Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC , implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine.To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine  modified phage genomes:  To gainphage 9g , α-putrephage 9g , N6-meth(MTases) , and 5-hand SPO1 . Non-canand SPO1 . Phage 9equences . In a liequences . The addequences . Phage Sequences . DNA dupequences .NedU) and 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) were recently discovered in the genomes of Pseudomonas phage M6 and Salmonella phage ViI (Vi1) deoxyuridine (5-iI (Vi1) . Hypermol groups . It has l groups .in vitro. We also analyzed the resistant sites for any conserved sequence motifs to shed light on possible modification site specificity. Furthermore, we introduced additional base modifications in their DNA by treatment with cytosine or adenine MTases to generate two types of base modifications . We also examined the ligation efficiency of phage DNA restriction fragments and tested two exonuclease activity on the modified DNA. This work provides basic information on restriction of T-modified DNA and further our understanding of the co-evolution relationship of host and hypermodified phage genomes. Study of highly modified phage genomes may have impact in phage therapy.The goal of this work is to examine Type II restrictions of modified DNA in phage M6, ViI, and phi W-14 genomes. We performed restriction digestions of these three gDNAs to verify their resistant level 3 extraction, and ethanol precipitation . Phage particles were purified by CsCl gradient method and phage DNA purified by phenol-CHClpitation . Due to pitation  was usedPhage DNA was methylated by treatment with excess DNA MTase and methyl-donor SAM in the recommended buffer for 2 h. Following Proteinase K treatment and spin column purification, the methylated DNA was digested by cognate or non-cognate REases to evaluate the degree of resistance to restriction.+ 228.1 and [M-H]- 226.2; dG [M + H]+ 268.1 and [M-H]- 266.1; dT [M + H]+ 243.1 and [M-H]- 241.1; dA [M + H]+ 252.1 and [M-H]- 250.1; 5mdC [M + H]+ 242.1 and [M-H]- 240.2; 6mdA [M + H]+ 266.1 and [M-H]- 264.1; 5hmdC [M-H]- 257.0; 5-NeOmdU [M + H]+ 302.1 and [M-H]- 300.1; α-putT [M + H]+ 329.2 and [M-H]- 327.2; putT-dC [M + H]+ 618.3 and [M-H]- 616.2; and putT-dG [M + H]+ 658.2 and [M-H]- 656.1. The relative abundance of each nucleoside was determined by dividing the UV absorbance by the corresponding extinction coefficient at 260 nm.Phage DNA was methylated by the frequent MTases M.EcoGII (adenine methyltransferase), M.SssI (CpG methyltransferase), M.CviPI (GpC methyltransferase) for 2–4 h with methyl donor SAM. After Proteinase K treatment, the DNA was precipitated in ethanol, dried and resuspended in a buffer for nuclease degradation. DNA samples (5 μg) were digested to nucleosides by treatment with the Nucleoside Digestion Mix  overnight at 37°C. Nucleoside analysis was performed on an Agilent LC/MS System 1200 Series instrument equipped with a G1315D diode array detector and a 6120 Single Quadrupole Mass Detector operating in positive (+ESI) and negative (-ESI) electrospray ionization modes. LC was carried out on a Waters Atlantis T3 column  with a gradient mobile phase consisting of 10 mM aqueous ammonium acetate (pH 4.5) and methanol. MS data acquisition was recorded in total ion chromatogram (TIC) mode. Each nucleoside was identified as follows: dC [M + H]+ modification) resistance to nearly 71% REases tested. The individual restriction results for three genomic DNAs are shown in FspI (TGCGCA) and SacI (GAGCTC) restriction, most likely due the modified T in TG and TC dinucleotide in both strands . Phage ViI DNA is resistant to restriction by BspHI (TCATGA), ClaI (ATCGAT), and NdeI (CATATG). Phi W-14 DNA is resistant to restriction by Hpy188III (TCNNGA) and HpyCH4V (TGCA) probably due to the modified bases in TG or TC dinucleotides in both strands. In some cases, phage DNA is also partially or completely resistant to REases that cleave target sites with 4–6 AT bp  protein to prevent self-restriction  and PspOMI (G/GGCCC) sites is only 0.25. Similarly, the probability of TG dinucleotide 5′ to the NarI (GG/CGCC) and PluTI (GGCGC/C) is 0.25. NarI and PluTI partially digested a single site plasmid pTYB2 as two sites are probably required for efficient digestion. This group of enzymes include Type IIE and IIF that requires a secondary site (effector site) and extensive looping and enzyme complex interaction (enzyme dimers or tetramers bound to two sites separated by a certain distance) , NgoMIV (G/CCGGC), NotI (GC/GGCCGC), or PspOMI digestion  DNA; but are unable to cleave M6 and phi W-14 DNA . We specistance) . Phi W-1igestion . The preIt has been proposed that M6, ViI, and phi W-14 phages utilize phage-encoded DNA polymerases and a 5hmdUTP, dATP, dCTP, and dGTP deoxynucleotide pool for DNA replication, thus replacing all T with 5hmdU . FurtherPseudomonas phage M6 DNA can be further modified by C5-cytosine and adenine MTases. This experiment has two possible outcomes: (1) if M6 DNA can be further modified by these MTases, the double-modified DNA may be resistant to more Type II restrictions; (2) 5mC- and N6mA-modified DNA may be subjected to 5mC-dependent restriction systems such as EcoK McrBC  strain  restriction. M.HpaII and M.MspI can also fully modify M6 DNA and render the DNA resistant to HpaII (CCGG) and MspI (CCGG) restriction, respectively. M.SssI and M.AluI can partially modify the M6 DNA and provide partial resistance to HpaII or AluI (ACGT) restrictions. For the adenine MTases, Dam methyltransferase, M.EcoGII, and M.TaqI can partially modify the M6 DNA and provide partial resistance to MboI (GATC) and TaqI (TCGA) restriction. The methylation and restriction results are summarized in Pseudomonas lytic phages to combat multi-drug resistant Pseudomonas infection.Next, we examined whether oK McrBC  and McrABC (McrA  or N6mA-EcoK Mrr  and Pgl ectively . Host-acectively . Over-ex) strain . In thisNeOmdU, 5-hmdU, and T in phage ViI genomic DNA were estimated at 43, 7, and 50%, respectively. In a control experiment, M.EcoGII-mediated A to 6mA conversion and M.CviPI-mediated GpC to Gp5mC conversion in phage λ DNA reached ∼93 and ∼30%, respectively (data not shown).To estimate the level of methylation in phage ViI and phi W-14 DNAs, we performed LC-MS analysis of the corresponding MTase-treated DNAs. EcoGII-treated phi W-14 genomic DNA indicated that 56% of adenosines were converted to 6mA  and FatI- (/CATG) partially digested, or RsaI (GT/AC) completely digested ViI restriction fragments were ligated at 16°C overnight. The sticky ends of NlaIII and FatI fragments were efficiently ligated, whereas the blunt-ended RsaI fragments were ligated at a lower efficiency  of phi W-14 restriction fragments were ligated efficiently indicated by the appearance of large concatenated DNA after ligation. Lower ligation efficiency was observed for blunt-ended RsaI fragments . The mechanism of phi W-14 DNA partial resistance to exonuclease digestion is still unknown. It was reported previously that the rate of DNA hydrolysis by non-specific endonuclease of modified phage PBS1 (dT substituted by dU) was decreased by 14.3-fold, and hypermodified phage T4 DNA also shows slow-down in nuclease degradation  and TDG to create AP sites (apurinic/apyrimidinic site), which can be further cleaved by AP endonucleases (5hmdU can be excised by DNA repair enzymes AlkA and Mug from ucleases . Since V+ found in phage 9g genome (N6-(1-acetamido)-adenine in phage Mu genome . We hypothesize that phages M6, ViI, and phi W-14 may be resistant or partially resistant to Type I restriction as long as the restriction sites of these enzymes contain one or more TS dinucleotide sequence. 5mC-dependent REases are not tested on the three phage DNA substrates.In bacterial host and phage coevolution, phage use extensive base modifications (nt substitutions) to protect its genome against host restrictions. The results presented here demonstrate that hypermodified T derived from 5hmdU can also efficiently protect phage genomes against Type II restrictions, in analogous manner to modified Gs, such as dGg genome  and 2′-dg genome , to modiu genome , to modiE. coli GmrSD endonuclease attacks 5hmC and 5gmC modified DNA  are recognized and methylated by the phage-encoded Dam MTase triggering cleavage of pac sites and phage packaging initiation  to specifically attack modified DNA . For exafied DNA . We havefied DNA . Anotherl capsid . In addil capsid . This haitiation . The effBacillus phage SPO1 genomic DNA wherein >98% of Ts are replaced by 5hmdU is resistant or partially resistant to over 50% of Type II REases with 0–4 Ts in the recognition sequences (Bacillus phage or prophage genomes encode frequent multi-specificity cytosine MTases (dam+) that methylates GATC sites to provide more resistance against REases with overlapping GATC sequence. T even phages provide examples of two types of base modifications (6mA+5hmC or 6mA+5gmC) in their genome  that remain to be characterized.The Pseudomonas lytic phages has been successfully used to treat P. aeruginosa infections in animal models  . For complete restriction digestion of hypermodified T phage DNA and cloning of certain genes (restriction fragments), Analysis of the resistant sites in the phage genomes revealed a conserved motif TG, TC, or TS, suggesting the modified Ts possess certain sequence specificity, which may have been conferred by phage DNA 5hmdU kinase that phosphorylates the base for further chemical modification. Understanding the enzymes involved in thymidine hypermodification in phage genomes is an active research topic in our lab  . In suppBoth ViI and phi W-14 encode a three-gene cluster with predicted function in restriction (phage against phage superinfection). ORFs Vi01_137, 138, and 139 encode putative RNA-DNA and DNA-DNA helicase/ATPase, CRISPR-Cas associated protein Cas4 nuclease , and ssDNA binding protein in the ViI genome. Similarly, a three-gene cluster in phi W-14 genome contains gp030, gp032, and gp031. But the exact function of the three proteins involved in DNA metabolism (restriction) is still unknown. Incidentally, phage ViI also encodes a superinfection exclusion protein (Vi01_111c) that may play a role in attenuation of other phage infections. ViI genome encodes one GIY-YIG superfamily endonuclease (Vi01_159c); and phi W-14 genome encodes two HNH endonucleases (gp143 and gp219). These endonucleases are probably homing endonucleases involved in insertion of intron into intronless target known as intron “homing” since there are no cognate MTase genes associated with the predicted endonucleases  in human pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania species, consisted of about 1% of total T in the genomes. The modified base J is an important regulatory epigenetic mark in trypanosomatids to influence gene expression. The JBP1/2 enzymes catalyze hydroxylation of thymine (E. coli, and hSMUG1 and TDG (Base J ( thymine , forming and TDG . When phThe datasets generated for this study are available on request to the corresponding author.KF, RT, MX, IC, AC, and S-YX performed experimental work. M-QX, Y-JL, IC, PW, and S-YX contributed with ideas. S-YX wrote the manuscript with input from all the authors.Y-JL, M-QX, IC, PW, and S-YX are employees of New England Biolabs, Inc. New England Biolabs commercializes reagents for molecular biological applications. 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.


