing 4% PFA and stained utilizing 0.1% crystal violet. Stained cells were evaluated in five randomly chosen fields under the optical microscope.For detecting cell migration, transwell chambers (pore size at 8 μm)  were applied. Non-serum medium with transfected cells  for luciferase reporter assay. The E2F1 3′UTR or LINC00461 containing miR-4478 binding sequences were inserted into the pmirGLO vectors to produce WT-LINC00461 and WT-E2F1. The Mut-LINC00461 and Mut-E2F1 (with mutated binding sites) were generated as well. MiR-4478 mimic or NC mimic were co-transfected with WT-LINC00461 or Mut-LINC00461, or WT-E2F1 or Mut-E2F1 into 293T cells  utilizing Lipofectamine 2000 (Invitrogen). The pGL3-LINC00461 promoter was co-transfected with pcDNA3.1/E2F1 or pcDNA3.1 into cells. 48 h following transfection, luciferase activity was assessed utilizing the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was applied as normalized control.Briefly, LINC00461 biotin probe and LINC00461 non-biotin probe were generated by GenePharma . Then, LINC00461 biotin probe and LINC00461 non-biotin probe were incubated with cell lysate and streptavidin-coated magnetic beads . The abundance of candidate miRNAs in biotin-coupled RNA complex was tested by quantitative real-time PCR (RT-qPCR).To evaluate the binding of E2F1 on LINC00461 promoter, a ChIP Assay Kit (Millipore) was used. The extracted chromatin DNAs were sonicated into 200–400 bp fragments and subjected to immunoprecipitation with the antibodies against IgG  or E2F1 . The precipitated DNAs were analyzed by RT-qPCR.The cell lysates were obtained by RIPA buffer  with 1% protease inhibitors . After separation on SDS-polyacrylamide gels (12%), the proteins were loaded onto polyvinylidene fluoride membranes  which were later sealed for 1 h with 5% skim milk. Subsequently, membranes went through overnight incubation with primary antibodies at 4°C and then 1 h incubation with secondary antibody . The following primary antibodies were used: anti-E2F1 , anti-N-cadherin , anti-E-cadherin , anti-Vimentin , anti-Slug , anti-Twist , and anti-GAPDH .In accordance with the protocols provided by the suppliers, PARIS™ Kit  was used for nuclear and cytoplasmic fractionation assay in NSCLC cells. NSCLC cells in fractionation buffer went through centrifugation, followed by collection of the supernatant. We washed the remaining lysates in fractionation buffer, and cell nuclei were obtained via cell disruption buffer. Thereafter, RT-qPCR was utilized to detect the cytoplasmic and nuclear LINC00461.Four percent paraformaldehyde was used to fix SPC-A1 and A549 cells maintained on glass slides, followed by being permeabilized with 0.5% TritonX-100. LINC00461 probe was utilized for hybridizing SPC-A1 and A549 cells by the ViewRNA™ ISH Cell Assay Kit (Thermo Fisher Scientific) referring to the manufacturer’s protocol, as described previously . At lastSPC-A1 and A549 cells were maintained in 6-well plates, and later transfected with shNC or shLINC00461#1/2 for 48 h. Cells were then fixed in 4% paraformaldehyde and incubated at 4°C with E-cadherin and N-cadherin primary antibodies overnight and anti-rabbit IgG which was fluorochrome-labeled. Prior to confocal microscopy analysis, DAPI was applied for staining cells.t-test or one-way/two-way ANOVA analyses were used respectively for evaluating two-group or multi-group variances. All assays were triplicated. P<0.05 was used to define the significance of differences.Data presentation was realized by mean ± S.D. Statistical analysis was accomplished on the SPSS 18.0 statistical software . Student’s n=90), and matched para-cancerous tissues (n=90) was taken as a reference. The results indicated that LINC00461 was up-regulated in NSCLC tissues  assay further corroborated that LINC00461 was a cytoplasmic RNA . In cytoplasm, lncRNAs could act as a ceRNA through competing with downstream mRNAs for miRNAs [http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-predicted). Among those potential LINC00461-associated miRNAs, we chose the top 200 miRNAs. Subsequently, RT-qPCR examined differential expression of these miRNAs in 16HBE  and three NSCLC cells . Results of RT-qPCR confirmed eight down-regulated miRNAs in NSCLC . RNA pull-down assay demonstrated that miR-4478 was significantly enriched in biotinylated LINC00461 probe while other seven miRNAs did not present significant enrichment . Consequently, miR-4478 was identified as a downstream miRNA of LINC00461 in NSCLC. The binding site between LINC00461 and miR-4478 was depicted in It was reported that the regulatory mechanism that lncRNAs mediated in cancer progression depended on their localization in cells . To decir miRNAs ; therefohttp://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8%2Findex). It was discovered that E2F1 was a putative target for miR-4478. E2F1 has been documented to present a high expression and could aggravate cell proliferation, invasion, and metastasis in NSCLC [Subsequently, we searched for the target mRNAs through TarBase , we found that E2F1 was a potential transcription factor at LINC00461 promoter  by acting as miRNA sponges . In our E2F1 is defined as a transcription activator and reported to regulate cell cycle, cell growth, and metastasis in cancers . The oncTo be concluded, E2F1-activated LINC00461 functioned as an oncogene in NSCLC. In addition, LINC00461 predicted a poor prognosis and promoted NSCLC proliferation, migration, and EMT. All results showed LINC00461/miR-4478/E2F1 positive feedback loop in NSCLC, indicating that LINC00461 could be a new prognostic marker. This finding provided a theoretic basis to explore the promising therapeutic strategies for NSCLC patients.Click here for additional data file.


A total of 367 minor nucleotide variations  were detected throughout the viral genome in both groups, while nucleotide variations at high frequencies (>10% abundance in relative read counts in a single sample) were more prevalent in ICC . Among the high-level variations found in ICC, six were located in the E1/E2 genes, and all of them were non-synonymous substitutions . In vitro functional analyses of these E1/E2 variants revealed that E1/M207I, E2/D153Y, and E2/R302T had reduced abilities to support viral replication, and that E2/D153Y and E2/R302T failed to suppress the viral early promoter. These results imply that some within-host variations of E1/E2 present at high levels in ICC may be positively selected for and contribute to cervical cancer development through dysfunction or de-stabilization of viral replication/transcription proteins.Human papillomavirus type 16 (HPV16) is the most common HPV genotype found in invasive cervical cancer (ICC). Recent comprehensive genomics studies of HPV16 have revealed that a large number of minor nucleotide variations in the viral genome are present in each infected woman; however, it remains unclear whether such within-host variations of HPV16 are linked to cervical carcinogenesis. Here, by employing next-generation sequencing approaches, we explored the mutational profiles of the HPV16 genome within individual clinical specimens from ICC ( Cervical cancer is the fourth most frequent cancer in women worldwide and is etiologically linked to persistent infections with high-risk genotypes of human papillomaviruses (HPVs) . Of the Recent advances in next-generation sequencing technologies have facilitated a detailed understanding of viral genetic diversity within and between infected individuals . AlthougE1/E2 genes resulted in a decreased ability to support viral replication and transcription. Based on these results, we propose that selective pressure is being exerted on these E1/E2 variants during cervical carcinogenesis.Here, by employing deep sequencing techniques, we focused our analysis on those within-host nucleotide variations in the HPV16 genome detected at high levels within individual specimens. Such high-level variations were more frequently found in ICC than in cytologically normal cervix. Of these, some non-synonymous variants in the n = 21) and ICC (n = 31), who visited the Keio University Hospital, Tsukuba University Hospital, or Showa University Hospital for cervical cancer screening or treatment of cervical diseases. Cervical smears were classified according to the Bethesda system. Histological diagnoses of ICC were made using hematoxylin and eosin sections of cervical biopsy specimens according to the World Health Organization classification. HPV16 positivity was determined by HPV genotyping as described previously  to cover the whole-genome sequence of HPV16. The sequences of PCR primers were as follows: HPV16-1744F (5′-TGT CTA AAC TAT TAT GTG TGT CTC CAA TG-3′) and HPV16-5692R (5′-GAT ACT GGG ACA GGA GGC AAG TAG ACA GT-3′); HPV16-5531F (5′-GGG TCT CCA CAA TAT ACA ATT ATT GCT G-3′) and HPV16-1980R (5′-TAT CGT CTA CTA TGT CAT TAT CGT AGG CCC-3′) . The ampipeline1 . The accipeline1 . The preRenilla luciferase expression together with 100 ng of the E1 expression plasmid and 50 ng of the E2 expression plasmid using the FuGENE HD reagent (Promega). The total quantity of transfected plasmid DNA was adjusted to 220 ng with the empty plasmid p3xFLAG-CMV10  as carrier DNA. At 72 h after transfection, firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase assay system (Promega) on an ARVO MX luminescence counter , and the level of replication was quantified as the ratio of the two luciferase activities.The HPV16 origin-containing plasmid and expression plasmids for N-terminal FLAG-tagged HPV16 E1 and E2 were described previously . ExpressRenilla luciferase activities were measured as described above, and the level of transcription was quantified as the ratio of the two luciferase activities.The reporter plasmid containing the HPV16 early promoter, pGL3-P97, was described previously . HPV18-p2) supplemented with a protease inhibitor cocktail (Roche), followed by centrifugation at 21,000 × g at 4°C for 10 min. The cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. The proteins were visualized by the ECL prime western blotting detection reagent  with the following antibodies: anti-FLAG  and anti-α-tubulin .C33A and HeLa cells were lysed on ice with RIPA buffer  were transfected with 5 μg of the E1 expression plasmids using FuGENE HD. At 48 h after transfection, total cell extracts