The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor—angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell–cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike–ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) structural proteins, such as spike, envelope, and membrane proteins, are transmembrane proteins synthesized in the perinuclear endoplasmic reticulum (ER), carried to the ER–Golgi intermediate compartment (ERGIC) between the ER and the Golgi apparatus, and mostly assembled into virions in the early secretory pathway, while some spike proteins are trafficked to the plasma membrane . The spiThe intracellular trafficking of transmembrane proteins synthesized in the ER through the secretory pathway is typically mediated by interactions between their cytoplasmic tails and the coat proteins COPI and COPII in the ER-to-Golgi apparatus . COPI-meVero cells were transfected with a spike-expressing plasmid to compare the cell fusion activity of C488A mutant and wild-type (WT) spike proteins. WT spike protein induced multinuclear syncytia formation in Vero cells, but the C488A mutant spike protein did not A,B. MoreTo confirm the impaired plasma membrane localization of the spike protein, C488R and C488F mutant spike proteins were examined on the Vero cell surface. Cell-surface C488 mutant spikes were not observed D. ConfocIntracellular spike localization in Vero cells was examined by confocal microscopy. Green signals are spike proteins probed with the anti-spike monoclonal antibody 1A9. The co-localization at the ER and ERGIC were visualized by co-staining with calnexin and ERGIC 53, respectively  with frequencies of 0.000002 and 0.000003, respectively. The Ser-686 cleavage in Wuhan-Hu-1, D614G, and Delta spike proteins were comparable, while those in Alpha and Omicron BA.1 were relatively low. Functional syncytium-inducing activity of Alpha variant spikes was observed in NCI-H522/ACE2 cells  protein induces spike and nucleocapsid (N) protein accumulation in the Golgi apparatus ,17. WT sBrefeldin A (BFA) is a fungal metabolite that disrupts intracellular membrane traffic at the ER–Golgi complex junction . HackstaSARS-CoV-2 infection causes cell–cell fusion to form cytopathic syncytia, which requires cell-surface expression of the viral spike protein and its binding to ACE2 expressed on the neighboring cell surface . The disThe SARS-CoV-2 spike protein is a type I transmembrane protein initially translocated into the ER, glycosylated in the ER and Golgi apparatus, and further processed into S1/S2 subunits by furin in the trans-Golgi network . The nonS1/S2 processing in Alpha- and Omicron (BA.1)-type spike proteins detected by Ser-686 cleaved spike antibodies was relatively decreased C. Recentv/v) fetal bovine serum (FBS) and 25 µg/mL kanamycin. NCI-H522  cells were cultured in RPMI1640 medium supplemented with 7.5% (v/v) FBS and 25 µg/mL kanamycin. NCI-H522/ACE2 cells were generated by isolating human ACE2-DYK-expressing plasmid -transfected stable cells. The plasmids used are listed in HEK293T, Vero , and VeroE6/TMPRSS2  cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 7.5%  for Western blot and immunofluorescence analyses, as described previously [Synthetic cDNA with human codon optimization to express SARS-CoV-2 spike and membrane proteins were purchased from GenScript and cloned into the expression plasmid pcDNA3.1. Plasmid DNA was transfected using PEIpro® 488, Cat#A-11001, and donkey anti-rabbit IgG (H + L) cross-adsorbed with Alexa Fluor® Plus 594, Cat#A-32754) were purchased from Thermo Fisher Scientific , and antifade  was purchased from Vector Laboratories Inc. . Conventional immunofluorescence images were captured using a BZ-9000 microscope . The green fluorescence protein (GFP) signal was measured using ImageJ software . Confocal microscopy was performed using a TCS SP8 confocal laser scanning microscope  and Leica Application Suite X software (Leica). TIFF images were merged using Adobe Photoshop CS4 Extended software . Anti-calnexin (Cat#PM060MS) and anti-GM130 (Cat#PM061MS) antibodies were purchased from Medical & Biological Laboratories Co., Ltd. . Anti-ERGIC53/LMAN1 (Cat#EPR6979) and anti-furin (Cat#EPR14674) antibodies were purchased from Abcam K.K. .Immunofluorescence analysis was performed as described previously . FluoresTwo 6-week-old female BALB/c mice were purchased from CLEA Japan  and housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all of the animal experiments (permit number: 2019NiA-001). Each BALB/c mouse was intraperitoneally (i.p.) immunized with 100 μg N-terminal His-tagged S1 spike protein of SARS-CoV-2  using Imject Alum . The procedure included three additional immunization procedures (100 μg/mouse), followed by a final booster intraperitoneal injection (100 μg/mouse) 2 days before harvesting the spleen cells, which were subsequently fused with P3U1 cells  using polyethylene glycol 1500 . The hybridomas were then grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection . The culture supernatants were screened using an enzyme-linked immunosorbent assay (ELISA) for detecting SARS-CoV-2 S1. Clone CvMab-6 culture supernatant using hybridoma-SFM medium (Thermo Fisher Scientific Inc.) was purified using Ab-Capcher .