Among these, activation of phosphoinositide 3-kinase (PI3K) has a critical effect on T cell proliferation, survival, migration, and effector/memory subset formation. Class I PI3Ks are composed of one of three isoforms of the p110 catalytic subunit , that constitutively associate with a p85 regulatory subunit. Class I PI3Ks catalyze the phosphorylation of phosphatidylinositol 4,5-bisphosphate, generating phosphatidylinositol -trisphosphate, which recruits proteins containing pleckstrin homology (PH) domains to the plasma membrane. PH domain-containing targets, including AKT, initiate signaling and activate downstream effectors of cellular differentiation and metabolism [During CD8tabolism . The costabolism .in vivo longevity. Long et. al. demonstrated that the presence of the CD28 costimulatory domain increased CAR-T cell exhaustion induced by persistent CAR self-signaling; the 4-1BB costimulatory domain had a lesser effect [While loss of PI3K activity is detrimental to immune function, constitutive activation of PI3K also impairs immunity because it preferentially promotes formation of short-lived terminally differentiated effector T cells at the expense of long lived memory T cells. Control of T cell activation and differentiation by PI3K is particularly relevant to Chimeric Antigen Receptor (CAR) T cell immunotherapy. CARs retarget genetically modified T lymphocytes through hybrid receptors that incorporate a tumor antigen-specific scFv, one or more costimulatory domains (most commonly 41BB or CD28), and the CD3-zeta domain. CAR-T cells have experienced a surge in interest due to the now proven effectiveness of CD19-specific CAR-T cells in the treatment of precursor B cell malignancies. CAR-modified T cells are not merely retargeted conventional T lymphocytes. The presence of a CAR on a T cell's surface alters its activation and differentiation, even in the absence of a complementary ligand. Constitutive self-signaling through CAR, related to both the scFv framework and the signaling domains, can lead to aberrant T cell behavior, including altered differentiation and decreased survival. This is significant as the effectiveness of CAR-T cells in patients is directly associated with their r effect .ex vivo expansion abrogated preferential effector T cell development and restored the CAR-T effector/memory ratio to that observed in empty vector transduced T cells. This improved in vivo T cell persistence and therapeutic activity in an AML model. Inhibition of p110δ PI3K has also been found to enhance efficacy and memory in tumor-specific therapeutic CD8 T cells, while inhibition of p110α PI3K increased cytokine production and antitumor response [Manipulation of PI3K signaling can be used to prevent altered CAR-T cell differentiation due to constitutive CAR self-signaling and foster long-lived memory T cell development. We demonstrated that pharmacologic blockade of PI3K during CAR-T manufacture and response , 7.+ T cell fate. Sustained AKT activation leads to T cell terminal differentiation. Its inhibition in CAR modified T cells results in an early memory phenotype and improved antitumor efficacy [ex vivo expansion of CAR-T cells promoted memory over effector cell formation. However, inhibition of these pathways also reduced CAR-T cell proliferative capacity, limiting therapeutic cell expansion [+CCR7+ T cells with TN and TCM phenotypes, and adoptive transfer of inhibitor-treated CAR T cells extended survival in an ALL model [et. al. found that the B cell adaptor for PI3-kinase (BCAP) is an important regulator of CD8+ effector and memory T cell differentiation, highlighting yet another potential target in the PI3K pathway to balance effector and long-lived memory T cell generation [Downstream targets of PI3K include AKT, mTOR, and FOXO1, and are important in determining CD8xpansion . These tLL model . We haveneration . It rema


Clock as a DENR target. Using Clock 5′ UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5′ UTR features predictive of DENR dependence. Among core clock genes, we identified  Initiation is considered the rate-limiting step for eukaryotic translation  and can Drosophila, DENR-MCTS1 was identified as a reinitiation factor required for efficient CDS translation on transcripts containing short and strong uORFs  and Malignant T-cell amplified sequence 1 (MCTS1) form a heterodimeric non-canonical initiation factor that has been described to bind to the small ribosomal subunit and to promote the eIF2-independent recruitment of the initiator tRNA to viral mRNAs, as well as to participate in the recycling of post-termination 40S subunits . In Drosng uORFs . Furtherng uORFs . HoweveruORF usage is pervasive in the mammalian transcriptome, with up to 40% of transcripts predicted to contain at least one uORF . It geneHere, we have used ribosome profiling in mouse NIH3T3 fibroblasts to identify and quantify DENR-mediated translational regulation transcriptome-wide, and in particular within core clock genes. We have used comprehensive regression analysis to characterize 5′ UTR features associated with DENR-dependent translational regulation and validate targets identified from our translatome-wide approaches. Finally, we show that DENR regulates efficient CLOCK biosynthesis involving a strong uORF and a so-far unannotated CDS initiation codon.2).NIH3T3 and HEK293FT cells were cultured under standard conditions  shRNAs served as controls.For the generation of lentiviral shRNA expression vectors, two different sequences targeting o. 10878 ): shRNA1Etaa1, forward (for): aaaggatccGACTTGCAAAGATGGCGCTGCAC, reverse (rev): tttggatccactagtGTCCTTCAGCTGCATTCACATTT; Map2k5, for: aaaggatccGGTTCCGGAGTAACAGCGGTCTAAC, rev: tttggatccactagtGGCCAGCCACAGCATTACAGGTTAA; Lrrc28, for: aaaggatccGCCGCTGAGTGCCGGTCAGCGGGC, rev: tttggatccactagtGATTTCGGATGCCATGACTGAACA; Klhdc8a, for: aaaggatccGTCTCCGACCCTGTAGACACTGCAG, rev: tttggatccactagtATTGGGCACTTCCATGGCAGCCCGG; Clock, for: aaaggatccGGGGAGGAGCGCGGCGGTAGCGGTG, rev: tttggatccactagtAACAATTGAGCTCATTTTACTACAGCTTACGG; Fth1, for: aaaaggatcCAGACGTTCTCGCCCAGAGTCGC, rev: tttggatcccatGGTGGCGGCGGGGCGAGGCGC; Rpl30, for: aaggatcCAGCGGCGGGTGGACAGAGATGCC, rev: tttggatccacgCGTCTTCTTTGCGGCCACCATCTTC; Deaf1, for: aaggatccGGGCGTCAGACTGCCTCTTCGGTC, rev: tttggatccacgcgtGGCAGCGGCCGCCACAGCTGCCGC; Cry1, for: aaaaagatctactagtCACCCGGGCAGCCTCGGGACCG, rev: tttagatctTCGGAACCAGTGCACGGCGTTCAC.For the generation of dual luciferase (Firefly/Renilla) reporter plasmids, fragments containing the 5′ UTR and first 5-14 codons of the selected DENR targets/controls were amplified by polymerase chain reaction (PCR) from genomic DNA or cDNA, cloned into the BamHI site of the prLV1 dual luciferase reporter plasmid , and valKlhdc8a-Mut, forward (for): GGCAGGGAGCCCGTGCAGCGCGgtaccGAGGCTGAGAGAGGGGACGCGCC, reverse (rev): GGCGCGTCCCCTCTCTCAGCCTCggtacCGCGCTGCACGGGCTCCCTGCC; Map2k5-Mut, for: CTCTTACTCACAAGGACTACAGgctagcTTGTGGGTTCTGTTTTGTCCAC, rev: GTGGACAAAACAGAACCCACAAgctagcCTGTAGTCCTTGTGAGTAAGAG; Etaa1-Mut, for: cgaaggatcCGACTTGCAAAGgtaccGCTGCACGGTGGCGCGCGGGCTC, rev: GAGCCCGCGCGCCACCGTGCAGCggtacCTTTGCAAGTCGgatccttcg; Clock-uORF-Stop-Mut, for: GTTATGGTGTTTACCGTcAGCTGTAGTAAAATGAG, rev: CTCATTTTACTACAGCTgACGGTAAACACCATAAC; Klhdc8a-uORF-Stop-Mut, for: GCCGACCGGCGCCGTGcacCCCGCGGGCCCCGGGGCT, rev: AGCCCCGGGGCCCGCGGGgtgCACGGCGCCGGTCGGC; Etaa1-uORF-Stop-Mut , for:TGCTCCGCTGTAGCAgcAAATGTGAATGCAGC, rev: GCTGCATTCACATTTgcTGCTACAGCGGAGCA; Clock-uORF1-Mut, for: CCCAAAATCACCAGCAAGAGTTgctagcGTCAGTCACACAGAAGACGGCC, rev: GGCCGTCTTCTGTGTGACTGACgctagcAACTCTTGCTGGTGATTTTGGG; Clock-uORF2-Mut, for: AAAGTGAAAGAGGAGAAGTACAggtacCTACCACAAGACGAAAACATAAT, rev: ATTATGTTTTCGTCTTGTGGTAGgtaccTGTACTTCTCCTCTTTCACTTT; Clock-AnnotCDS-Mut, for: CAAGACGAAAACATAATGTGTTtcGGTGTTTACCGTAAGCTGTAG, rev: CTACAGCTTACGGTAAACACCgaAACACATTATGTTTTCGTCTTG; Clock-AlterCDS-Mut, for: GTTTACCGTAAGCTGTAGTAAAcaGAGCTCAATTGTTactagtg, rev: cactagtAACAATTGAGCTCtgTTTACTACAGCTTACGGTAAAC.5′ UTR mutants were generated by site-directed mutagenesis of the corresponding plasmids, using the following primers : Denr cloned into a FLAG-Venus-pLenti6 vector (Addgene 36948). Cells were grown to confluency in a 15-cm dish. One dish was used for overexpression, and six for non-transfected experiments.For iCLIP experiments, we used either NIH3T3 cells without overexpression to immunoprecipitate the endogenous DENR or NIH3T3 cells transiently transfected with 2 in a Stratalinker 2400. After cell lysis , 0.1% sodium dodecyl sulphate (SDS), 0.5% Na deoxycholate, ethylenediaminetetra acetic acid (EDTA)-free protease inhibitors), RNA was partially digested with 0.2 U RNase I (Thermo Fisher). DENR–RNA complexes were pulled down with anti-DENR antibody (Abcam 108221), pre-immobilized on protein G-coated magnetic beads . An IgG immunoprecipitation was used as control. Immuno-complexes were washed twice with High Salt Buffer  and twice with PNK Wash Buffer . An infrared adaptor was ligated to RNA 3′ ends, and protein–RNA complexes were visualized in a LiCor CLx Imager (Odyssey), after denaturing PAGE and nitrocellulose membrane transfer. About 60–130 kDa DENR–RNA complexes were excised from the membrane and liberated by proteinase K (Roche) digestion. This size range likely reflects DENR–MCTS1 complexes  bound to RNA, rather than DENR–RNA alone, indicating that the DENR–MCTS1 interaction is strong. During library preparation, cDNAs were ligated on their 5′ end to a 5-nt sample barcode to allow demultiplexing. The barcode is preceded by a 4-nt and followed by a 3-nt random sequence that serves as unique molecular identifier (UMI) to mark individual cDNA molecules. cDNA libraries were then PCR amplified (24 cycles) and subjected to high-throughput sequencing (Illumina HiSeq2500 platform). Experiments were done in triplicate for DENR-overexpression, and in duplicate for non-transfected and IgG IP conditions.iCLIP was performed as described ,14. RNA–https://github.com/tomazc/iCount), as described  with minor modifications as described below. Cells were treated with cycloheximide (100 μg/ml) for 2 min at 37°C to arrest translation elongation, and immediately harvested, followed by lysate preparation. For the generation of ribosome-protected fragments, cell lysates were treated with 5 units RNase I (ART-Seq) per OD260, and monosomes were purified in MicroSpin S-400 columns . For both RPF- and RNA-seq, 5 μg of RNA was used for rRNA depletion following the Ribo-Zero Magnetic Kit instructions from Epicentre-Illumina (Cat.no. MRZH11124). Finally, libraries were amplified using 12 PCR cycles, and sequenced (HiSeq2500 platform).One 15-cm dish of confluent NIH3T3 cells was used per replicate. Ribosome profiling and parallel RNA-seq were performed in triplicate for Initial processing, mapping and quantification of mRNA and footprint abundance were performed as previously . BrieflyRPF- and RNA-seq read counts were normalized separately with the upper quartile normalization