were prepared in RIPA buffer as described above. N-terminal 6xHis-tagged HPV16 E2 (His-E2) was bacterially expressed and purified as previously described (Human embryonic kidney 293 (HEK293) cells  were prepared by PCR using the HPV16 whole-genome plasmid as a template with the following primers: HPV16-bio7791F (5′-biotin-TAC ATG AAC TGT GTA AAG GTT AGT CA-3′), and HPV16-120R (5′-TGT GGG TCC TGA AAC ATT GCA GTT CTC TTT-3′). The biotinylated DNA probes were coupled to Dynabeads/M-280 streptavidin  at room temperature for 20 min in coupling buffer . Total cell extracts were prepared from HEK293 cells that had been transfected with the E2 expression plasmids as described above, and incubated with the DNA-coupled or uncoupled magnetic beads at 4°C for 2 h. The beads were washed three times with RIPA buffer, and the bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer and analyzed by western blotting with anti-FLAG antibody.2 Mann–Whitney U test was used to evaluate the average variation number between the NILM and ICC samples. Welch’s t-test was used to evaluate a difference in variant frequency or reporter activity between different groups. A value of p < 0.05 was regarded as statistically significant.All statistical analyses were performed using R version 3.6.3.DRA009226.Short-read sequencing data are available from the DNA Data Bank of Japan, Sequence Read Archive, under accession number n = 31) and 200 from NILM samples (n = 21)  and from 0 to 46 in NILM  . As shown in p = 0.02, Welch’s t-test). These nucleotide variations were almost completely evenly distributed throughout the viral genome in both NILM and ICC samples (E5 and L2 (NC) showed the highest density of nucleotide variations , it became apparent that the E1, E2, and L2 regions contained more nucleotide variations at high frequencies in ICC than in NILM  . As we p(n = 21) , non-synge, 9.5) . There w samples , althougriations . When th in NILM . The E7 p = 0.03, Welch’s t-test), whereas no such differential trend was apparent for synonymous and non-coding region substitutions . The distributions of frequencies of non-synonymous versus synonymous substitutions were also examined according to individual gene regions. As shown in E1, E2, and L2 regions were more enriched for non-synonymous substitutions at high frequencies  compared to the other regions. The one E7 and one L1 variation detected at high levels , and only one in the non-coding region between E5 and L2. Of the 10 coding-region variations, nine were non-synonymous or missense substitutions, generating variant forms of viral proteins: E1 Q142K, E1 M207I, E1 L262V, E2 D153Y, E2 R302T, E2 A357T, E7 L65F, L1 E52∗ , and L2 N147D. Among these substitutions, E1 L262V, E2 D153Y, E2 R302T, and L1 E52∗ were not found by BLAST search, and thus were newly identified variations. Regarding the substitution pattern, as reflected in Highly abundant within-host variations, which we defined as nucleotide variants with >10% abundance in relative read counts in a single sample, were more frequently detected in ICC than in NILM: 10 variations in ICC and one variation in NILM . Of thesE1/E2 genes prompted us to test whether the resulting E1/E2 variants had altered activities in supporting viral replication. To this end, the variant and prototype E1/E2 proteins were transiently expressed from expression plasmids in HPV-negative C33A cells, together with a viral origin-containing plasmid that included the firefly luciferase gene. Three days after transfection, replication levels of the origin plasmid were evaluated as a readout of elevated reporter activities. Although the transient replication assay does not reflect a replication mode of viral persistence, it has often been used to evaluate inherent activity of E1/E2 to induce viral replication. Two E1/E2 variants, E1 K483A , but the length of a reading frame alone cannot explain the enrichment of E1/E2 mutations because the late L1 and L2 regions of similar lengths  harbored only one high-frequency non-synonymous substitution in L1. The enrichment of high-level, non-synonymous substitutions in E1/E2 among the ICC samples prompted us to explore any functional changes in these gene products. Indeed, we found that of six E1/E2 variants tested, three  had a reduced ability to regulate viral replication or transcription.Interestingly, the non-synonymous substitutions detected in ICC were more prominent in the E2 is often disrupted by integration into the host genome or transcriptionally silenced by epigenetic modifications  . Moreove protein . Enrichmer study . These oAmong the E2 variants found in this study, D153Y seems to be less stably expressed in cells, which likely leads to the failure of this variant to regulate viral replication and transcription. Three-dimensional structural projection of the transactivation domain of E2 indicates that the D153 residue is exposed on the surface of the molecule, positioned spatially near residues that were previously reported to be subject to intrahost variation . The celConsistent with the important role in DNA interaction suggested from structural inspection, R302T completely lost binding activity to the viral origin DNA containing three E2-recognition sites, and this defect nicely explains the inability of R302T to regulate viral origin-dependent replication and early promoter-driven transcription.Regarding E1 variants, M207I showed a reduced ability to support viral origin-dependent replication, although it was expressed at a similar level to the prototype E1 and retained the ability to interact with E2. Such replication defect may be attributed to some change in DNA interaction during DNA unwinding by M207I because this residue is included in the DNA-binding domain of E1 . Our preE1/E2 genes are a hotspot for high-level within-host variations, it is also clear that not all the variations of E1/E2 cause functional changes or reduced expression. Because HPV-infected cervical lesions generally undergo two-way processes of progression and regression during a long period of persistent infection, it is very likely that a small viral population in the regression phases experiences a bottleneck for random selection of a minor variant genome . Enrichment of variant viral genomes in cervical cancer specimens may reflect such neutral selection processes. In this regard, the presence of enriched nucleotide variations in the HPV genome is not a prerequisite but rather episodic for individual cases to eventually progress to ICC, as recently demonstrated for APOBEC3 mutagenesis in a range of human cancer cell lines  can be found below: The studies involving human participants were reviewed and approved by the Institutional Review Boards at Keio University Hospital, Tsukuba University Hospital, Showa University Hospital, and the National Institute of Infectious Diseases. The patients/participants provided their written informed consent to participate in this study.in vitro experiments. YH, YT, MY-N, and IK analyzed and interpreted the data. MO, NT, TS, TI, AS, and KM collected the clinical specimens. All authors read and approved the final manuscript.IK conceived and designed the study, and wrote and revised the manuscript. YH and YT obtained the sequencing data. MY-N, KT, and IK performed 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.


Escherichia coli expressing the engineered chimeric PHA synthase PhaCAR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaCAR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1PsSTQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaCAR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in  PHAs hamaterial , composimaterial , or blenmaterial . Therefoco-3HB) is a semitransparent material with pliable property (co-3HB). Chemically synthesized polyglycolide acid and poly(lactide-co-glycolide) are known as highly hydrolytic degradable materials, and they are used in biomedical fields as bioabsorbable materials (co-3HB) exhibits an intermediate hydrolytic degradability between P(3HB) and poly(lactide-co-glycolide), which potentially manipulates the bioabsorption rate of PHAs (The glycolate (GL) unit is an unusual component of PHA, because it does not exist in naturally occurring PHAs . As a typroperty . In addiaterials . Althougaterials . Artific of PHAs .PsSTQK, which belongs to class II PHA synthases and has two point-mutations, possesses extremely broad substrate specificity and plays a central role in the biosynthesis of lactate-based and other 2-hydroxyalkanoate-based PHAs  are desired to expand the variety of GL-based PHAs.PHA synthases are the key enzymes that determine the monomeric unit composition of a polymer . The engsed PHAs . The finased PHA . NeverthAR. PhaCAR is a chimeric PHA synthase composed of the N-terminal region of PhaCAc derived from Aeromonas caviae and the C-terminal region of PhaCRe derived from Ralstonia eutropha (Cupriavidus necator) (AR is the first known class I PHA synthase that can efficiently incorporate 2-hydroxybutyrate (2HB) units (Re exhibits very little activity toward 2HB-CoA (AR possesses a unique function for synthesizing block copolymers (Escherichia coli expressing PhaCAR and propionyl-CoA transferase (PCT) spontaneously produced P(2HB-b-3HB) from the mixture of 2HB and 3HB precursors supplemented in the medium. P(2HB-b-3HB) is the first structure-proven block PHA. Therefore, the monomer sequence of the obtained polymer is of interest in the attempt to incorporate GL units using PhaCAR. Indeed, we found PhaCAR is the first known class I GL-CoA-polymerizing PHA synthase and that the obtained polymer possesses a unique block sequence consisting of a random segment and a homopolymer segment.The present study aims to examine the GL-incorporating capacity of an engineered PHA synthase, PhaCnecator) . The junB) units , whereas 2HB-CoA . Moreovepolymers . EscheriRephaCARpct harboring the phaCAR and PCT genes from Megasphaera elsdenii under the control of the R. eutropha phb operon promoter and pBSPRephaC1PsSTQKpct harboring the PsSTQKphaC1 instead of the phaCAR were constructed in a previous study  medium containing 2 wt% glucose, 100 mg/L ampicillin, 5 g/L sodium -3HB and varied concentrations of sodium GL in a 500 mL shake flask at 30°C for 24 h with reciprocal shaking at 120 rpm.pBSPus study . pBSPRep’-phbCAB , which chaCARpct . E. coliThe concentration of monomer precursors in the medium was measured using Agilent 7100 CE, a capillary electrophoresis (CE) system, equipped with a capillary tube . The sample was injected for 4 s under a pressure of 50 mbar. The electrophoresis was performed at 25°C and -15 kV, with α-AFQ108 used as the running buffer . The sample was detected by a diode array at 400 nm.The lyophilized cells (approximately 10 mg) were treated in a solution of 0.5 mL chloroform and 0.5 mL 15% sulfuric acid in ethanol at 100°C for 2 h. The obtained ethyl esters were analyzed by gas chromatography (GC) as described previously .The polymer was extracted from lyophilized cells with chloroform at 60°C for 48 h in a test tube with a screw cap. The cell debris was removed through a 0.2-μm-pore-size polytetrafluoroethylene (PTFE) membrane filter. The chloroform extract was concentrated under air flow in a fume hood. The excess amount (approximately 10 times) of methanol was added to the extract at room temperature to precipitate the polymer. This purified polymer was applied in further analyses.in vacuo for 24 h at room temperature. The DSC data were recorded on a DSC3+ differential scanning calorimeter (Mettler Toledo). The samples were heated from -50 to 210°C at 20°C/min (the first heating scan). After rapid quenching at -50°C and isothermal incubation for 5 min, the samples were heated to 210°C at 20°C/min for a second heating scan.A differential scanning calorimetry (DSC) sample was prepared on an aluminum pan. A polymer chloroform solution containing approximately 3 mg polymer was applied to the pan and air dried for 2 h, then subsequently 3 containing tetramethylsilane, which was dissolved at 60°C and passed through a 0.2-μm-pore-size PTFE membrane filter. The NMR data were recorded on JEOL ECX-400 and ECS-400 spectrometers . The same solution was analyzed using size-exclusion chromatography equipped with two tandem high-performance liquid chromatography columns of Shodex GPC K-806L . The flow rate was 0.8 mL/min; the column oven was kept at 40°C; and the sample volume was 100 μL.Nuclear magnetic resonance (NMR) analysis of the polymers was performed using approximately 5 mg/mL of a polymer solution in CDClco-3HB) and P(3HB) solutions, respectively. Then, the suspension was filtrated through a 0.1-μm-pore-size PTFE membrane filter. The precipitated fraction refers to the polymer on the filter that was recovered in chloroform, whereas the soluble fraction was the polymer solution that dried up after passing through the filter.The purified polymer samples were fractionated into two fractions via solubility to distinguish a polymer blend and block copolymer . The 10 co-3HB) in E. coli JM109 tested the GL-incorporating capacity of PhaCAR. Two 3HB-CoA supplying pathways were used: the CoA transferring pathway catalyzed by PCT from the 3HB supplemented in the medium (pBSPRephaCARpct) and the well-known dimerization pathway of acetyl-CoA catalyzed by β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) (pBSPRephaCARpctAB). First, the cells harboring the pBSPRephaCARpct were grown on the GL and 3HB. The PsSTQKphaC1 gene served as a control. As a result, a polymer containing GL units was obtained in both constructs  with an increase in the GL concentration was due to the toxicity of the GL in the medium and a decrease in polymer production. A GL concentration of 4 g/L was used in further study, because a greater increase in the GL concentration (12 g/L) exhibited no significant effect on the GL molar ratio. E. coli did not grow at 20 g/L GL concentration (data not shown).The attempts to synthesize P from the glucose and GL  (∗-GL (a), GL-GL∗-3HB or 3HB-GL∗-GL [(b) or (c)], and 3HB-GL∗-3HB (d), respectively  , these fectively . Notably , (c), and (d)  .x (mol/mol) over the total polymer, the abundance of the four triad sequences a–d is calculated by the following equations. Here, 1-x indicates the 3HB rate (mol/mol) over the total polymer.Given an ideal random copolymer with a GL rate of 1H NMR of P(40 mol% GL-co-3HB) synthesized by PhaCAR, the a∼d values were experimentally determined (x = 0.71). This means that the P(40 mol% GL-co-3HB) contains a considerably rich GL region, which is a nearly ideal random copolymer with a local GL molar ratio of 71 mol%. Thus, the GL-rich region is referred to as the P(GL-ran-3HB) segment. Consequently, it was rationally presumed that the copolymer contains a region(s) composed of mostly 3HB units, which is referred to as the P(3HB) segment. The molar ratio of the P(3HB) segment over the total polymer was calculated by subtracting the amount of 3HB units in the P(GL-ran-3HB) segment from the total 3HB units as shown in the following equation. Here, function [n] indicates the molar ratio of n. x is the local GL rate (mol/mol) in the P(GL-ran-3HB) segment; and 1-x is the local 3HB rate (mol/mol) in the P(GL-ran-3HB) segment.Based on the termined , No. 1. x, 0.6 for [total 3HB], and 0.4 for [total GL], the [P(3HB) segment] was estimated to be 0.44 (mol/mol). The high intensity of the resonance occurring at 5.2 ppm, which is ascribed to the methine proton of the 3HB units in the 3HB-3HB∗ dyad, is consistent with the interpretation. Overall, it was concluded that the copolymer synthesized by PhaCAR is composed of two segments, P(71 mol% GL-ran-3HB) and P(3HB), and the ratio of these segments is 56:44 (mol/mol)  .AR, PhaC1PsSTQK synthesized the copolymer with a large d, which indicates an abundant 3HB-GL-3HB triad in the polymer chain  (No. 4). The calculated value (18 mol%) was slightly greater than the actual GL molar ratio (11 mol%). These results indicate that the copolymer synthesized by PhaC1PsSTQK is a nearly random copolymer, but the monomer sequence is not ideally random and contains a slightly GL-rich region.In contrast to PhaCer chain , No. 3. 1H NMR analysis indicated that P(GL-co-3HB)s synthesized by PhaC1PsSTQK and PhaCAR has distinct structures in terms of the monomer sequence. The interpretation was verified by the thermal properties of the polymers. The whole polymer without solvent fractionation was used for the analysis. P(11 mol% GL-co-3HB) synthesized by using PhaC1PsSTQK exhibited a small melting peak at 126°C  at 7.6°C, which is consistent with the previous result , which was estimated using the enthalpy of fusion of 100% P(3HB) crystal (146 J/g) (PsSTQK (16%). The copolymer synthesized by PhaCAR exhibited a complicated Tg shift  film synthesized by PhaC1PsSTQK with similar GL molar ratio  , was 1.5Tg shift , which iar ratio , and a lar ratio .AR contained two distinguishable segments. To determine whether these segments could be a block copolymer or polymer blend, the copolymer was subjected to solvent fractionation. Because no sufficient difference in the solubility was found between P(GL-ran-3HB) and P(3HB), the polymer solution in chloroform was partially precipitated by adding methanol. This fractionation method is based on the principle that the solubility of polymers in organic solvents is dependent on their molecular weight. In fact, the insoluble fraction of the partially precipitated P(3HB) possesses a higher molecular weight than that of the soluble (non-precipitated) fraction (The copolymer synthesized by using PhaCfraction .co-3HB) synthesized by PhaCAR was separated into two fractions by solvent fractionation: precipitated and soluble fractions. The precipitated fraction showed higher total and local GL molar ratios, which was determined based on the 1H NMR  was lower than that of the soluble fraction . The postulation that the sample is a blend of P(GL-ran-3HB) and P(3HB) leads to a contradicting interpretation that P(3HB) with relatively low Mw was readily precipitated. Therefore, it is likely that the P(3HB) segment in the precipitated fraction is covalently linked to the P(GL-ran-3HB) segment.After the addition of methanol, P-b-P(3HB)  . CurrentAR is the first known GL-incorporating class I PHA synthase. It was concluded that PhaCAR possesses a higher GL-incorporating capacity than that of PhaC1PsSTQK, because PhaCAR synthesized the P(GL-ran-3HB) segment, which was estimated to contain 71 mol% GL, whereas PhaC1PsSTQK can incorporate a much lower GL molar ratio -b-P(3HB). PhaCAR was previously shown to synthesize P(2HB-b-3HB), which is composed of homopolymer segments. These results raise a question about why a random segment was generated by the same set of enzymes PhaCAR and PCT. One possible factor for the generation of a random segment is the copolymerization kinetics of PHA synthase. It was previously demonstrated that the in vitro activity of PhaC1PsSTQK toward lactyl-CoA was lower than that toward 3HB-CoA under single substrate conditions. However, when two substrates (each 0.2 mM) were combined, lactyl-CoA was consumed faster than 3HB-CoA  biosynthesis is efficient when the cultivation temperature is higher than the Tg of the polymer (Tg than that of polyglycolate (35–40°C), P(GL-ran-3HB) chains are more readily synthesized than the homopolymer. In other words, cultivation temperature could be a parameter for regulating GL ratio in P(GL-ran-3HB) segment.Moreover, the obtained polymer was a unique block PHA, P(GL- 3HB-CoA . A simil polymer . Given tran-3HB)-b-P(3HB) was processed into a stiff film as was P(3HB)  . The stiphb operon of R. eutropha, which expresses in E. coli without induction under the conditions used in this study. The use of inducible promoter might be useful for optimizing the polymer production. The expression levels of PHA biosynthetic genes reportedly influenced the production and molecular weight of P(3HB) (lac promoter for production of P(lactate-co-3HB) in E. coli. Interestingly, the highest polymer production was achieved with relatively low expression conditions (co-3HB) production. Further study will be needed for efficient production of the block PHAs.The promoter used in this study is derived from f P(3HB) . In our nditions . AlthougAR was found to be the first known class I GL-CoA-polymerizing PHA synthase. PhaCAR synthesized the unique random-homo block PHA consisting of P(GL-ran-3HB)-b-P(3HB). The finding of the random segment-containing block PHA has the potential to expand the molecular design of polymers with a variety of properties. In addition, the GL-rich structure synthesized by PhaCAR