®  and recorded with an ImageQuant LAS4000 mini-image analyzer .Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer -containing protease inhibitors. Proteins were resolved by electrophoresing on a 5–20% gradient sodium dodecyl sulfate polyacrylamide gel and electrotransferred to an Immobilon-P membrane . The primary antibodies against SARS-CoV-2 anti-spike , anti-nucleocapsid , and anti-membrane  proteins were purchased from GeneTex Inc. . Cleaved SARS-CoV-2 spike protein (Ser686) antibodies (Cat#84534) and anti-mouse IgG–HRP (Cat#7076) were purchased from Cell Signaling Technology, Inc. . Anti-rabbit IgG–HRP (Cat#NA934) was purchased from Cytiva™ . Anti-glyceraldehyde 3-phosphate dehydrogenase  was purchased from Medical & Biological Laboratories Co., Ltd. . Immunoblot signals were developed using EzWestLumi plusn ≥ 3). The Student’s t-test or one-way ANOVA was used to evaluate the significance of differences between the experimental and control groups with similar variances. Differences were considered statistically significant at p < 0.05. Each experiment was independently repeated at least twice, and similar results were obtained. The statistical analysis and graphing were performed using GraphPad Prism 9 software.Quantitative results are presented as mean ± standard deviation (SD) from a minimum of three samples (


Our results challenge the stem cell theory in hematology and EC-derived mast cells may contribute to the pathogenesis of postnatal mast cell disorders.Recent advances in developmental immunology have revealed a hematopoietic stem cell (HSC)-independent origin for various innate immune lineages, including mast cells (MCs). It is now established that adult bone marrow (BM) long-term HSCs do not regenerate MCs but, instead, the physiological production of MCs starts before the emergence of HSCs in the aorta-gonad-mesonephros (AGM) region and is mostly completed before birth. However, while the AGM region represents a major site of MC generation during ontogeny, whether the first emerging HSCs in the AGM or fetal liver (FL) possess the potential to regenerate MCs is unknown. Here, we combined three fate-mapping mouse models with detailed HSC transplantation assays to determine the potential of AGM and FL HSCs to produce MCs. We show that HSCs from E11.5 AGM and E12.5 FL efficiently repopulated MCs in recipients. In stark contrast, HSCs from ≥E14.5 FL failed to reconstitute MCs. An Endothelial (EC) fate-mapping study confirmed the EC origin of the majority of MCs. Additionally, our HSC-labeling showed that HSCs do not produce MCs in a physiological setting. Hence, although most MCs are generated and maintained  Howev culture , 8, post culture . These copoiesis , 9. Althh hosts , 7, 9. Hh hosts , suggest+ ECs in the extraembryonic yolk sac (YS) possibly through EMP. Subsequently, the second wave of MCs is derived from the ECs in the aorta-gonad-mesonephros E10.5 (AGM) region , c-Kit+β7-integrin+ cells in the E12.5 FL  neonate, we show that the first emerging HSCs in the developing AGM region and E12.5 FL HSCs retain the potential to produce MCs. In clear contrast, FL HSCs at E14.5 and later including adult BM HSCs have almost no contribution to producing MCs in both transplantation and fate-mapping assays. Thus, our studies underpin the divergence of MC lineage and HSC in the early embryonic period and highlight the unique property of the first emerging HSC.In this study, we asked whether strictly defined fetal HSCs could give rise to MCs in the peritoneal cavity (PerC) and skin under physiological (fate-mapping) and transplantation settings. Using complementary EC- and HSC-fate mapping assays in addition to highly purified HSC transplantation into NOD/SCID/IL2γcZsGreen-ERT2Cre, B6.RosaCAG-LSL-tdTomato (RosaLSL-Tom), Gt(ROSA)26Sortm4Luo/J (RosamT/mG), B6.IgHa mice, and NOD.Cg-PrkdcscidIL2rgtm1 (NSG) mice were purchased from Jackson Laboratory. B6-Tg(Cdh5-cre/ERT2) Mice were purchased from