from edgeR  and thenTranslation efficiencies were then calculated per sample as the ratio of RPF RPKM to RNA RPKM of the region of interest , log2 transformed and averaged over all replicates.bins=10000, RiboDiff  pipelines. Babel relies on edgeR for parameter estimation and uses an errors-in-variables regression to test for significant changes in translation within and between conditions.Differential TE was set at FDR < 0.1 for all four methods and performance for false positive discovery rate was further assessed by randomly label-shuffling knockdown and control datasets, which should not yield differential TE genes.N = 4637). In order to include as many transcripts as possible for the annotation, genes with multiple protein-coding isoforms were also considered if (i) one isoform represented >65% of all expressed  or (ii) all expressed isoforms had the same CDS start, in which case the transcript with the highest expression was selected (N = 1826). For the selected transcripts, uORFs were annotated and considered as translated with the following criteria: (i) started with AUG, CUG, GUG or UUG, (ii) had an in-frame stop codon within the 5′ UTR or overlapping/within the CDS, (iii) were at least 9 nt long (including stop codon), (iv) had a coverage >25%, (v) showed a frame preference and (vi) the preferred frame was the one corresponding to the uORF 5′ end/start codon. Importantly, we considered only uORFs coding for at least two amino acids , as for shorter uORFs frame bias assessment would be highly prone to false positives, and Kozak context scoring would be compromised .For uORF detection, a set of transcripts was used for which reads can be unambiguously assigned to the 5′ UTR, i.e. transcripts that are the only protein-coding isoform expressed (Denr knockdown or control cells were included (N = 4755) and the following 5′ UTR features were used as predictors of the fold change in CDS TE across conditions: (i) the number of uORFs, (ii) the uORFs length, using the median length in transcripts with several uORFs, (iii) the proportion of AUG uORFs, (iv) the uORF Kozak context, scored relative to the consensus GccA/Gcc###G, where upper-case letters denote highly conserved bases (scored +3), lower-case letters indicate the most common nucleotides (scored +1) and ### represents the start codon (not scored). The median Kozak score was used when several uORFs were present on a transcript. (v) The CDS Kozak context, scored in the same manner, (vi) the distance of the first uORF start codon from the 5′ cap, (vii) the distance of the last uORF stop codon to the CDS start, (viii) the uORF GC content, using the median for transcripts with several uORFs, (ix) the presence of overlapping uORFs in the transcript (binary predictor). Predictors with skewed distributions  were log2 transformed. Partial regression plots were generated with the R package ‘visreg’. Absence of multicollinearity between predictors was assessed with the variance inflation factor using R function vif (car package).For the modelling, all transcripts containing translated uORFs in P-values were corrected by Benjamini–Hochberg FDR. Categories with FDR <0.05 were considered as significant and were used for visualization with Cytoscape v.3.5.1 EnrichmentMap tool . Firefly/Renilla luciferase (FL/RL) activities were first normalized to that of the empty vector in scramble shRNA-transduced cells. For DENR target experiments, FL/RL signals were also normalized to the scramble shRNA-transduced, non-mutated construct. For Clock mutational experiments, FL/RL signals were secondly normalized to the scramble shRNA-transduced Clock-WT construct.NIH3T3 cells were first lentivirally transduced with the dual luciferase reporters containing the 5′ UTR construct of interest and then transduced with in vitro transcription at the ‘ivt mRNA production and formulation platform’ in Zurich (http://www.cancer.uzh.ch/en/Research/mRNA-Platform.html). The 5′ end consisted of a CleanCapTM (Trilink) followed by the WT or mutated Klhdc8a 5′ UTR and by a codon optimized firefly luciferase open reading frame. The 3′ end consisted of a tandem repeat of the mouse β-globin 3′ UTR and a poly-A tail (in vivo (mRNAs were produced using y-A tail . Uridine(in vivo ).in vivo bioluminescence imaging was performed on an IVIS Lumina instrument (PerkinElmer). Immediately before each measurement, D-luciferin (Synchem) dissolved in phosphate-buffered saline (15 mg/ml stock) and sterile filtered was injected . Emitted photons from live animals were quantified 10-20 min post luciferin injections, with an exposure time of 3 min. Regions of interest (ROI) were quantified for average radiance (photons s-1 cm-2 sr-1) (IVIS Living Image 3.2).Animal procedures were approved by the Veterinary Office and its research ethics review committee of the University of Zurich . Animals were purchased from Envigo (Netherlands). Four- to eight-week-old mice were injected intravenously with 1 μg of mRNA formulated with TransIT transfection reagent following the manufacturer’s suggestions (Mirus). The procedure for one mouse was as follows: the nucleic acid was diluted in 38 μl of Opti-MEM (Gibco) before the addition of 0.72 μl of the ‘mRNA Boost Reagent’. After mixing, 1.12 μl of the TransIT reagent was added. The solution was thoroughly homogenated and injected immediately: 40 μl per mouse. At different time points after mRNA injection, Total protein extracts from NIH3T3 cells were prepared according to the NUN procedure . AntibodDenr in mouse NIH3T3 fibroblasts, a cell line in which we had previously observed that Denr depletion led to a shortening of the period of circadian oscillations  or control RNAs , grown to confluency and harvested in the presence of cycloheximide (CHX) to stabilize elongating ribosomes. After preparation and sequencing of ribosome-protected mRNA fragment (RPF-seq) and total RNA libraries (RNA-seq) as previously described , whereas the abundance of Mcts1 mRNA, encoding the DENR heterodimerization partner, or of Eif2d, encoding a structurally and functionally related homologue of the DENR-MCTS1 dimer  on their CDS when DENR is absent Figure . We therllations . Cells wescribed , we obtaescribed , which pescribed  and thatescribed . Across S1 dimer , remaineS1 dimer .Denr levels that alters reinitiation efficiencies should engender differences in ribosome occupancies on the CDS and, possibly, on uORFs. First, we compared CDS footprint levels for all expressed genes    of DENR  (iCLIP) . Over 85 (iCLIP) , with th (iCLIP) . This ou (iCLIP) ,33. More (iCLIP) . In contDenr-deficient cells . Importantly, differential TE genes detected by Riborex, RiboDiff and Xtail completely overlapped, while only about half of the genes detected by Babel were also found by any of the other methods , but genes with uORFs overlapping the CDS , or that had both overlapping and non-overlapping uORFs  were frequent, too  is known to respond to cellular stress signals via two uORFs, the first of which is reinitiation-permissive whereas the second overlaps the main CDS  remained unchanged compared to control cells, while translation of the second uORF and the CDS were decreased  from the same bidirectional promoter  , indicating that DENR was indeed involved in their regulation. Mutating uORF start codons increased reporter signal and abolished most DENR-dependence  fused to Firefly luciferase CDS and carrying a synthetic polyA tail. We then monitored expression by bioluminescence live imaging in mice. Bioluminescence signal was primarily detected in the livers of the animals, and was 2–4.3 times higher in the uORF-less compared to the WT constructs  in a lentiviral vector that also expresses e Figure , bottom,e Figure . uORF-det Figure , bottom.nstructs , demonstTaken together, we concluded that DENR was involved in the regulation of protein biosynthetic output, and that a set of >200 transcripts containing uORFs was particularly sensitive to loss of this reinitiation factor.Drosophila S2 (uORFs can operate through a bewildering diversity of mechanisms that act on CDS translation (by affecting start site selection/leaky scanning/re-initiation/ribosome shunting), transcript abundance (by eliciting nonsense-mediated mRNA decay) or in some cases through uORF-encoded bioactive peptides (reviewed in ). Howevephila S2  and humaphila S2  cells, vDenr knockdown using nine relevant transcript features, namely the number of uORFs, uORF start codon identity (AUG versus near-cognate) and context, the length, GC content and location of the uORF within the 5′ UTR, the presence of overlapping uORFs and CDS start codon context  and yielded several features with significant predictive value for DENR-sensitivity of TE . Three of the identified features reflected the expected positive relationship between efficient uORF translation and a greater need for reinitiation to subsequently translate the CDS as well. These features were: the number of uORFs present on a transcript , the presence of AUG, rather than non-canonical uORF start codons , and the strength of Kozak contexts around uORF start codons . Relatedly, DENR targets were enriched for AUG uORFs as compared to the ensemble of translated uORF-containing transcripts , suggesting no DENR involvement in its recognition. Moreover, the presence of CDS-overlapping uORFs was not associated with DENR-dependent TE change , as expected for a reinitiation factor, given that overlapping uORFs would be reinitiation-incompetent unless the transcripts contained a downstream alternative CDS start codon.We used multiple regression analysis to determine whether DENR-dependent translation events were associated with specific transcript features. We modelled the CDS TE change observed upon t Figure . The modnt cells . By contb = 0.0006, P = 0.81), whereas the uORF–CDS intercistronic distance had predictive value for DENR-sensitivity of CDS TE . Briefly, already early studies on a model transcript had suggested that the location of a uORF with respect to the main ORF could dictate the efficiency of reinitiation, as longer spacing would generally allow the reacquisition of initiation factors to the scanning ribosome . Of note, the uORF–CDS distance was only weakly correlated with 5′ UTR length (Spearman rho = 0.16), whereas the correlation of the 5′ cap-first uORF distance with 5′ UTR length was more prominent , in agreement with previous results in human cells using synthetic uORFs encoding >2 amino acids (9 nt)  . Of note , suggesting that potential weaker secondary structures (low GC) favoured reinitiation involving DENR.Finally, the uORF GC content showed a significant relationship with TE fold change , the observed changes occurred already at the level of mRNA abundance and subsequently led to comparable changes at the footprint level as well  was associated with marked TE reduction  across human and mouse datasets, and elongating ribosome profiling