is useful as hydrolytically degradable material.PhaCAll datasets generated for this study are included in the article/SA performed the experiments and wrote the original draft. SS and RM performed the experiments. TO and MZ contributed to the scientific discussion and the manuscript writing. KM conceived and designed the study and wrote the manuscript. All authors revised the manuscript and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These demonstrate CBP blockage can be developed for the treatment of CML independent of BCR-ABL mutation status including T315I.The treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, has yielded clinical success. However, the direct targeting of BCR-ABL does not eradicate CML cells expressing mutant BCR-ABL, especially the T315I mutation in BCR-ABL. Moreover, increasing mutations were identified in BCR-ABL domain, resulting in TKIs resistance recently. It is necessary to find BCR-ABL-independent target for treating CML patients with various mutations, including T315I mutation in BCR-ABL. The dichotomous behavior of CREB binding protein (CBP) and E1A protein (p300), recruited by β-catenin associated with self-renewal and differentiation, have been identified in hematopoietic stem cells, respectively. In this study, CBP was aberrantly expressed in CML cells on the basis of Oncomine dataset. The β-catenin bound with much more CBP than p300 in CML cells. Down-regulation of CBP inhibited cell proliferation capacity and increased the binding of β-catenin to p300, thus promoting cell differentiation and p53-dependent cell senescence in CML cells with either wild type or T315I mutant BCR-ABL  Chronic myeloid leukemia (CML) is a clonal malignant disorder of pluripotent hematopoietic stem cells (HSCs), characterized by the presence of Philadelphia chromosome (Ph) translocation t9;22)q34;q11), leading to the formation of the novel BCR-ABL fusion gene Despite the high degree of homology and similar patterns of expression, CBP and p300 play unique and distinct roles in gene regulation , 9. Distin vitro and in vivo models.In the current study, more p300 is bound to beta-catenin when CBP is knock-down. Down-regulation of CBP increased the binding of β-catenin to p300, thus promoting cell differentiation and p53-dependent cell senescence and inhibiting cell proliferation capacity in CML cells with either wild type or T315I mutant BCR-ABL Nitrobluetetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich . Antibodies against β-catenin (#8480), p21(#2947S), phosphorylated Rb (#9307S), and CyclinD1 (#2922S) were purchased from Cell Signaling Technology . CBP (sc-369), p300 (sc-584), JUN (sc-45) were purchased from Santa Cruz Biotechnology Inc. . p16 (ab117443) and recombinant mouse Wnt3a protein was purchased from Abcam . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (822051) was purchased from Kangchen Co. . Cell protein Extraction Kit and Nucleoprotein Extraction Kit was purchased from Beyotime . The senescence detection kit was purchased from Beyotime .The human CML cell line K562 was obtained from ATCC and the murine 32D myeloid cells stably expressing either wild-type BCR-ABL (32D-WT) or T315I mutant BCR-ABL (32D-T315I) were obtained from Professor Pan JX . BCR-ABL expression in these cells was checked by Western-blot for verification every 3 to 4 months routinely. Cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum , 1 unit/ml penicillin and 1 mg/ml streptomycin at 37°C and 5% CO2, 32D-WT, and 32D-T315I cells were additionally supplemented with 0.5 ng/ml WNT3a to imitate the feature of activated Wnt signaling in CML cells.For depletion of CBP and p300, the pSIH-H1-puro-construct  containing CBP or p300 short hairpin RNAs (shRNA) was generated by cloning the following CBP specific or p300 specific RNAi target sequences into pSIHF-H1:CBP shRNA: 5-GATCCCGTTTACCATGAGATCCTTATCTTCCTGTCAGAATAAGGATCTCATGGTAAACGTTTTTG-3’.P300 shRNA:5’- GATCCTAACCAATGGTGGTGATATTACTTCCTGTCAGATAATATCACCACCATTGGTTATTTTTG-3’.Briefly, CMLs silencing CBP or p300 were generated by infection with CBP shRNA lentiviruses or p300 shRNA lentiviruses, and selected by treatment with 0.5 μg/ml puromycin for 10 days, beginning from 48 h after infection.K562, 32D-WT, and 32D-T315I cells receiving different treatment were cultured in 96-well plates for 72 h. The NBT reduction assay was performed as Collins’description .For the SA-ß–galactosidase assay, the SA-ß-galactosidase activity was determined using a senescence detection kit (Beyotime) according to the manufacturer’s instructions. Senescent cells were identified as blue-stained cells under an Olympus IX71S8F inverted microscope.Cell specimens were washed twice with PBS and total cellular protein was extracted with RIPA. Nuclear proteins were extracted by nucleoprotein extraction buffer and were used when detecting expression of ß-catenin. Equal amounts of protein (50–100 ug) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to PVDF membranes, blocked and incubated overnight with primary antibodies at 4°C. After the secondary monoclonal antibodywas applied to visualize proteins using Kodak X-AR film, the results were analyzed by Gel-Pro Analyzer.3) was calculated as L×W2×0.5 (L indicates the length-diameter and W the width-diameter of the tumor) . To test the incidence of tumors and proliferation ability, 32D-WT or 32D-T315I cells with CBP or p300 knockdown were subcutaneously injected into left subaxillary of mice, respectively. Tumor volume (mme tumor) .via boiling and centrifugation. The immnoprecipitated proteins were then analyzed by standard immune blotting , and the supernatant of cell lysate corresponding to1mg of total protein was precleared by Protein G Plus/Protein A-agarose beads  to minimize nonspecific binding, followed by the addition of either mouse IgG  or β-catenin overnight. The bound proteins were washed thrice with lysis buffer and dissociated with the beads blotting .p<0.05 was considered statistically significant.Data analysis was performed using the SPSS 16.0 software package. All experiments were repeated at least three times, and data are presented as Means ± SEM. The mean values were compared using unpaired student’s t-test (two groups). A value of www.researchdata.org.cn), with the approval RDD number as RDDB2020000942.The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform (p=0.002) and β-catenin (p=0.001) expression was significantly up-regulated in 76 cases of CML samples compared with 74 cases of normal samples (p=0.900), suggesting that β-catenin/CBP is preferentially expressed in malignant proliferative CMLs. To further evaluate the significance of CBP and p300 on cell growth or differentiation in CMLs, we silenced endogenous CBP and p300 using lentiviral short -hairpin RNA (shRNA) in K562, 32D-WT, and 32D-T315Icells, respectively  showed that CBP  was performed and indicated that the down-regulated expression of CBP increased the binding of β-catenin to p300 in both BCR-ABL wild-type and T315I mutant CMLs , has been successful in 5%–10% AML patients but not in CML patients . Under tWnt signaling pathways regulate a variety of processes including cell growth, oncogenesis, and development. Upon Wnt signaling, β-catenin accumulates in the nucleus and binds to transcription factors of the TCF/LEF family to activate transcription and constitutive activation of downstream target genes, such as surviving, c-myc and cyclinD1 by the TCF/LEF-β-catenin complex, which is essential for balancing cell survival and differentiation , 23, 24.Our results indicate that β-catenin recruits more p300 after CBP expression is silenced in CMLs expressing wild-type and T315I mutant BCR-ABL. The repression of CBP decreased the expression of cyclinD1 , which ivia a switch to β-catenin binding to p300 by targeting CBP, and to ultimately eliminate CMLs, including expressing T315I mutant BCR-ABL CMLs.Cellular senescence disables proliferation in damaged cells and is relevant for cancer and aging. This involvement of tumor-suppressor pathways led to the identification of senescence as potent tumor-suppressor mechanism. Several studies have revealed that, adult stem cells could escape cellular senescence by avoiding both differentiation and the activation of checkpoint responses that arrest the cell cycle in cell homeostasis. The key proteins that induce senescence are members of the p53/p21 and p16/p-Rb tumor suppressor networks that function to arrest proliferation and contribute to the irreversibility of the senescent state. In our study, we used SA-β-Gal staining to identify senescent cells because it is a proven and reliable marker of senescence in many settings. To determine whether p53/p21 or p16/p-Rb were required for β-catenin/p300 mediated senescence, we examined these markers and mediators of senescence. The results indicated that the increased β-catenin/p300 mainly activated p53/p21 signaling mediated cell senescence. This offers a possibility to promote cell differentiation and senescence and limit proliferation These results suggest the binding of β-catenin to CBP or p300 decides CML cell fate, proliferation or differentiation, which is independent of BCR-ABL mutation status. The inhibition of CBP could be a promising strategy for the treatment of CMLs including BCR-ABLT315I mutant CMLs Figure 5The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Sun Yat-Sen University.in vitro experiments and analyzed data. XKW, LC, XS, XPW, and QH performed the animal experiments. ZC analyzed data and provided technical assistance. KY wrote the paper. LF and Z-SC provided technical assistance and revised the manuscript. All authors contributed to the article and approved the submitted version.LF designed the research. KY, FW, and HZ performed the This work was financially supported by the National Science & Technology Major Project Key New Drug Creation and Manufacturing Program, China (2018ZX09711002), the Natural Scientific Foundation of China , the Guangdong Provincial Special Fund for Marine Economic Development Project (GDNRC [2020]042), the Science and Technology Foundation of Guangdong Province , and the Scientific and Technological Leading Talent Project of Guangdong Province . Author QH was employed by Guangzhou Handy Biotechnological Co., Ltd.The remaining 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.