Taconic (No:13073). Enhanced green fluorescent protein (pCx-eGFP)   and red Cx-eGFP)  transgenant mice  were pro-c-KithiSca1hi(LSK)CD150+CD48- . As we did not use CD135 Ab, we defined adult BM MPP as LSKCD150-CD48+ cells, which is corresponding to MPP3/4 or MPPLy+MPPG/M elsewhere (-CD48+ cells or KSL CD201-(EPCR)-CD48+ cells were referred to as MPP in the FL (Embryos were harvested and dissected in YS and AGM regions. Each tissue was dissected and digested in 0.25% collagenase (Stemcell Technologies) for 15–30 minutes at 37°C, followed by cell dissociation buffer (Life Technologies) to stop the reaction and make a single cell suspension. The fetal liver was dissociated and applied to mononuclear cell gradient centrifuge separation with Histopaque 1083 (Sigma) to obtain blood mononuclear cells (MNCs). Cell number was counted by trypan blue, and cells were stained with antibodies (Listed below) based on lsewhere . FL MPP n the FL . All ant5 BM rescue cells from 8 wks old pCx-eGFP mice. In separate experiments, B6.IgHa mice received 1.5 x 106 cells from unsorted fetal liver or adult BM. LT-HSCs were sorted on Emory Pediatric/Winship Flow Cytometry Core or Stanford Shared FACS Facility BD FACSAria II instruments.Day 1-3 NSG neonatal or adult mice (expressing CD45.1) were used as recipients and were irradiated (neonate: 150 or adult: 200 rad) before transplantation. Donor cells (CD45.2) were injected into the facial (neonate) or tail (adult) vein. Blood was taken periodically from the 4th week after transplantation and donor cell types were examined by flow cytometry . Successful MC repopulation was defined as more than 0.1% donor-derived MCs among the total host MCs in PerC. Transplant experiments using RFP+ donor cells were performed as described . BrieflyZsGreen-ERT2Cre : RosaLSL-Tom (iFgd5) and Cdh5ERT2Cre : RosaLSL-Tom (iCdh5) mice were created by crossing RosaLSL-Tom with the respective Cre-bearing animals. Runx1MER/Cre/MER and RosamT/mG were crossed as described previously to create iRunx mice. Details of labeling were performed , CD45.1 (A20), CD19 (1D3), CD4 (GK1.5), CD8 (53-6.7), CD3 (145-2C11), IgM (RMM-1), CD5 (53-7.3), CD11b (M1/70), Gr-1 (RB6-8C5). CD144 (clone:11D4), CD45 (30-F11), NK1.1 (PK136), CD41 (MWReg.30), c-Kit (2B8), Ter119 (TER-119), CD201 (1560), Sca-1 (D7), CD48 (HM48-1), CD150 (TC15-12F12.2), β7-integrin (FIB27), FcERI (MAR-1), F4/80 (BM8), CD127 (A7R34).+CD48- LSK cells) or multipotent progenitors  were sorted from CD45.2+ C57BL/6 mice and transplanted into lethally irradiated F1 mice (CD45.2+CD45.1+) with competitor cells (CD45.1+), and MCs engraftment in the recipient PerC was examined. As previously reported, we confirmed the lack of MC repopulation . Based on this, we decided to evaluate the feasibility of neonatal NSG recipients for the MC repopulation assay, by transplanting E12.5 FL c-Kit+β7-integrin+ cells into NSG neonates instead of in-utero transplantation because NSG neonates still maintain the fetal environment that accepts immature fetal cells for the physiological hemato-immunological development compared to adult recipients  c-Kit+EPCR++  and Fgd5+ HSCs, using iCdh5 (Cdh5ERT2Cre : RosaLSL-Tom) and iFgd5 (Fgd5ZsGreen-ERT2Cre : RosaLSL-Tom) mice, respectively. Cdh5 encodes VE-cadherin, specifically expressed in the ECs, and Fgd5 is exclusively expressed in HSCs (+ of a defined cell population divided by the %Tom+ of HSCs in the same host (ZsGreen-ERT2Cre mouse) showed the brightest signal in the E12.5 FL HSC compartment defined by CD48-EPCR++LSK, but also dim positive signal in CD48+EPCR- MPP population  mice. TAM injection into iRunx1 mice at E8.0 marked only MCs in adult mice without labeling other lineages in PerC  , 24, andOne of our intriguing findings is that host MC eradication is not required to detect the MC potential of donor cells. As + MCps. In our data, whether β7-integrin+ MCps in the FL are derived through EMP or HSCs remains unclear. E10.5-11.5 AGM contains a variety of HSC-independent progenitors capable of regenerating innate and adaptive immune cells, including macrophages, MCs, B-1 B-cells , NIH R01AI123126 (EG), and Lowance Center for Human Immunology (EG).