studies showed good footprint coverage on uORF-2 and the characteristic accumulation of ribosomes at the start and stop codons  in frame and upstream of Firefly luciferase in our dual luciferase vector  to display the isolated effects of the Clock variants.In order to investigate uORF usage, effect, and DENR-dependence for CLOCK biosynthesis from the annotated and alternative CDS, we cloned the r Figure , and comDenr depletion resulted in an almost 30% reduction in expression for the Clock WT construct   , whereasockouts) ,7,47,48 ecycling .per se may simply not be a good proxy for translation time  likely reflect a straightforward logic between a stronger requirement for reinitiation when the usage of uORFs is efficient. This outcome is expected and fully consistent with human  and fly ion time ,8,51.R2 of 0.057). This is reminiscent of analogous models that have been developed to predict, for example, miRNA target sites, whose adjusted R2s range from 5 to 13%, but that have nevertheless proven biologically useful  most of them show TE downregulation and few upregulation in the absence of DENR, that (ii) they are depleted for CDS-overlapping and enriched for non-overlapping uORFs, and that (iii) a DENR-dependent inhibitory effect of their 5′ UTRs can be rescued by uORF start codon mutations in reporter assays, all make a direct activity of DENR on these transcripts very likely. Moreover, these observations are all compatible with a role of DENR in reinitiation, but not in leaky scanning  was expressed in NIH3T3 cells. Tmem60 was predicted to undergo a 25% translational downregulation upon Denr loss-of-function and indeed showed a decrease in that range in reporter assays in HeLa cells  were indeed downregulated upon Denr knockdown, though to a lesser extent. Conceivably, DENR targets are species- and cell type-specific; moreover, it is likely that the differences in the magnitude of translational regulation observed across studies are the result of different experimental and analytical approaches.A comparison of DENR targets found in our and in previous studies returned little overlap. Of the eight targets validated in HeLa cells , only onLa cells , whereasan cells , >90% weClock was a DENR target just below the significance threshold. Follow-up experiments confirmed Clock regulation through DENR and allowed mapping the responsible uORF. These findings establish uORF translation and re-initiation as a novel, potentially regulated mechanism acting on core clock gene expression, which opens several intriguing questions. First, is it possible that reduced biosynthesis of Clock would be responsible for the observed phenotype? Loss-of-function of a general gene expression machinery component typically goes along with sizeable distortions in global RNA and protein profiles, and Denr knockdown is no exception in this respect. Any phenotype thus likely represents a composite effect, rather than the result of a single misregulated gene expression event. We therefore do not view reduced CLOCK biosynthesis as a satisfactory hypothesis that alone would be able to explain the substantial period shortening. Nevertheless, it is reassuring that Clock−/ − fibroblasts have recently been shown to have shorter periods as well . Code for ribosome profiling pre-processsing, aligning and quantification, and for uORF annotation, is available online at gkz261_Supplemental_FilesClick here for additional data file.


Dear Editors,1. CV-A16 belongs to the Enterovirus (EV) A species and is one of the major serotypes that causes hand, foot, and mouth disease (HFMD). CV-A16 can be classified into three genogroups, A–C, based on the VP4 and VP1 gene sequences5. Genogroup B can be further divided into B1 (B1a–B1c) and B26. CV-A16 Genogroup D is a novel recombinant genogroup. The epidemiological origin can be traced to Peru7. Hassel et al. reported its first emergence and circulation in France during 2010–20141 and provided further detailed evidence of an intertype recombinant origin of CV-A16 genogroup D. To the best of our knowledge, no more report of the novel genogroup D could be found in the PubMed database.We report the first outbreak and spread of one Coxsackievirus A16 (CV-A16) novel Genogroup D recombinant strain in Shanghai, China, in 2016, 2 years after an initial report in FranceThe genogroup D Shanghai strain, designated SH-HP-16-51, was isolated from a mild HFMD case collected by our laboratory surveillance system on 5 October 2016. The patient was a 3-year-old girl. The epidemiological origin was traced to an aggregated case of 14 suspected HFMD patients reported in Pudong CDC. Between 2 and 5 October, 11 and 3 students among these 14 patients belonged to two adjacent classes in one kindergarten, respectively. The first case occurred on 2 October with fever (high temperature 38.9 °C), sore throat, and oral ulcers, and the child was diagnosed with HFMD on the next day. Unfortunately, clinical samples of only two patients were successfully collected in this outbreak because the kindergarten was quickly closed before we could collect other patient samples. However, no EV RNA was detected in the first case sample. In addition, although the guardians of both the first and second cases denied any history of contact with foreigners or travel abroad 2 weeks before the onset, we cannot exclude the possibility that the SH-HP-16-51 strain originated from other geographic locations, like France, because of increasingly frequent international business and communication.8 and submitted to GenBank (accession number: MG948605). The SH-HP-16-51 genome was 7231 bp long. Phylogenetic trees were constructed by using nucleotide sequences of the VP1, P1, P2, and P3 regions of the reference strains representing all genogroups with complete genome sequences. Phylogenetic analyses based on the complete VP1 sequence (891 bp) provided evidence that the isolate SH-HP-16-51 belonged to CV-A16 genogroup D and was most closely related to the strains isolated from France and Peru . A human rhabdomyosarcoma cell line was then used for virus isolation. When typical cytopathic effects occurred (data not shown), culture medium and cells were harvested for purification. The complete genome was determined as describederu Fig. . These seru Fig. . Further1 (data not shown) between clades B and D. Hence, the high nt identity between the SH-HP-16-51 strain and the French strains strongly indicated the potential common evolutionary origin. Moreover, we performed similarity plot and bootscan analyses using whole-genome sequences. The Shanghai isolate showed a greater similarity to the sequences of clade D sampled in France, with more than 93% nt identity  were compared Table . Sequencity Fig. , stronglity Fig. .C) and the SH-HP-16-51 strain were further conducted as previously described9. CV-A16C  was isolated from a Shandong HFMD patient in 2007. One hundred serum samples from healthy children  were collected in 2017. The overall seropositive rates (NtAb titer more than 8) against CV-A16C and SH-HP-16-51 were 67% and 23%, respectively. Among these, 40% of healthy children had a high CV-A16C NtAb titer (more than 16), while none of them had a high SH-HP-16-51 NtAb titer. Except for one case, all of these children with NtAb-positive SH-HP-16-51 had high CV-A16C NtAb titer, indicating the common susceptibility of children to SH-HP-16-51 infection, as well as its potential circulation and outbreak in China in the future.Seroprevalence of neutralizing antibody (NtAb) titers against one common CV-A16 clinically isolated strain . Although the CV-A16 NtAb in the population induced low immune responses against the new CV-A16 recombinant genogroup, the low-titer NtAb did not produce effective protection against recombinant genogroup D or the onset of herpangina. Further studies emphasize the need for improving novel recombinant CV-A16 genogroup D surveillance. Genetic and antigenic characterization studies will no doubt assist in efforts to prevent and control potential transmission and epidemics.HFMD causes a substantial disease burden in the Asia-Pacific region, especially in children below 5 years of age. CV-A16 is one of the most common causative pathogens for HFMD. Recombination is a common phenomenon in EV evolutionFigure S1Table S1Supplementary files


Infectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Potato virus S (PVS) is a carlavirus with a worldwide distribution. Although the complete genome sequences of many PVS isolates have been reported, the construction of an infectious cDNA clone of PVS is yet to be reported. The aim of this study is the development and molecular characterization of an infectious cDNA clone of PVS.Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of cDNA clone pPVS-H-FL-β was determined as the genome sequence of PVS-H00 and compared with the consensus sequence of PVS-H95 genome.A full-length cDNA clone pPVS-H-FL-AB was constructed by connecting eight cDNA clones of PVS isolate H95. Capped RNA transcripts from pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, proved to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating a single local lesion isolation in Nicotiana occidentalis plants inoculated with ≥0.2 μg capped RNA transcripts from pPVS-H-FL-β developed symptoms on upper leaves, as observed with PVS-H00 inoculation. Similar levels of viral genomic and subgenomic RNAs and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed 370 nucleotide polymorphisms (4.4% of the entire genome sequence), causing 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of chimeric RNAs derived from recombinants between the two cDNA clones revealed that the lack of infectivity of pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared with pPVS-H-FL-β. Approximately 71.3% amino acid substitutions in replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains.All This is the first report of the development of an infectious cDNA clone of PVS. Our analyses suggest that PVS population within a plant exists as quasispecies and the replicase sequence diversity of PVS obstruct the construction of a full-length infectious cDNA clone.The online version of this article (10.1186/s12985-019-1124-x) contains supplementary material, which is available to authorized users. Solanum tuberosum L.) with a worldwide distribution. PVS alone typically causes mild or no symptoms in most potato varieties; however, potato yield losses as high as 20% have been reported in the event of secondary infection [O (ordinary) and PVSA (Andean), have been identified, based on the ability to systemically infect Chenopodium quinoa and C. amaranticolor plants. PVSO induces chlorotic local lesions on the inoculated leaves of C. quinoa and C. amaranticolor without systemic infection, whereas PVSA induces systemic chlorotic mottling in C. quinoa and C. amaranticolor plants after inducing chlorotic lesions on the inoculated