To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4 CD4+ T cell antigen receptors recognize proteolytically processed protein fragments (peptides) bound to major histocompatibility complex class-II (MHCII)+ T cells . Relative peptide occupancy of these pockets within the binding cleft designates peptide anchor residues at positions 1 (P1), 4 (P4), 6 (P6), 7 (P7), and 9 (P9). Stable peptide-MHCII complexes are formed when anchor pockets are occupied with chemically and structurally compatible side chains. These optimal amino acid residues define the peptide-binding motif of the MHCII.HLA-DM is structurally homologous to classical MHCII molecules. Despite the similarity, DM is unable to associate with peptide antigens due to the near-complete occlusion of the peptide-binding cleft . Classic+ T cells for activation or tolerance induction. It is through this effect that HLA-DM is said to edit the pMHCII repertoire that is presented on the cell surface.HLA-DM encodes an endogenous signal that directs steady-state localization into late endosomal MHC class II compartments (MIIC) where antigen processing occurs , 16. ClaAlthough a large body of evidence demonstrates a central role for HLA-DM in shaping T cell immunity, neither DM's catalytic mechanism nor the pMHCII structural or chemical determinants that govern differential susceptibility to DM editing are well-understood , 8–10. E8–AFig. S1)  , 29–32.  S1A) 29––35. ReceS1A) 29–––38. This1A) 29–––, 39, 40.––et al. , two co-––et al. , 41, 42.––et al. , 43, 44.kobs) with pMHCII intrinsic stability (kin), DM binding affinity (mK), and catalytic turnover (kcat). Analyzing multiple pMHCII complexes, we find that the intrinsic stability of a pMHCII is linearly correlated with DM catalytic turnover and nonlinearly correlated with DM-binding affinity. We evaluated the impact of prominent pMHCII structural and chemical features on the catalytic mechanism and discovered that interactions throughout the peptide-binding cleft contribute to the binding affinity of DM and to the rate of peptide dissociation from the catalytic complex. We provide evidence that dissociation of the peptide N terminus is not a necessary precondition for binding of HLA-DM to pMHCII complexes.In the current study, we find that DM-mediated peptide dissociation rates are saturable. This observation allowed us to investigate pMHCII susceptibility to HLA-DM editing by resolving the independent kinetic parameters in the enzymatic activity of DM-mediated peptide exchange reactions. We developed a fluorescence anisotropy assay that measured the real-time dissociation of peptides from MHCII molecules and derived a Michaelis-Menten kinetic model that equates the peptide's observed dissociation rate (kobs) were calculated by fitting each dissociation curve to a single phase exponential decay formula . With a highly stable peptide binder (OPT1) that contains optimal amino acids in the major anchor positions P1, P4, P6, P7, and P9, we observed little to no peptide dissociation over days with HLA-DM concentrations up to 2 μm (A). Compared with OPT1, a wide range of DM-mediated peptide dissociation rates was observed when amino acids at anchor positions P1, P4, or P9 had been substituted (B–D). This varied by 1 order of magnitude at P9 for an isoleucine to glutamic acid (I9E) substitution (B), 2 orders of magnitude at P4 for a leucine to glycine (L4G) substitution (C), and 4 orders of magnitude at P1 for a tyrosine to alanine (Y1A) substitution (D). The observed dissociation rates (kobs) increased with increasing concentrations of HLA-DM, consistent with previous reports . We further assayed a DR1 complex bound with a highly stable peptide derived from the HA protein of influenza A virus and were able to observe an upper rate limit as DM concentrations approached 80 μm , suggesting that DM-catalyzed peptide dissociation rates are saturable for all pMHCII complexes given sufficient concentrations of DM. Moreover, this finding supports the conclusion that DM-catalyzed peptide dissociation reactions follow Michaelis-Menten kinetics, as previously reported in early studies , followed by the rate-limiting dissociation of the peptide from the catalytic complex defined by the catalytic turnover rate constant, kcat . Consistent with Michaelis-Menten kinetics, the model equates the upper rate limit (maximum) of peptide dissociation with kcat and the DM concentration at half-maximum with the Michaelis constant mK. Moreover, we derived an experimental specific equation to model the Michaelis-Menten reaction scheme, relating the observed peptide dissociation rates (kobs) from anisotropy assays to the kinetic parameters of the HLA-DM–catalyzed reactions . The derivation describes the catalytic turnover kcat as a measure of the stability of peptide bound in the DM-pMHCII catalytic complex, the Michaelis constant mK as DM-binding affinity of the pMHCII substrate, and the intrinsic peptide dissociation rate kin as a measure of the intrinsic stability of the pMHCII complex.These observations formed the experimental basis for a comparative study of the independent kinetic parameters of DM's enzymatic activity. We formulated a Michaelis-Menten kinetic model of the catalytic reaction involving a rapid steady-state binding of DM to a pMHCII substrate, assembling the DM-pMHCII catalytic complex (Michaelis complex) defined by the equilibrium dissociation constant kobs rate curves for greater than 30 individual DR1-peptide complexes  and resolved the kinetic parameters of DM enzymatic activity for each pMHCII substrate to facilitate assessment of kinetic correlations in DM-catalyzed peptide dissociation reactions. The parameters spanned 6 orders of magnitude in intrinsic pMHCII stability (kin), 2 orders of magnitude in DM-binding affinity (mK), and 5 orders of magnitude in DM catalytic turnover (kcat)  .r = −0.7177) between mK and kin , suggesting a nonlinear association between decreased pMHCII stability and increased DM-binding affinity. A robust Pearson correlation (r = 0.9342) between kin and kcat was observed , indicating a direct relationship between the intrinsic stability of the pMHCII and the stability of the peptide bound in the DM-pMHCII catalytic complex. Furthermore, an inverse Spearman correlation (r = −0.5992) between DM's catalytic turnover and binding affinity (B) suggests a nonlinear coupling between DM-binding affinity and the peptide dissociation rate from the catalytic complex. Together, these results support the idea that a significant number of peptide sequence-specific structural interactions that exist in the resting pMHCII complex are retained in the DM-bound catalytic intermediate.Pearson correlation analysis was used to assess monotonic linear relationships and Spearman rank correlation was used to evaluate nonlinear relationships between the kinetic parameters. We found a significant inverse Spearman correlation  was evaluated as a measure of pMHCII susceptibility to DM enzymatic activity. A robust Pearson correlation (r = 0.9903) was observed between the specificity constant and intrinsic stability (kin) . We observed an inverse Spearman correlation (r = −0.7581) between the specificity constant and DM-binding affinity , and a strong Pearson correlation (r = 0.9589) with kcat . Thus, susceptibility to DM is linearly related to kin and to the rate of peptide dissociation from the catalytic complex, but nonlinearly correlated with DM binding affinity. DM catalytic potency (kcat/kin) did not correlate strongly with any of the independent kinetic parameters (D). Thus, pMHCII intrinsic stability cannot be used to predict the fold-enhancement of peptide dissociation rates in the presence versus absence of DM. Overall, however, the findings indicate that the pMHCII intrinsic stability correlates strongly with DM-binding affinity, the rate of peptide dissociation from the catalytic complex, and catalytic specificity.The specificity constant (ty (kin) C, left. affinity C, middleith kcat C, right.rameters D. Thus, kcat) because these interactions do not exist in the catalytic complex  (kin for DR1-HA peptide complexes (A), underscoring the importance of this feature to the intrinsic stability of the pMHCII complex  >3600-fold, suggesting that the two hydrogen bonds mediated by Asn-82 are retained in the active DM-pMHCII catalytic complex, serving as potent stabilizers of the catalytic intermediate.The Asn-82 residue of the DRβ1 domain forms highly-conserved bidentate hydrogen bonds with the peptide main chain straddling the P2 residue in MHCII-peptide complexes (ig. S1A) , 32, 48.omplexes A, unders complex –33, 40. AFig. S1). Consistent with the previous observation, DRβ1 H81A decreased the intrinsic stability of the pMHCII (i.e. greater kin) and it increased DM-binding affinity (i.e. reduced mK) (B). Notably, we observed an ∼5-fold enhancement in peptide dissociation from the catalytic complex (kcat), suggesting that the His-81–mediated hydrogen bond was also retained in the catalytic complex. The impact of truncation of two N-terminal residues (2NΔ) of the HA peptide, functionally removing three hydrogen bonds between the MHCII and the peptide main chain and occupancy of the P-2 and P-1 residues  was evaluated. This resulted in a ∼5-fold increase in kcat (C), indicating that the two N-terminal peptide residues influence the stability of the catalytic complex.We explored the possibility that additional peptide N terminus interactions with the MHCII may be retained in the catalytic complex. We compared a pair of DR1-HA complexes varying in a histidine to alanine mutation at DRβ1 position 81 (DRβ1 H81A) that eliminates a single hydrogen bond between the MHCII and the peptide main chain on the carbonyl group of the P-1 residue (uced Km) B. Notabl in kcat C, indicakin), increased DM-binding affinity (lower mK), and a significant ∼8-fold enhancement in kcat (D). In the context of the OPT1 peptide, we observed a lesser impact on the stability of the pMHCII complex by the alanine substitution (Y1A′), yet an increased >2600-fold enhancement in kcat (E). This observation suggests that the energetic contribution to the kinetics of DM activity by specific anchor-pocket