leaves. In addition, PVSA causes more severe symptoms on potato leaves, and it is more readily transmitted by aphids and contact with infected plants than PVSO [O and PVSA strains have been reported from Tasmania, Australia [O, 13 out of 44 Tasmanian isolates cause local but asymptomatic infection only on inoculated leaves of C. quinoa. By contrast, nine isolates, unlike PVSA, cause systemic infection without the typical symptoms in C. quinoa. Of these nine isolates, five induce symptoms on inoculated leaves only, and four cause asymptomatic systemic infection on inoculated and uninoculated upper leaves [Potato virus S (PVS) is one of the most common viruse of potato  isolates from Central Europe are genetically distinct from PVSA isolates, and they are closely related to European PVSO isolates. Cox and Jones [O-CS for isolates that systemically infect Chenopodium spp. but do not group within the PVSA clade, according to phylogenetic analysis based on coat protein (CP) gene sequences. However, there is no evidence that CP is involved in systemic infection on Chenopodium spp. For instance, the CP gene sequence of Vltava isolate from Czech Republic groups within the PVSA clade [C. quinoa [O and PVSA isolates [C. quinoa; phylogenetic analysis of CP gene sequences shows that the US isolate clusters with many PVSO isolates, whereas both Chilean isolates cluster within the PVSA clade [Matoušek et al.  suggest nd Jones  proposedSA clade , althoug. quinoa . Recombiisolates . Lin et isolates  have repSA clade .Chenopodium spp., it is necessary to construct an infectious PVS cDNA clone; infectious clones are the most powerful genetic tool for reverse genetics of RNA viruses. Although the complete genome sequence of PVS was first reported in 2005 for a German isolate Leona [To elucidate the genetic factors involved in the pathogenicity of PVS, e.g. systemic infection to te Leona , the conCarlavirus [Quinvirinae, family Betaflexiviridae, and order Tymovirales. PVS particles are slightly flexuous filaments, with a length of ca. 650 nm and a width of ca. 12 nm [2-Cys - X10–12 - Cys - X4 - Cys) and possesses nucleic acid-binding properties [PVS belongs to the genus rlavirus , subfamia. 12 nm . Of thesa. 12 nm . Downstroperties . The CRPoperties –18.O was determined in 2001 [Among carlaviruses, an infectious RNA transcribed in vitro from the full-length cDNA clone was first reported in BlScV in 1994 , PVM 2121, chrys in 2001 . HoweverNicotiana debneyi, a systemic host of PVS, in 1995. PVS-H95 isolate was identified as an ordinary strain of PVS by reactivity on C. quinoa. Subsequently, N. occidentalis was identified as a symptomatic systemic host of PVS-H95, and determined to be suitable for the maintainance and purification of the virus. The full-length sequence of PVS-H95 genome extracted from purified virus particles was determined in 2001 [C. quinoa inoculated with PVS-H95 in 2000. In the meanwhile, the virus from the single local lesion on a C. quinoa leaf was propagated on N. occidentalis once and then inoculated on C. quinoa again. After three cycles of transmission from C. quinoa lesion to N. occidentalis, the isolate, designated as PVS-H00, was maintained on N. occidentalis. No biological and serological differences were observed between PVS-H95 and PVS-H00.The PVS isolate used in this study was originally detected on the potato cultivar Astarte in Hokkaido, Japan, in 1993 using enzyme-linked immunosorbent assay (ELISA). The virus was transmitted from a singly infected potato plant to  in 2001 . AnotherEx Taq . The long cDNA corresponding to most parts of ORF1 (ca. 5.4 kb) was amplified using a pair of PVS-11P and PVS-11 M primers  tail comprising 42 adenine nucleotides  by connecting eight clones, as shown in Fig. CP gene fragment in pPVS-H-FL-D was replaced by the clone pPVS-1P1M-6 to produce pPVS-H-FL-G with Gly-54  and KOD-Plus-  with specific primers designed against the consensus sequence of PVS-H95 genome , resulting in the introduction of a unique SpeI restriction site immediately after the poly(A) tail comprising 66 adenines at the 3′ terminus  from dried leaves of Replacement of the cDNA fragment was performed at the restriction enzyme digestion sites using T4 DNA ligase. Nucleotide substitutions were performed using a QuikChange II XL Site-Directed Mutagenesis Kit , or according to the protocol of KOD-Plus-Mutagenesis Kit .MluI or SpeI for transcription. Digested DNA was purified by phenol/chloroform extraction and ethanol precipitation. Capped RNA was transcribed in a 20 μL reaction mixture, containing 40 mM Tris-HCl (pH 8.0), 25 mM NaCl, 8 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol, 2 mM each of ATP, UTP, and CTP, 0.2 mM GTP, 0.5 mM cap analog m7G(5′)ppp(5′)G , 40 U of human placenta RNase inhibitor , 5 μg of linearized plasmid DNA, and 50 U of T7 RNA polymerase , at 37 °C for 25 min. Subsequently, 2 μL of 20 mM GTP was added to the reaction to a final concentration of 2 mM. After 50 min incubation at 37 °C, plasmid DNA template was digested with 5 U of RNase-free DNase I  at 37 °C for 15 min. The reaction volume was then increased to 100 μL with ultrapure water, and 10 μL of a solution containing 5 M ammonium acetate and 0.1 M EDTA (pH 8.0) solution was added to the reaction. The reaction products were purified by phenol/chloroform extraction, followed by precipitation with 1 volume of 2-propanol. The products were resuspended in THE RNA Storage Solution  containing 1 mM sodium citrate (pH 6.4). The RNA yield was estimated using a NanoDrop 1000 spectrophotometer . Subsequently, 0.5 and 0.2 μg of RNAs were electrophoresed on formaldehyde-denaturing 1.2% agarose gel, and RNA transcripts were evaluated by comparing with a single-stranded (ss) RNA ladder  and RNA transcripts of known size, and by comparing the intensity of ethidium bromide staining. For inoculation, RNA transcripts were mixed with a buffer containing a final concentration of 0.1 M Tris-HCl, 10 mM EDTA (pH 7.5), and 0.5 mg/mL bentonite. Two leaves per plant in N. occidentalis plants at the 4–5 leaf stage grown on a shelf under controlled conditions (16 h fluorescent light and 24 °C temperature) were dusted with carborandom (600 mesh) and rub-inoculated with 0.1–5 μg of RNA transcripts in 2.5 μL of inoculum per plant using a finger covered in a finger cot. Plants of C. quinoa grown under a controlled temperature of 25 °C in the greenhouse were rub-inoculated with the sap of N. occidentalis, containing PVS generated by inculation with RNA transcripts, using carborandom and cotton swabs. The appearance and development of symptoms on plants were observed and compared with those obtained by inoculation with the original virus.The plasmid containing full-length PVS cDNA was linearized with p-nitrophenyl phosphate was measured at 405 nm using Wallac 1420 ARVO MX multilabel plate counter .To detect viral CP, upper leaves of plants inoculated with RNA transcripts or virus were used for double antibody sandwich (DAS)-ELISA  approximN. occidentalis 14 or 15 days post inoculation (dpi) using TRIzol Plus RNA Purification Kit . The isolated total RNA (10 μg) was electrophoresed on formaldehyde-denaturing 1.2% agarose gel, and then transferred onto Amersham Hybond-N nylon membrane . The RNA was fixed to the nylon membrane by exposure to ultraviolet light, and hybridized with a digoxigenin (DIG)-cRNA probe (ca. 800 nucleotides) complimentary to the 3′ terminal region of PVS-H00 genome, excluding the last 9 nucleotides and poly(A) tail at the 3′ end. RNA hybridization was detected with the DIG detection system and chemiluminescent substrate CDP-Star , and visualized using LAS-4000mini luminescent image analyzer .For northern blot hybridization, total RNA was extracted from the upper leaves of The cloned cDNAs were sequenced using an automated DNA sequencer, Li-Cor Model 4000 L , with Thermo Sequenase fluorescent labeled primer cycle sequencing kit , or ABI PRISM 310 Genetic Analyzer  with BigDye Terminator v1.1 cycle sequencing kit . Primers used for cDNA sequencing are listed in Additional file N. occidentalis plants. Next, pPVS-H-FL-AB was modifed to obtain an infectious cDNA clone. pPVS-H-FL-V with Ser-44 and pPVS-H-FL-C with Ile-660 and Ser-908 were constructed by the modification of ORF1; however, capped RNA transcripts derived from the two clones also failed to infect N. occidentalis. Then, pPVS-H-FL-D with Arg-128, pPVS-H-FL-E with Asn-21, and pPVS-H-FL-F with Asn-21 and Arg-128 were constructed by the conversion of TGBp1 encoded in ORF2 of pPVS-H-FL-C  of pPVS-H-FL-AB compared with the consensus sequence of PVS-H95 genome . Symptom onset in plants inoculated with RNA transcripts was approximately 5 days delayed compared with virus inoculation, implying that the delay was caused by the nature of inoculum, i.e. copies of viral RNA genome or virus particles, because no delay was observed when the sap of N. occidentalis infected with RNA transcripts was used as inoculum (data not shown). Except for the delay, no significant difference between PVS-H00 and pPVS-H-FL-β RNA transcript inoculation was observed in symptom development .The infectivity of RNA transcripts prepared from pPVS-H-FL-β was further investigated on N. occidentalis by rub-inoculation of plasmid DNA linearized with SpeI; however, the infection rate was much lower (18.75%) than the RNA transcripts, despite inoculating with 6 μg of plasmid DNA per plant (data not shown).In addition to pPVS-H-FL-β under control of T7 promoter, full-length cDNA from pPVS-H-FL-β was cloned downstream of the two copies of the cauliflower mosaic virus 35S promoter. The resulting p35S-PVS-H-FL-β construct proved to be infectious to The sequence of infectious cDNA clone pPVS-H-FL-β, which originated from PVS-H00 genome, was determined and compared with the previously determined consensus sequence of PVS-H95 genome. Both complete genome sequences were 8485 nucleotides long, excluding the poly(A) tail at the 3′ end, and were deposited to the DDBJ nucleotide sequence database under the accession numbers LC375227 (PVS-H95) and LC375228 (PVS-H00). Six ORFs were identified in both PVS genomes: ORF1 (nucleotides 63–5990), ORF2 (6025–6708), ORF3 (6686–7012), ORF4 (6976–7176), ORF5 (7218–8099), and ORF6 (8102–8383) , followed by ORF3 , ORF2 , ORF4 , and ORF6 , and the lowest in ORF5 . No substitutions were detected in the non-coding regions at the 5′ or 3′ termini of the genome, intergenic regions, or overlapping regions of TGB.Surprisingly, numerous sequence polymorphisms were identified between the two PVS genomes Table . A totalMore than 80% of the nucleotide substitutions in each ORF were transitions (Ti), and transversions (Tv) were detected only in ORFs 1, 2, and 3, and not in ORFs 4, 5, and 6; the Ti/Tv ratio in the entire genome was 4.44  scoring system, based on structural similarities and interchangeability of amino acids, on a scale of 0–5, where 0 represents