interactions is influenced by the overall sequence of the bound peptide. Nonetheless, these results support the conclusion that the identity of the peptide P1 anchor residue influences the stability of the DM-pMHCII catalytic complex. This conclusion was further supported by a tyrosine to isoleucine substitution (Y1I) that enhanced kcat by 18-fold (F). Altogether, the results provide strong evidence that interactions between DR1 and the N terminus of bound peptides can have a considerable influence on the rate of peptide dissociation from the catalytic intermediate, supporting the conclusion that these elements are conserved in the DM-pMHCII catalytic complex.These results suggested that peptide occupancy of the P1 anchor pocket might be conserved in the catalytic complex. The P1 anchor is the deepest and most hydrophobic pocket within the binding cleft of HLA-DR1, optimally binding large aromatic residues , 49. Pep in kcat D. In the in kcat E. This o 18-fold F. Altogekin), but had no significant impact on DM-binding affinity (mK) (A). The glycine substitution had a marked >120-fold increase in kcat, however, indicating that interactions involving the P4 anchor help to stabilize the DM-DR1 catalytic complex. The P4 pocket interaction was further destabilized by replacing leucine with arginine (L4R), a charged residue that is chemically incompatible with the properties of the P4 pocket. This substitution resulted in a marked >1300-fold increase in kcat, but again no significant effect on DM-binding affinity was observed (B). These results were additionally mirrored in a separate leucine to glycine substitution (L4G′) that was evaluated in the context of an OPT1 peptide that has an unfavorable glutamic acid residue at the P9 anchor position (C). Notably, the glycine substitution at P4 with complexes containing a disfavored P9 anchor had a greater impact on kcat (510-fold increase) than those containing an optimal P9 residue (A and C), again demonstrating that the energetic contributions of specific anchor-pocket interactions can be substantially modified by interactions elsewhere in the peptide-binding cleft.The contribution of interactions within the central region of the MHCII-binding cleft on the DM catalytic mechanism was assessed by evaluating the relative change in kinetic parameters with single amino acid substitutions in the peptide P4 and P6 anchor positions. The P4 pocket in HLA-DR1 is hydrophobic and relatively shallow, preferring moderately sized aliphatic residues , 49. Theity (Km) A. The globserved B. These position C. Notabl residue , A and Ckcat with no significant impact in kin nor mK (D). Replacement with the chemically unfavorable glutamic acid, however, substantially increased kcat by >100- or 200-fold, respectively (E and F). Altogether, the results of substitutions in P4 and P6 anchor residues indicate that interactions in the central region of the MHCII peptide-binding cleft have negligible influence on DM-binding affinity but considerable impact on the stability of the DM-pMHCII catalytic complex.The P6 pocket in HLA-DR1 is hydrophobic and the most shallow of the dominant pockets, preferring small aliphatic residues , 49. Repn nor Km D. Replacectively , E and Fkin) of the pMHCII and an increase in the catalytic turnover kcat (A). A trending ∼9-fold decrease (p = 0.119) in mK was observed, implying increased DM-binding affinity, although the effect did not reach the level of statistical significance. However, in comparing two additional pairs of DR1 complexes containing related peptides in which an unfavorable charged acidic glutamic acid (I9E′) or a bulky aromatic phenylalanine residue (I9F) replaced an optimal isoleucine in P9, we observed significant ∼7- and 6-fold increases, respectively, in DM-binding affinity (B and C). These substitutions also resulted in increases in kin and kcat, reflecting destabilization of both the resting and catalytic complexes.The impact of interactions at the peptide C terminus on the kinetic parameters of DM activity was also evaluated. The P9 pocket in DR1 is hydrophobic and the second largest pocket, with a preference for moderate sized aliphatic residues , 49. We ver kcat A. A trenaffinity , B and CAFig. S1), resulted in a significant ∼8-fold increase in DM-binding affinity and concomitant increases in kin and kcat (D) as compared with the WT DR1-HA complex. The finding that mK is influenced by interactions between the MHCII and peptide C terminus provides evidence that chemical and structural features distal to the DM-binding site have a long-range effect on DM-binding affinity.The effect of interactions at the C terminus of the bound peptide on DM-binding affinity was further addressed by evaluating a tryptophan to alanine substitution at amino acid position 61 of the β1 domain (W61A) of DR1. This mutation, which eliminates a single hydrogen bond with the main chain of the bound peptide between P8 and P9  between pMHCII stability and susceptibility to DM editing, as gauged by the specificity constant kcat/mK, which quantifies the catalytic efficiency of DM when the pMHCII substrate concentration is much less than mK  and instability of the N-terminal segment of the bound peptide can have a large impact on DM-binding affinity and catalytic turnover . In addition, the P1 anchor pocket is not occluded by the conformational change associated with DM binding . Instead, the dimensional and structural features are largely retained when compared with the P1 pocket of the resting DR1 complex, including the capacity to bind large hydrophobic amino acid residues (C and D). Together, this strongly suggests that the conformational state of the DM-DR1 co-crystal structure at pH 6.5 may better represent the functional catalytic complex. In this model, DM can bind pMHCII with full occupancy of the peptide-binding cleft, and interactions involving the peptide N terminus can influence the stability of the catalytic complex.The data presented herein are consistent with the DM-DR1 structure at pH 6.5 as a depiction of an active catalytic complex . In contFig. 8kcat, but did not generally impact DM-binding affinity  was kindly provided by Dr. Elizabeth Mellins (Stanford University), and generated as previously described (Drosophila S2 cell lines expressing soluble recombinant WT or mutant HLA-DR1 (DRA*0101/DRB1*0101) were generated as previously described  293T cell line expressing soluble recombinant HLA-DR1 (DRA*0101/DRB1*0101) was generated as previously described . BrieflyDrosophila S2 cell lines were purified as previously described (m CuSO4 in BaculoGold Max-XP (BD Biosciences) serum-free cell culture media. After 7 days, supernatant was cleared of cellular debris by centrifugation and 0.45-μm vacuum filtration (Corning). Filtrate was concentrated in a stirred 30-kDa MWCO PES ultrafiltration concentrator (Millipore) under N2 pressure at 4 °C. Concentrate was buffer exchanged on a HiPrep 26/10 desalting column  into PBS prior to protein isolation. Soluble HLA-DR1 produced in HEK 293T cells was purified as previously described (2 prior to −80 °C storage.Soluble HLA-DM and HLA-DR1 proteins produced in escribed . Brieflyescribed . Brieflyescribed . AggregaN-(9-fluorenyl)methoxycarbonyl) chemistry. Briefly, synthesis was initiated on 25 μmol of preloaded Wang resin. Peptide chains were cleaved from resin with 82.5% TFA, 5% thioanisole, 5% H2O, 5% phenol, and 2.5% EDT. Cleaved peptides were precipitated overnight in diethyl ether and air dried prior to purification. Crude peptides were resuspended in DMSO and purified on an Agilent 1200 series by RP-HPLC using a Jupiter 4μ Proteo 90Å C12 (Phenomenex) column in a multistep gradient elution from 10 to 60% solvent B . Peptides synthesized or purchased from Celtek Peptides were verified for >95% purity by RP-HPLC and molecular weight by MALDI-MS. Peptides were quantified by UV 205 nm absorbance using the calculated peptide main chain-specific extinction coefficient. Peptide stocks were lyophilized and stored at −80 °C.N-terminal acetylated peptides were synthesized in-house or commercially acquired from Celtek Peptides (Celtek Bioscience). In-house peptides were synthesized on a PS3 peptide synthesizer (Protein Technologies) using standard solid-phase Fmoc (m triethylamine in DMSO) at a 4:1 molar ratio of peptide to fluorophore. The reaction proceeded on a rotator in the dark at room temperature for 1 h and overnight at 4 °C. Labeled peptides were separated from unlabeled on an Agilent 1200 series by RP-HPLC using a Vydac 218TP52 C18 column (Grace). Fractions containing labeled peptides were identified by retention-time shift and fluorophore UV 494 nm absorbance peak. Fluorophore-labeled peptides were verified for molecular weight by MALDI-MS. Peptides were quantified by averaging the concentration derived from UV absorbance at 205 nm using the calculated peptide main chain-specific extinction coefficient, and fluorophore UV absorbance at 494 nm. Labeled stock peptides were lyophilized and stored at −80 °C.Peptides were labeled on the ϵ-amine of lysine residues with Alexa Fluor 488 5-TFP (Molecular Probes) following a modified manufacturer's protocol, and purified as described . Brieflym citrate-phosphate buffer, pH 5.0, 150 mm NaCl) were incubated on a rotator in the dark at 37 °C. After 24–72 h, endogenous peptide exchange was terminated by pH neutralization  and temperature reduction (4 °C). Labeled pMHCII complexes were separated from aggregates and unlabeled complexes on a Superdex 75 10/300 GL gel filtration column  using a Shimadzu HPLC System equipped with a RF-10AxL florescence detector. Fractions containing labeled peptide-DR1 complexes were identified by retention-time shift and fluorophore UV 535 nm peak emission. Labeled complexes were quantified by averaging the concentration derived from UV absorbance at 280 nm using the calculated protein sequence-specific extinction coefficient, and fluorophore UV absorbance at 494 nm. Protein stocks were concentrated on 30-kDa MWCO spin concentrators (Amicon) and snap frozen in liquid N2 prior to −80 °C storage.Labeled peptide-MHCII complexes were generated as previously described . Brieflym) with DM concentrations up to 90 μm and 400-fold excess unlabeled, high binding-affinity peptide (20 μm) to prevent rebinding of signal (fluorophore-labeled) peptide. Assayed concentrations of HLA-DM were chosen to extend above mK, achieve reaction equilibrium within 14 days, and produce a curvature of observed dissociation rates approaching saturation. A prereaction mix was prepared at room temperature in citrate/phosphate buffer  containing HLA-DM, unlabeled competitor peptide, and 25 μl of mineral oil (surface overlay) to prevent evaporation. Prereactions were incubated at 37 °C for 15 min prior to initiation by addition of 1 μl of labeled peptide-DR1 complexes. Anisotropy of peptide dissociation from HLA-DR1 complexes was recorded over a 1–14 day span at 37 °C and terminated when at least one peptide dissociation curve achieved reaction equilibrium. Anisotropy of OPT1-DR1 complex was recorded for ∼34 days without attaining reaction equilibrium. Background was measured by unbound labeled peptide.Real-time HLA-DM–catalyzed peptide dissociation reactions were measured by fluorescence anisotropy. Anisotropic peptide dissociation curves were generated for fluorophore-labeled peptide-DR1 complexes (50 nA = (G I‖ − I⊥)/(G I‖ + 2 I⊥), where A is the unitless anisotropy, I‖ and I⊥ are the intensity of the polarized emission parallel and perpendicular to the excitation source, respectively, and G is a correction factor for variance in emission detection of perpendicular versus parallel components. Anisotropy measurements were taken every 30 s for the first 1000 measurements and were incremented to 3, 5, 10, 15, and 20 min every 1000 measurement interval.Anisotropy measurements were performed in black polystyrene 384-well NBS microplates (Corning) on a Tecan Infinite F200 plate reader equipped with light polarizing filters at 485 ± 20 nm for absorbance and 535 ± 25 nm for excitation, set for a 20-μs integration of 25 excitation flashes. Tecan i-Control software was used to calculate the fluorescence anisotropy defined by kobs were obtained by fitting dissociation curves to a single-phase exponential decay equation defined by A = A0kte + A∞, where A is unitless anisotropy, A0 is anisotropy of the peptide-MHCII complex at time zero, k is the observed peptide dissociation rate constant kobs, and A∞ is anisotropy of the dissociated peptide at time infinitum (reaction equilibrium). Anisotropy values were background subtracted using the free fluorescence peptide signal prior to rate modeling. Peptide dissociation rates for OPT1-DR1 complex were determined by initial velocity measurement. Rates were calculated using Prism 7.0 (GraphPad Software).Observed dissociation rate constants Fig. S2),kobs are the saturating dissociation rate curves obtained from anisotropy peptide dissociation assay, kcat is the maximal dissociation rate constant  of peptide from the catalytic complex (Michaelis complex), [p*MHC]0 is the initial concentration of the fluorescence peptide-DR1 complex, [DM]0 is the HLA-DM concentration of the corresponding observed peptide dissociation rate constant, mK is the Michaelis constant characterizing the binding affinity of HLA-DM for the pMHCII complex, and kin is the intrinsic peptide dissociation rate constant characterizing the intrinsic stability of the pMHCII complex. Kinetic parameters of OPT1-DR1–labeled complex were determined by Lineweaver-Burk plot analysis.The independent kinetic parameters of DM-catalyzed peptide dissociation reactions were determined by modeling the observed peptide dissociation rates to an experimental specific kinetic model .r method to identify linear correlations and compared with the Spearman rank method to differentiate curvilinear correlations. The null hypothesis was rejected when the p value was <0.05. Correlations were calculated by Prism 7.0 (GraphPad Software). Statistical significance between mean values in the comparative analysis of homologous pMHCII was determined by Student's independent two-sample t test. The null hypothesis was rejected when the p value was <0.05. Student's t test was calculated by Stata/IC 15.0 (StataCorp). The p values of the analyses are as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.Correlation between kinetic parameters were calculated by the Pearson E. R.-V., A. P. B., Z. Z., and P. E. J. conceptualization; E. R.-V., A. P. B., Z. Z., and P. E. J. resources; E. R.-V., A. P. B., and P. E. J. data curation; E. R.-V., A. P. B., and P. E. J. formal analysis; E. R.-V., A. P. B., X. H., and P. E. J. supervision; E. R.-V. and A. P. B. validation; E. R.-V. and A. P. B. investigation; E. R.-V. visualization; E. R.-V., A. P. B., Z. Z., and P. E. J. methodology; E. R.-V. writing-original draft; E. R.-V., Z. Z., X. H., and P. E. J. writing-review and editing; A. P. B. and P. E. J. project administration; P. E. J. funding acquisition.


A deeper understanding of PEDV attenuation will aid in the rational design of a live attenuated vaccine (LAV) using reverse genetics system. Our results showed that replacing the S gene from the highly virulent iPEDVPT-P5 led to complete restoration of virulence of the attenuated iPEDVPT-P96, with nearly identical viral shedding, diarrhea pattern, and mortality rate as the parental iPEDVPT-P5. In contrast, substitution of the S gene with that from the attenuated iPEDVPT-P96 resulted in partial attenuation of iPEDVPT-P5, exhibiting similar viral shedding and diarrhea patterns as the parental iPEDVPT-P96 with slightly severe histological lesions and higher mortality rate. Collectively, our data confirmed that the attenuation of the PEDVPT-P96 virus is primarily attributed to mutations in the S gene. However, mutation in S gene alone could not fully attenuate the virulence of iPEDVPT-P5. Gene (s) other than S gene might also play a role in determining virulence.Porcine epidemic diarrhea virus (PEDV) has continuously caused severe economic losses to the global swine industries; however, no successful vaccine against PEDV has been developed. In this study, we generated four autologous recombinant viruses, including the highly virulent iPEDVPT-P5, attenuated iPEDVPT-P96, and two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) with the reciprocally exchanged  Alphacoronavirus. PEDV is the causative agent of porcine epidemic diarrhea (PED), a historic, highly contagious enteric swine disease characterized by diarrhea, dehydration and poor growth performance in pigs at all ages [Porcine epidemic diarrhea virus (PEDV) is an enveloped, positive-sense and single stranded RNA virus belonging to the genus all ages ,2. PED wall ages . For decall ages ,4. Moreoall ages . Phylogeall ages .The complete genome of PEDV is approximately 28 kilobase pairs and consists of seven open reading frames (ORF). Out of the seven, four ORFs are responsible for encoding structural proteins, namely the spike (S) glycoprotein as well as envelop (E), membrane (M), and nucleocapsid (N) proteins, which are crucial for viral morphogenesis and establishment of infection establishment . Among aS gene [S gene exchange had no influence on the virulence of the highly virulent G2b BJ2011C strain and avirulent G1a CHM2013 strain [LAVs have been shown to be the most effective way to elicit protective immunity against PEDV infection . TherefoS gene . While t3 strain .S gene in the attenuation process of PEDV using the highly virulent and attenuated G2b PEDV Pintung (PEDVPT) 52 strains, the PEDVPT-P5 and PEDVPT-P96 viruses. Nonetheless, our findings were discordant with previous reported data [S gene with the iPEDVPT-P5 virus enabled iPEDVPT-P96 to regain its virulence. A reciprocal approach revealed that iPEDVPT-P5 virus became partly attenuated after the S gene was exchanged to iPEDVPT-P96. Collectively, we concluded that the S gene is of critical importance to the attenuation process of the PEDVPT 52 strain. However, mutation in the S gene alone could not completely attenuate the iPEDVPT-P5 virus. Thus, gene(s) other than S gene could also play a role in determining the virulence.In the present study, we utilized reverse genetics to examine the role of the ted data . We founAll procedures involving animal experiment were reviewed, approved and conducted in strict accordance with the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University  with the approval No.: NTU105EL-00160.Vero C1008 cells (ATCC No. CRL-1586) were maintained in growth medium containing Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine protein (FBS), 250 ng/mL Amphotericin B, 100 U/mL Penicillin and 100 μg/mL Streptomycin. The recombinant viruses (iPEDVPT-P5 and iPEDVPT-P96), and the chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S), were propagated in post-inoculation medium (PI medium) containing DMEM supplemented with 0.3% tryptose phosphate broth (TBP), 0.02% yeast extract, and 10 μg/mL trypsin as described previously .E. coli cells  despite propagation in LB broth supplemented with 10% SOC medium and being incubated at 30 °C. To generate chimeric viruses carrying heterologous spike (S) genes, namely the iPEDVPT-P5-96S and iPEDVPT-P96-5S, cDNA clones of iPEDVPT-P5 and iPEDVPT-P96, respectively, were used as the backbones. The sequences covering the complete S gene of each virus were exchanged without disruption to the remaining genomic structure. Sequence differences in the S genes of iPEDVPT-P5 and iPEDVPT-P96 viruses are summarized in 7 cells/mL Vero cells in RNase-free phosphate buffered saline (PBS) along with 5 μg of PEDV nucleocapsid (N) transcripts. After electroporation, the cells were allowed to recover in growth medium for approximately 16 h and then maintained in PI-medium until cytopathic effects involved over 90% of cell monolayers. The whole flasks were subjected to one freeze-and-thaw cycle and the rescued viruses were passaged once to generate viral stocks (P1). The viral stocks were titrated on Vero cells in 96-well plates to determine the viral titer (see below).The strategy used to recover iPEDVPT-P96 has been described previously . The app4 cells/well) were infected with the designated P1viruses at a multiplicity of infection (MOI) of 0.005. At 18 h post-infection, cells were fixed with 80% ice-cold