the most drastic (rarest) change and 5 represents the most frequent substitution  Table 2. Across Amino acid substitutions present in replicase, which accounted for approximately 88% of the total substitutions, were further investigated in each functional domain  tail length (59–70 adenines in the newly constructed clones compared with 66 adenines in pPVS-H-FL-β) . It wasSpeI restriction site was created immediately after the poly(A) tail of 66 adenines. Additionally, we used the genome of a pure isolate PVS-H00, selected from PVS-H95 populations by repeating a single local lesion isolation in C. quinoa three times, as a template for the RT-PCR. The chlorotic local lesion induced in C. quinoa and C. amaranticolor by rub-inoculation with PVS is considered to be caused by a hypersensitive response (HR), which is one of the most notable resistance responses. The single local lesion isolation of plant virus in a host plant is considered as a genetic bottleneck, which leads to a reduction in genetic variation in the viral population [N. glutinosa infected with tobacco mosaic virus, approximately 103 virus particles were present per cell, which is two to four orders magnitude lower than that in systemically infected cells [C. amaranticolor has been shown to require cell-to-cell movement of the virus via plasmodesmata outside an initially infected cell [C. quinoa inoculated with the soil-borne wheat mosaic virus [trans-acting genes or elements responding to host shifting and changes in growth conditions. Thus, our data are consistent with Miyashita and Kishino [N. occidentalis and C. quinoa. The existence of PVS-H00 genome in PVS-H95 populations was partially verified by direct sequencing of RT-PCR products amplified from PVS-H95 genome as a minor signal  was observed in replicase encoded by ORF1, which occupies ca. 70% of the entire PVS genome length. Experiments on the infectivity of chimeric RNAs derived from recombinants between pPVS-H-FL-H and pPVS-H-FL-β revealed that the lack of infectivity of RNA transcripts derived from pPVS-H-FL-H was due to the ORF1 sequence , resulting in 91 amino acid substitutions (3.3%), were identified between PVS-H95 (consensus) and PVS-H00 (pPVS-H-FL-β) genome squences. Phylogenetic analysis of global PVS isolates based on the complete genome sequence shows that PVS-H95 and PVS-H00 are genetically distinct; however, both isolates cluster with six PVS.4%, resuhttp://web.expasy.org/protscale/) with the scale of Hphob./Kyte & Doolittle  Additional file 2:Figure S1. Comparison of amino acid sequences of functional domains within replicase between PVS-H95 and PVS-H00 genomes. (PDF 275 kb)Additional file 3:Figure S2. Direct sequencing of RT-PCR products amplified from PVS-H95 genome. (PDF 138 kb)Additional file 4:Figure S3. Phylogenetic tree calculated by the maximum-likelihood method from complete genome sequences of global PVS isolates. (PDF 77 kb)Additional file 5:Figure S4. Hydropathy plot of PVS-H95 and PVS-H00 replicases. (PDF 190 kb)Additional file 6:Figure S5. Predicted secondary structure of PVS-H95 and PVS-H00 replicases excluding RNA-dependent RNA polymerase domain. (PDF 271 kb)


Administration of CD300c-Fc protein attenuates graft-vs.-host disease (GVHD) in mice. Our results suggest that therapeutic interaction with the CD300c inhibitory pathway may represent a new strategy to modulate T cell-mediated immunity for the treatment of GVHD and autoimmune disease.T cell responses are fine-tuned by co-stimulatory and co-inhibitory molecules. Among the T cell regulators, the B7 family members are of central importance. The recent success in targeting the B7 family molecules for the treatment of immune-related diseases has attracted intense interest in identifying additional B7-related molecules. In this study, we describe CD300c as a novel T cell co-inhibitory molecule that shares significant sequence homology with existing B7 family members. CD300c protein is expressed on professional antigen-presenting cells (APC), including B cells, monocytes, macrophages, and dendritic cells (DCs). The putative CD300c counter-receptor is expressed on CD4 and CD8 T cells, and the expression levels are upregulated upon activation. Soluble human and mouse CD300c-Fc fusion proteins significantly inhibit the proliferation, activation, and cytokine production by CD4 and CD8 T cells  T cell immune responses are tightly controlled by co-stimulatory and co-inhibitory molecules. The co-stimulatory molecules contribute to the development of immune responses against cancers and foreign pathogens, while the co-inhibitory molecules are critical for peripheral tolerance to avoid autoimmunity, GVHD and transplant rejection.A number of T cell co-stimulatory and co-inhibitory ligands and receptors have been identified. Among them, B7 family members are of central importance. The B7 family ligands include B7-1 , PD-L1 , 4, PD-LThe unprecedented success by targeting the ligands or receptors of the B7 family members for the treatment of cancer and autoimmune disease has attracted considerable interest in identifying additional T cell regulators. In this study, we identify CD300c as a novel T cell inhibitory molecule. Human CD300c (hCD300c), also known as CMRF-35A or CMRF-35, was originally isolated from human leukocytes –20. It hin vitro. Administration of hCD300c-Ig protein ameliorates GVHD in mice. Therefore, this unique T cell inhibitory pathway may provide a new strategy to modulate T cell-mediated immunity to treat immune-related diseases.We show here that hCD300c is homologous to known B7 family members in amino acid sequence. CD300c is expressed on APCs, and its putative counter-receptor is expressed on CD4 and CD8 T cells. Both soluble human and mouse CD300c-IgG2a Fc (CD300c-Ig) fusion proteins significantly inhibit the proliferation and activation, and cytokine production of CD4 and CD8 T cells http://www.cbs.dtu.dk/services/SignalP), TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and InterPro (https://www.ebi.ac.uk/interpro).Sequence alignments of CD300c and known B7 family members, and CD300c orthologous proteins were analyzed via the Clustal W program in MacVector 16.0.5 . The leader peptide, transmembrane, and Ig-like domain were predicted with SignalP 4.0 (The extracellular domains of hCD300c (aa29-183) and mCD300c2 (aa22-193) were cloned and fused into a pCMV6-AC-FC-S expression vector containing the constant region of mouse IgG2a (ORIGENE). The vectors were transfected into HEK293F cells. The fusion proteins were purified for supernatant using Protein G Sepharose 4 Fast Flow according to the manufacturer's instructions . Purified proteins were verified by SDS-PAGE, Coomassie Staining and Western blot. Protein was quantified using the Pierce™ BCA Protein Assay Kit . Control Ig (recombinant mouse IgG2a Fc protein) was purchased from BXCell .Purified CD300c-Ig was loaded on a 12% SDS-PAGE, and stained with Coomassie blue or transferred to a polyvinylidene fluoride membrane. The protein containing membrane was incubated with HRP conjugated anti-mouse IgG2 antibody, or anti-hCD300c antibody  followed by HRP conjugated second antibody, and then developed with Super Signal® West Pico chemiluminescent Substrate (Thermo Scientific).b, Annexin V, Ki67, CD44, CD62L, CD69, CTLA-4, CD28, PD-1, BTLA, and ICOS and mCD300c2 . mCD300c2-Ig and hCD300c-Ig were biotinylated with sulfo-NHS-LC-Biotin (Pierce). The samples were analyzed on a FACSCalibur or LSRFortessa X-20 Cell Analyzer (BD Biosciences). Data analysis was done using FlowJo software .Single cell suspensions of organs were stained with the fluorochrome-conjugated antibodies protein as described –35. For The endotoxin level in the purified proteins was determined by the endpoint chromogenic LAL test according to the manufacturer's instructions  .+ Pan T Cells that were negatively isolated from mononuclear cells using an indirect immunomagnetic Pan-T labeling system were purchased from ALLCELLS, LLC . Murine CD3+ T cells were purified from C57BL/6 mice by an immunomagnetic system , and the purity of the cells was usually >95%. T cells were stimulated with anti-CD3 and/or anti-CD28 antibodies (Biolegend) in the presence of CD300c-Ig or control Ig. Proliferative response was assessed by pulsing the culture with 1 μCi of [3H] thymidine  12 h before harvest. Incorporation of [3H] thymidine was measured by liquid scintillation spectroscopy . For carboxyfluorescein diacetate succinimidyl ester (CFSE) assay, splenocytes were labeled with CFSE (ThermoFisher Scientific), and stimulated with anti-CD3 in the presence of CD300c-Ig or control Ig. The cells were analyzed by flow cytometry.Normal human peripheral blood CD3Four-week-old female C57BL/6 and BALB/c mice were purchased from Jackson Laboratory. The mice were used in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Connecticut.BALB/c recipients received 900 cGy total body irradiation from a 137Cs source . Two to four hours later, the mice were injected intravenously (i.v.) with BM and spleen cells from C57BL/6 mice. The recipients were injected i.p. with hCD300c-Ig, or control Ig. The severity of GVHD was evaluated with a clinical GVHD scoring system. In brief, GVHD recipients in coded cages were individually scored every week for five clinical parameters on a scale from 0 to 2: weight loss, posture, activity, fur texture and skin integrity. A clinical GVHD index was generated by summation of the five criteria scores (maximum index = 10).GVHD target organs were harvested for histopathological analysis. The organs were formalin-preserved, paraffin-embedded, sectioned and hematoxylin/eosin (H&E)-stained. Assessment of tissue damage was performed based on scoring systems previously described . BrieflyP-values were based on the two-sided Student's t-test. A confidence level above 95% (p < 0.05) was determined to be significant.Recognizing the importance of the B7 family in controlling immune responses, we performed a series of genome-wide database searches to find molecules that are homologous to known B7 family members. We discovered that hCD300c shares varying levels of amino acid identity and similarity with B7-1 (17 and 13%), B7-H2 (16 and 12%), B7-H3 (13 and 12%), B7-H4 12 and 15%), PD-L1 (14 and 19%), and PD-L2 (13 and 15%) Figure . It has %, PD-L1 It has been reported that the mouse orthologs of hCD300c are mouse CD300c (mCD300c) [also called CMRF-35-like molecule-6 (CLM-6)] and mCD300c2  –28. hCD3To investigate whether like the known B7 family members, CD300c protein can affect T cell function, we produced an hCD300c-Ig fusion protein by cloning the extracellular domain of the hCD300c gene into an expression vector containing the constant region of the mouse IgG2a. The expression vector was then transfected into human HEK-293 cells to produce hCD300c-Ig fusion protein that was then purified from the supernatant of the cells. A relatively high purity of hCD300c-Ig protein was obtained, as determined by Coomassie blue-stained SDS-PAGE  for 3 days. Since the molecular weight of hCD300-Ig fusion protein is ~1.5-fold higher than that of control Ig protein, we used equimolar amounts of the control Ig as a control. T cell proliferation was measured by [3H] thymidine incorporation. As shown in Figure We then determined whether hCD300c-Ig protein affected T cell proliferation. To do this, CD3To confirm the effect on T cell proliferation and to determine whether hCD300c affects CD4 and/or CD8 T cells, we performed a carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay. Murine splenocytes were labeled with CFSE, and then cultured with anti-CD3 antibody in the presence of graded doses of hCD300c-Ig or control Ig. T cell proliferation was measured by CFSE fluorescent dilution in CD4 and CD8 T cells. As shown in Figures in vitro. CD69 is an early activation marker. After splenocytes were cultured with anti-CD3 antibody and hCD300c-Ig or control Ig, the expression of the CD69 on CD4 and CD8 T cells was analyzed 24 h later. As shown in Figures We next determined whether hCD300c-Ig affects the activation of T cells in vitro, we examined whether hCD300c-Ig affected human T cells. Purified human T cells were cultured with anti-CD3 antibody in the presence of graded doses of hCD300-Ig or control Ig, and T cell proliferation was measured by [3H] thymidine incorporation. Similarly, hCD300c-Ig markedly inhibited human T cell proliferation with ~53 and 77% inhibition by 1,500, and 3,000 ng/ml hCD300c-Ig, respectively . Although the manufacture's manual indicates that this antibody recognizes mCD300c and mCD300d, this antibody was generated by using mouse MAIR-II (mCD300c2)-transfected cells as an immunogen. As indicated in the Discussion Section, the nomenclature for mCD300c is still confusing and MAIR-II is also known as mCD300c2/CD300d. Therefore, we believe that this antibody reacts to mCD300c2 protein. We found that resting splenic CD42. Althou+ and CD8+ T cells, and the binding was increased when CD4+ and CD8+ T cells were activated by anti-CD3 and anti-CD28 antibodies  → BALB/c (H2d)] GVHD mouse model to validate the effect of CD300c on T cells in vivo. BALB/c mice were lethally irradiated and injected i.v. with BM and splenic cells from allogeneic C57BL/6 mice. The recipients were then injected i.p. with hCD300c-Ig, or control Ig. The development of GVHD was monitored over time. As shown in Figures in vivo.Although bone marrow (BM) transplantation (BMT) has been widely used in the treatment of many diseases, GVHD remains a major complication after allogeneic BMT. Acute GVDH is primarily caused by T cells in donor transplants attacking recipient's tissues. We used a well-defined MHC-mismatched  thymidine incorporation and cytokine production assays, we used splenocytes that include T cells and other immune cells for the T cell activation studies. The possibility that the inhibition of T cell activation by CD300c-Ig may be caused by its indirect effect on other immune cells cannot be excluded at this stage.It has been reported that CD300c has effects on other types of immune cells –30. Crosferation , 35. In feration . Furtherferation , 37. In feration . It is uin vitro. Administration of hCD300c-Ig protein attenuates GVHD in mice. Therefore, CD300c protein has the potential to be used in the treatment of GVHD, autoimmune disease, and transplant rejection.In summary, we have identified CD300c as a B7 family-related molecule. CD300c protein is expressed on APCs, and its counter-receptor is expressed on T cells. Soluble human or mouse CD300c-Ig fusion proteins significantly inhibit T cell proliferation, activation, and cytokine production CC performed experiments and analyzed data. MS and YL performed experiments. LL designed experiments, analyzed data, supervised the study, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium abscessus complex (MAB) is a rapidly growing mycobacterium(RGM) whose clinical significance as an emerging human pathogen has been increasing worldwide. It has two types of colony morphology, a smooth (S) type, producing high glycopeptidolipid (GPL) content, and a rough (R) type, which produces low levels of GPLs and is associated with increased virulence. However, the mechanism responsible for their difference in virulence is poorly known. By ultrastructural examination of murine macrophages infected, we found that MAB-R strains could replicate more actively in the macrophage phagosome than the S variants and that they could escape into cytosol via phagosomal rupture. The cytosolic access of MAB-R strains via phagosomal rupture led to enhanced Type I interferon (IFN) production and cell death, which resulted in their cell-to-cell spreading. This behavior can provide an additional niche for the survival of MAB-R strains. In addition, we found that their enhancement of cell death mediated cell spreading are dependent on Type I IFN signaling via comparison of wild-type and IFNAR1 knockout mice. In conclusion, our data indicated that a transition of MAB-S strains into MAB-R variants increased their virulence via enhanced Type I IFN production, which led to enhanced survival in infected macrophage via cell death mediated cell-to-cell spreading. This result provides not only a novel insight into the difference in virulence between MAB-R and -S variants but also hints to their treatment strategy. Mycobacterium abscessus complex (MAB) is now recognized as a major pathogen leading to pulmonary infection within the rapidly growing mycobacteria (RGMs) (a (RGMs) –3 and isa (RGMs) –6. In Soa (RGMs) , 8. MAB a (RGMs) . MAB infa (RGMs) , 11.M. abscessus subsp. abscessus  and M. abscessus subsp. bolletii. M. abscessus subsp. bolletii was proposed to include the two former species M. massiliense (S-Mas) and M. bolletii (S-Bol) (hsp65 genotypes (Type I and Type II)  , 13. S-MType II) .MAB consists of two phenotypes: smooth colony (MAB-S) and rough colony (MAB-R), with or without glycopeptidolipid (GPL) gene synthesis , 16. In Mycobacterium tuberculosis (Mtb) strains have ability to escape into the cytosol from the phagosome via the type VII secretion system ESX-1 : M. abscessus type strain ATCC 19977 rough strain, Asan 52550 and Asan 58116; S-Mas type I-Smooth (S-Mas_I-S): type strain, Asan 15, Asan 51312, and Asan 51843; S-Mas type I-Rough (S-Mas_I-R): Asan 16, Asan 22, and Asan 34; and S-Mas type II-Rough (S-Mas_II-R): Asan 4, Asan 50594, and Asan 62188] in murine macrophage J774A.1 cells (1 × 106 cells) as a function of the 10 multiplicity of infection (M.O.I.) (1 × 107 bacteria)  . The suracteria) . Of noteTo check the capacity of MAB-R and -S strains to induce the innate immune response, we measured cytokine production from supernatants of infected cells with 10 M.O.I at 24 h.p.i by enzyme-linked immunosorbent assay (ELISA). Both of the pro-inflammatory cytokines TNF-α and IL-6 were more induced in two different macrophages (J774A.1 cells and BMDMs) and in BMDCs infected with MAB-R strains than in those infected with MAB-S strains . In addiTo better understand the mechanism underlying the different growth kinetics during macrophage infections of MAB-R and -S strains . We condhsp65 DNA in the cytosol fraction of macrophages with either one of two MAB-R strains [S-Abs_R (M. abscessus type strain ATCC 19977 rough strain) and S-Mas_II-R (Asan 50594)] or with M. marinum, which is known to be capable of phagosomal escape (M. abscessus type strain ATCC 19977 smooth strain) and S-Mas_I-S (Asan 51843)], M. smegmatis or BCG , and S-Mas_II-R (Asan 50594), induced IRF-3 activation in infected BMDMs. We also found increased levels of the activated forms of STING (33–35 kDa) and cGAS (62 kDa) in these two MAB-R strains, consistent with the previous result that cytosolically exposed DNA induced Type I IFN secretion via the cGAS -STING axis (M. abscessus type strain ATCC 19977 rough strain), and S-Mas_II-R (Asan 50594)] and M. marinum, and not by infection of other mycobacterial strains ([S-Abs_S (M. abscessus type strain ATCC 19977 smooth strain) and S-Mas_I-S (Asan 51843)] and M. bovis BCG), further proving the enhanced Type I IFN signaling by MAB-R strains. But, there were no significant differences in IRF3 transcription levels between MAB-R strains and other mycobacteria , no significant difference in cell death-inducing capacity between live and HK bacteria was found, suggesting that the active multiplication of MAB-R strains may contribute to enhanced cell cytotoxicity.Phagosomal rupture in macrophage by virulent mycobacteria has been reported to induce cell death followed by cell-to-cell spread of cytosolic bacteria into neighboring cells via cell membrane rupture , 25. To M. smegmatis in all the infected time points than MAB-R strains  and 7-AAD (for necrotic cell death). Our data showed that two MAB-R strains more induced both apoptotic and necrotic cell death than other mycobacterial strains in a time dependent manner . In cont strains .Furthermore, we also found that live MAB-R strains but not HK strains induced apoptotic and necrotic cell deaths . TogetheM. tuberculosis H37Rv has been reported to contribute to bacteria cell-to-cell spreading, thus leading to increased bacterial growth  with different M.O.I. . We found that the cell spreading of MAB-R, but not M. smegmatis was gradually increased in a time and in an M.O.I dependent manner  led to significantly lower survival capacity in infected BMDM cells from IFNAR1 KO mice than in infected BMDMs from wild mice , and M. bovis BCG (rEGFP_BCG) after their infection into BMDMs from wild mice or IFNAR1 KO mice. We found that MAB-R but not M. smegmatis and BCG led to significantly less cell-to-cell spreading in BMDMs of IFNAR1 KO mice at the 24 and 48 h.p.i. than in BMDMs of wild type ] strain, ild type , suggestM. tuberculosis strains have been reported to survive better than attenuated strains because of their cell-to-cell spreading and bacterial cytosol access via ESX-1-derived active phagosomal rupture  leads to the production of a higher level of CFUs and TNF-α secretion from human monocytes than the Type I genotype (smooth type) . It was  rupture –22. In t rupture . These prophages , consequrophages  and cellrophages –7.M. tuberculosis or M. marinum strains harboring the ESX-1 locus   supporting the presence of cytosol stages of MAB-R strains in infected macrophage. First, we also found that irrespective of the types of their subspecies (S-Abs or S-Mas) or genotypes (Type I or Type II of S-Mas), MAB-R strains but not MAB-S strains induced IFN-β secretion , ISRE-ded BMDMs) .M. tuberculosis  were purchased from Orient-Bio . All mice were housed and maintained in a specific pathogen-free facility at Seoul National University College of Medicine. All mice were used for experiments at 8~12 weeks of age.2 in humid incubator.J774A.1 (murine macrophage) and L929 cells  