acetone, air-dried, and then incubated with an in-house anti-PEDV S antibody, P4B [Immunofluorescence assay (IFA) was performed to detect PEDV antigens as previously described with modifications . Brieflyody, P4B , at a diS gene, which contained both naturally occurred and artificially introduced marker mutations  gene, which contained a naturally occurred mutation (G19470T) were used to verify the identities of the four P1 viral stocks and the recombinant viruses shed in feces per group at the time point of peak fecal viral shedding.Sequence analysis was conducted as described previously ,19 and tutations , as well5 cells/well) and infected with each virus at a MOI of 1 and 0.001 at 37 °C for 1 h in triplicates. The cells were then washed twice with Dulbecco’s phosphate-buffered saline (DPBS) and maintained in PI medium. The supernatants at indicated time points were collected and proceeded for viral quantification on Vero cells in 96-well plates using the standard 50% tissue-culture infectious dose (TCID50) assay. In brief, Vero cells in 96-well plates were washed twice with DPBS and then incubated with a 10-fold serially diluted culture supernatant acquired from the aforementioned six-well plate at 37 °C for 1 h. After absorption, the inoculum was removed and replaced with fresh PI-medium following one wash step. The titers were determined at 72 h post-infection using the Reed–Muench method [Confluent monolayers of Vero cells were seeded onto six-well plates  at RT for 1 h and color was developed afterward with 3, 3′-diaminobenzidine (DAB) chromogen . The slides were counterstained with hematoxylin , mounted in Entellan  and cover slipped. Positive signals were visualized under an inverted light microscope .Immunohistochemistry was performed to evaluate the distribution of PEDV antigen. Briefly, formalin-fixed paraffin-embedded tissues were sectioned at 4 μm, deparaffined in xylene, rehydrated in serially diluted ethanol, and proceeded to epitope retrieval with the Trilogy antigen retrieval system . After being washed three times with Tris-buffered saline plus 0.1% Tween 20 (TBST), tissue slides were treated with 3% hydrogen peroxidase  and 10% normal goat serum  to block the endogenous peroxidase activity and non-specific signals, respectively. For antigen detection, an in-house anti-PEDV N antibody, DE-1, at a dilution of 1:1000 in 10% normal goat serum was applied to the slides for 1 h at RT followed by three times wash with TBST. The first antibodies were then captured using the polyclonal anti-rabbit/mouse immunoglobulin, EnVision-DABp < 0.05 was considered statistically significant.All values were expressed as the mean standard ± deviation (SD). Comparison of syncytia size and villous height to crypt depth (VH:CD) ratio were analyzed using statistical software GraphPad Prism 6.0 . Variables were compared using the non-parametrical Kruskal–Wallis test; S gene in the attenuation mechanism of PEDVPT-P96, we further generated the recombinant, highly virulent Taiwan PEDVPT 52 strain, iPEDVPT-P5, and two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) by replacing the complete sequence of the S gene reciprocally , and the presence of PEDV was confirmed by detection of PEDV S protein by immunofluorescence assay A and seqS gene exhibited similar fusogenic ability as suggested by the comparable number of nuclei per syncytium; representative micrographs used to count the number of nuclei in syncytia are shown in S gene derived from iPEDVPT-P96, induced 3.5 to 4 times larger syncytia than the other two viruses containing S gene derived from iPEDVPT-P5  of 1 and 0.001, respectively. Interestingly, although replacement of the ro cells C.S gene between iPEDVPT-P5 and iPEDVPT-P96 affected the fusogenicity and, to a lesser extent, replication kinetics in Vero cells.We observed that plaque morphologies of the four recombinant viruses correlated to the results of syncytia size. At 72 h post-infection, both iPEDVPT-P5 and iPEDVPT-P96-5S induced barely visible viral plaques in Vero cells macroscopically, whereas iPEDVPT-P96 and iPEDVPT-5-96S generated distinct and comparable viral plaques with the plaque size of iPEDVPT-P5-96S being slightly smaller than that of the iPEDVPT-P96 D. These S gene replacement in PEDVPT 52 strain, we orally inoculated 2 mL of 5 × 102 TCID50/mL of iPEDVPT-P5, iPEDVPT-P5-96S, iPEDVPT-P96, iPEDVPT-P96-5S viruses and with PI medium in seven-day-old crossbred piglets assigned in the corresponding groups. Parameters used to assess the pathogenicity of different recombinant viruses were summarized in S gene derived from the highly virulent iPEDVPT-P5, namely the iPEDVPT-P5 itself and iPEDVPT-P96-5S, had an early onset of clinical symptoms including diarrhea, anorexia and decreased activity, and peak viral shedding at 1 d post-inoculation (DPI). At 3 DPI, piglets inoculated with iPEDVPT-P5 (n = 3) and iPEDVPT-P96-5S (n = 3) both exhibited extensive PEDV-induced villous blunting and atrophy in the jejunum and, to a lesser extent, in the ileum; histological changes in the duodenum were not obvious  at 3 DPI revealed a much milder degree of villous atrophy in the jejunum with conspicuous villous hyperplasia compared to the other two groups. Notably, iPEDVPT-P5-96S-treated piglets exhibited statistically significant severer villous atrophy in jejunum than iPEDVPT-P96-treated piglets  with exchanged S gene. We found that the iPEDVPT-P96 virus fully regained the virulence after the exchange of S gene derived from the highly virulent iPEDVPT-P5 virus, showing comparable patterns of viral shedding, diarrhea and histopathological changes. Our data confirmed that the S gene is the primary attenuating determinant of the iPEDVPT-P96 virus and the genetic backbone other than the S gene involving the attenuation of the iPEDVPT-P96 might be of less importance. However, the iPEDVPT-P5-96S still exhibited partial virulence resulting in severer villous attenuation and higher mortality rate compared to those caused by the iPEDVPT-P96 virus. This result supports that the virulence of PEDV might be a multigenic event. Porcine epidemic diarrhea (PED) to date remains a colossal burden to the global swine industries owing to the lack of successful vaccine in the field ,13. MoreS gene was reciprocally replaced. However, this finding is in disagreement with previously published results by Wang et al. [S gene substitution between the highly virulent G2b strain, BJ2011C, and the avirulent G1a strain, CHM2013. There could be a few reasons for the discrepancy between our observations that of previous studies. First, the autonomy of the viruses is different. Viral attenuation in serial cell-culture or animal passage is a progressive process that involves a series of gene mutations and the subsequent alternated cooperative interplay between gene products and mechanisms influencing cell-virus interaction. Although the patterns of attenuating mutation are similar among G2b PEDV strains, the asynchronous mutations between different PEDV strains could lead to loss of the cooperative or complementary function(s) for other gene products. In the previous study, the used viral strains belonged to different genogroups [S gene exchange failed to reverse the virulence of the avirulent G1a CHM2013 strain might be due to, at least in part, the suboptimal cooperation between structural proteins in terms of PEDV morphogenesis. Second, the degree of attenuation differed between the CHM2013 virus and PEDVPT-P96 virus. Comparing to the avirulent CHM2013 virus that induced no detectable viral shedding in two-day-old piglets, iPEDVPT-P96 virus retained some levels of virulence as it caused viral shedding, observable clinical symptoms, and even mortality in the seven-day-old conventional piglets. Besides the S gene [S gene they carry. Therefore, variations of NSPs in the CHM2013 virus might have a critical effect on its virulence, and the effect might be stronger than that contributed by the S gene derived from the highly virulent BJ2011C virus. Similarly, we also speculated that the IFN-suppressive function provided by NSP(s) might also contribute to virulent differences between the iPEDVPT-P5-96S and iPEDVPT-P96 since nine mutations were identified in NSPs in the iPEDVPT-P96 as compared with that of PEDVPT-P5 [In the present study, the first pair of cDNA clones of a virulent G2b PEDV and its derived attenuated strain were generated. Since these two viruses were closely related to each other, this established platform would allow for easier manipulate in studying the effect of single nucleotide polymorphism for identifying potential virulent/attenuating determinate(s). In the present study, we demonstrated that reversion of virulence or attenuation occurred after the g et al. , indicatnogroups ,28. Effie S gene ,28, the e S gene ,27. ThatEDVPT-P5 . RegardlS gene  and S1B (T554C) domains compared to the iPEDVPT-P5 virus. For PEDV, S10 and S1B domains are known to contain sialic acid-binding and receptor-binding domains, respectively; thus, are crucial for viral entry [0 domain of PEDV resulted in attenuation in piglets, a more specific epitope has yet to be identified. Therefore, we are curious about whether only these two mutations in iPEDVPT-P96 can attenuate the highly virulent iPEDVPT-P5 virus or if the S2 domain must be primarily accounted for the attenuation. Future studies will be conducted to elucidate these questions.Sequence comparison between the autologous highly virulent PEDVPT-P5 and attenuated PEDVPT-P96 revealed several amino acid substitutions , especiagene see . Consistg et al. , Suzuki g et al.  reportedal entry . Althougal entry  previousS gene in PEDV pathogenesis. We confirmed that the S gene is a crucial virulent/attenuating determinant for the iPEDVPT-P96, but its importance varies among different PEDV strains. Thus, other studies’ results alongside our results will provide valuable information for the future generation of novel chimeric or multivalent vaccines.In this study, the first pair of cDNA clones for a virulent G2b PEDV, and its derived attenuated strain were generated, allowing us to use both gain-of-function and lose-of-function approaches to studying the role of the