were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and penicillin/streptomycin (100 U/ml) and kept at 37°C and 5% COg, 5 min), and the supernatant was discarded. BMDMs were differentiated from bone marrow precursors cultured for 4 days in complete DMEM containing 10% FBS, 1% sodium pyruvate, 4 mM glutamine, and 1% penicillin-streptomycin (100 units/ml) supplemented with 10% L929 cell-conditioned media. Total BM cells were cultured in microbiological 12- or 24-well plates (Sterilin) and kept at 37°C and in 5% CO2 in a humid incubator. BMDCs were differentiated with complete IMDM supplemented with 10% FBS , recombinant mouse GM-CSF  and mouse IL-4 , penicillin , streptomycin , gentamicin , L-glutamine , and β-mercaptoethanol  and seeded at a concentration of 1 × 106 cells per well in a 24-well plate in a final volume of 2 ml of complete IMDM. Half of the medium was replaced every other day with an equal volume of complete IMDM for 6 days. All the cells expressing CD11C+ in the different wells were isolated using appropriate magnetic-activated cell sorting (MACS) kits according to the manufacturer's instruction (Miltenyi Biotec). CD11c+ cells were collected and used in all experiments.BMDMs and BMDCs were generated from the bone marrow (BM) of 8 to 12-week-old C57BL/6J mice. BM cells were obtained from femurs and tibia of mice and flushed out with serum-free DMEM media  or serum-free Iscove's modified Eagle's medium . The single-cell suspension was then filtered through a nylon cell strainer  and washed twice with HBSS media. The cells were pelleted by centrifugation , and the supernatant was aspirated. The pellet was resuspended with red blood cell lysis buffer (Sigma) and incubated for 3 min at room temperature; the reaction was stopped by diluting the Lysis Buffer with V/V ml of serum-containing DMEM (BMDMs) or complete IMDM (BMDCs). The cells were spun (350 × Mycobacterium abscessus (ATCC 19977 T rough and smooth type), Mycobacterium abscessus subsp. massiliense (CIP 108297T), Mycobacterium smegmatis ATCC 19420T, Mycobacterium bovis BCG Tokyo strain (BCG), and Mycobacterium marinum JCM 17638T. Thirteen clinical isolates of Mycobacterium abscessus smooth  and rough , Mycobacterium abscessus subsp. massiliense Type I smooth , type I-Rough , type II-Rough . All the strains were cultured from low-passage frozen stocks (at −70°C) to exponential phase and subcultured in 7H9 broth (supplemented with 10% ADC) or on 7H10 agar plates (supplemented with 10% OADC) at 37°C for each experiment. RGM were grown in Middlebrook 7H9 broth supplemented with Tween 80 and 10% ADC (BD) by shaking incubation for 3–5 days  at 37°C. To obtain single-cell bacterial suspensions, all the strains were washed and resuspended in phosphate-buffered saline (PBS) with 0.05% Tween 80 (PBS-T) and passed through a 27-gauge needle three to five times. The concentration was determined by measuring the optical density at 600 nm (OD 600) as a function of CFU/ml. To inactivate various mycobacteria strains, we heated them at 100°C in a boiling-water bath for 30 min.The mycobacterial strains used in this study are as follows: 5 type strains of M. abscessus type strain ATCC 19977 rough strain) MAB-S  and incubated for 18 h at 4°C and then labeled with Goat anti-Rat IgG (H+L), Alexa Fluor® 633 conjugate, dilution 1:1,000 for 45 min at room temperature. Nuclei were stained with 300 nM DAPI  for 5 min at room temperature. The samples were washed with PBS and mounted on a glass slide with Vectashield solution (Vector Laboratories) and observed using a confocal laser scanning microscope system (Olympus-FV1000).To evaluate co-localization with bacteria containing phagosome and lysosome, we prepared J774A.1 cells on chamber plates (Nunc™ Lab-Tek™ II Chamber Slide™ System). The cells infected for 24 h.p.i. with CFSE  stained bacteria [MAB-R , and the hsp65 promoter gene was amplified from genomic DNA of M. bovis BCG. The EGFP gene with the hsp65 promoter was amplified from the pMyong2-EGFPh vector and ligated into pAL5000-TOPO using HindIII and BamHI restriction sites. The hsp65promoter and EGFP gene were amplified by overlapping PCR. To generate three different types of recombinant strains expressing EGFP , the pAL5000-TOPO-EGFP plasmid was electroporated into competent bacteria  using the Gene Pulser II electroporation apparatus . Transformants were selected on Middlebrook 7H10 medium    suppleme6 cells), BMDMs (1.0 × 106 cells), and immature BMDCs (1.0 × 106 cells) were infected with mycobacterial strains at an M.O.I. of 10 (1 × 107 bacteria) and incubated for 2 h to allow the phagocytosis of the bacteria. The infected cells were washed three times with PBS and to remove extracellular mycobacteria, cells were incubated with fresh culture medium containing 50 μg/ml amikacin , and the plates were then placed on a dryer with their smeared surface upwards, where they were air-dried for approximately 30 min and then heat-fixed. The specimens were covered with carbol fuchsin stain and then heated until vapor just began to rise . Additional stain was added if necessary, and the heated stain remained on the slide for 5 min. The stain was removed by washing with clean water. The smear was covered with 3% v/v acid alcohol (or 20% sulfuric acid) for 20 s and washed off with clean water. Methylene blue was then applied to counterstain any cells that had been decolorized. The smear was then examined with microscope using the 100 × oil immersion objective , and the smear was systematically scanned.Infected cells were washed with PBS and fixed with 2% paraformaldehyde (v/v) and 2% glutaraldehyde (v/v) in 0.05 M Na cacodylate-HCl buffer (pH 7.2) at 4°C for 2~4 h. Primary fixed samples were washed three times with 0.05 M Na cacodylate-HCl buffer (pH 7.0) at 4°C for 10 min. The fixed samples were post fixed with 1% osmium tetroxide (w/v) in Na cacodylate-HCl buffer (pH 7.2) at 4°C for 2 h. Then, fixed samples were washed two times with distilled water at room temperature. The pelleted cells were block-stained with 0.5% uranyl acetate (w/v) at 4°C for 30 min and dehydrated with 30, 50, 70, 80, 90, 100, 100, and 100% at room temperature for 10 min each. The samples were transferred two times with 100% propylene oxide at room temperature for 15 min, 1:1 propylene oxide/Spurr's resin (2 h), 1:2 propylene oxide/Spurr's resin (4 h or overnight), and 100% Spurr's resin (2 h) to infiltrate. In the following step, samples were embedded in molds filled with a mixture of Spurr's epoxy resin and polymerized for 24 h at 70°C. The polymerized blocks were cut on a ultramicrotome , and sections were stained with 2% uranyl acetate (7 min) followed by Reynolds' lead citrate (7 min) and then examined on a JEOL transmission electron microscope (Japan) at 80 kV accelerating voltage.Infected cell lysates were collected for protein analysis, and cells were lysed in RIPA buffer  containing complete EDTA-free protease inhibitor (Roche) and phosphatase inhibitor cocktail (Roche). Protein concentrations were determined using a bovine serum albumin (BSA) protein assay (Bio-Rad). Cellular proteins separated by SDS–polyacrylamide gel electrophoresis were electrotransferred to nitrocellulose (NC) membranes and subjected to immunoblot analysis with various primary antibodies. Immunoblot analysis was done with various primary antibodies: anti-rabbit IRF3 , anti-rabbit pIRF3 , anti-rabbit cGAS , anti-rabbit STING , anti-rabbit Actin , and anti-rabbit GAPDH  antibodies.Supernatants from infected cells were overlaid on top of L929 IFN reporter cells containing the ISRE-luciferase construct  and incuCells were seeded in 12-well plates and infected the following day with the different strains as explained before. The supernatant was completely aspirated and washed with PBS 2 times, trypsinized cells were collected, and cells were used to analyze apoptosis by flow cytometry. Phosphatidylserine (PS) exposure and membrane integrity were analyzed by using Annexin V and 7-AAD (BD Biosciences) and flow cytometry according to the manufacturers' instructions. Briefly, cells were washed with PBS and incubated with Annexin V and 7-AAD in Annexin V Binding buffer for 15 min. Then, 400 μl of Annexin V Binding buffer was added to each tube, and the samples were analyzed by flow cytometry (FACS LSRFortessa X-20).g at 4°C for 10 min; this process was repeated three times. The supernatant was carefully removed and discarded, and the pellets were resuspended in Protease Inhibitor Solution lysis buffer, vortexed, and incubated at 4°C for 10 min on an end-over-end shaker. The lysate was centrifuged at 1,000 × g for 10 min at 4°C, and the supernatant was carefully transferred into a new microcentrifuge tube, where it was stored on ice. This supernatant primarily contained cytosolic proteins. To extract DNA from the fractionized supernatant, we performed the PCI method (hsp65 gene (BMDM and J774A.1 cells were infected in 6-well plates. Cytosolic cell fractions were isolated using the manufacturer's protocol for the Qproteome Cell Compartment Kit (QIAGEN). Briefly, cell pellets were resuspended in ice-cold PBS by pipetting up and down and then centrifuged three times at 500 × I method . To detep65 gene  and applp65 gene . A set oCells were infected in 6-, 12-, and 24-well plates. Post infection cell culture supernatants were collected from infected cells and stored at −70°C. Paired antibodies and standard recombinant mouse IL-1β, TNF-α, IL-6, IL-10 (eBioscience), and IFN-β (BioLegend) were used to determine cytokine concentrations according to the manufacturers' instructions.CtΔΔ) was obtained by normalizing its level to that of the β-actin gene in each experiment. Specific primers with the following sequences were used  according to the manufacturer's instructions. For quantitative real-time PCR analysis, cDNA was amplified using specific primers for IFI204, IFNβ, STING, cGAS, IRF3, and SYBR green PCR Master Mix  and processed using an ABI 7500 . The results were acquired as cycle threshold (Ct) values and represented the average of at least two independent experiments. The relative amount of mRNA .Statistical analysis was performed using GraphPad Prism 5 software and Microsoft Excel software. All experiments were performed at least two times and the data are presented as the mean ± standard deviation (SD). Asterisks indicate significance as determined by the ANOVA with Tukey's correction and the Student's All animal treatment was in accordance with the institutional recommendations in the National Guidelines for the care and use of laboratory animals. The protocol was approved by the Institutional Animal Care and Use Committee  of the Institute of Laboratory Animal Resources at Seoul National University.B-RK and By-JK performed the experiments and analyzed the data. Bu-JK and Y-HK designed and interpreted the experiments. Bu-JK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.