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ceacam1 deletion developed plaque-like aortic lesions even on C57BL/6J background in the presence of increased endothelial cell permeability and insulin resistance. Loss of endothelial Ceacam1 gene caused endothelial dysfunction and reduced vascular integrity without affecting systemic insulin sensitivity. Because endothelial cell injury precedes atherosclerosis, we herein investigated whether the loss of endothelial Ceacam1 initiates atheroma formation in the absence of insulin resistance. (2) Methods: Endothelial cell-specific Ceacam1 null mice on −/−C57BL/6J.Ldlr background (−/−VECadCre+Cc1fl/flLdlr) were fed an atherogenic diet for 3–5 months before metabolic, histopathological, and en-face analysis of aortae were compared to their control littermates. Sirius Red stain was also performed on liver sections to analyze hepatic fibrosis. (3) Results: These mice displayed insulin sensitivity without significant fat deposition on aortic walls despite hypercholesterolemia. They also displayed increased inflammation and fibrosis. Deleting Ceacam1 in endothelial cells caused hyperactivation of VEGFR2/Shc/NF-κB pathway with resultant transcriptional induction of NF-κB targets. These include IL-6 that activates STAT3 inflammatory pathways, in addition to endothelin-1 and PDGF-B profibrogenic effectors. It also induced the association between SHP2 phosphatase and VEGFR2, downregulating the Akt/eNOS pathway and reducing nitric oxide production, a characteristic feature of endothelial dysfunction. Similarly, hepatic inflammation and fibrosis developed in −/−VECadCre+Cc1fl/flLdlr mice without an increase in hepatic steatosis. (4) Conclusions: Deleting endothelial cell Ceacam1 caused hepatic and aortic inflammation and fibrosis with increased endothelial dysfunction and oxidative stress in the presence of hypercholesterolemia. However, this did not progress into frank atheroma formation. Because these mice remained insulin sensitive, the study provides an in vivo demonstration that insulin resistance plays a critical role in the pathogenesis of frank atherosclerosis. (1) Background: Mice with global  Togethe−/−VECadCre+Cc1fl/flLdlr mice was marked by higher LDL-C levels likely resulting from reduced clearance instead of synthesis. Despite the adverse effect of CEACAM1 deletion on endothelial cell integrity and increased cell permeability in −/−VECadCre+Cc1fl/flLdlr mice, consistent with previous reports ; ApoB100 and ApoB48 ; PCSK9 ; nitric oxide (NO) ; NAD/NADH ; Glutathione (GSH) ; 8-isoprostane ; TNFα ; IL-6 ; endothelin-1 ; prostaglandin E2 ; PDGF-B . Hepatic triacylglycerol was determined as previously described  and aort5 cells/well in 6-well plates and allowed to grow for 24 h before being serum-starved for 2 h in phenol red-free medium  supplemented with 25 mM HEPES and 0.1% BSA before being incubated in the presence or absence of 40 ng/mL VEGF-A  for 5 or 20 min for Western blot and media analysis, respectively. Primary heart endothelial cells were isolated from ketamine/xylazine-anesthetized 2-to-3–month-old mice, as previously described . BrieflyAs previously described , hearts 2.As in , aortae Following sedation with ketamine and xylazine (100/10 mg/kg), mice were fixed on a 15-cm cell-culture lid in a supine position. As previously described , the rigAs previously described , formaliTissues and cells were lysed and proteins analyzed by SDS-PAGE followed by immunoprobing with polyclonal antibodies  against: phospho-Akt (Ser473), Akt, phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, phospho-eNOS (Ser1177), eNOS, phospho-Smad2Ser465/467, Smad2, phospho-Smad3 (Ser423/425), Smad3, phospho-Stat3 (Tyr705), Stat3, phospho-NF-ϏB (Ser536), NF-ϏB, phospho-PKCζ (phospho-Thr410/403), PKCζ, phospho-VEGFR2 (Try1175), VEGF-R2, VCAM1 and Tubulin. A custom-made rabbit polyclonal antibody (Ab 3759) were used against mouse CEACAM1 extracellular domain and phospho-CEACAM1 (α-pCC1) . Blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody  and proteins were visualized using ECL (Amersham). Polyclonal antibodies against SHP2, and Shc  were used in co-immunoprecipitation experiments. These membranes were incubated with light chain specific horseradish peroxidase-conjugated IgG monoclonal mouse anti-rabbit  to prevent detection of the antibody heavy chain. Total RNA was isolated from aortae with RNeasy fibrous tissue kit . cDNA was synthesized by iScript cDNA Synthesis Kit , using 1 μg of total RNA and oligodT primers. qRT-PCR was performed using Fast SYBR Green Master Mix by the ABI StepOnePlus Real-Time PCR System , as described . All prip < 0.05 was considered statistically significant.Data were analyzed by one-way analysis of variance (ANOVA) with Tukey’s for multiple comparisons using GraphPad Prism 7 software.


Currently, little is known about the mechanism that DNA vaccines employ to induce adaptive immune responses. Prior studies have demonstrated that stimulator of interferon genes (STING) and conventional dendritic cells (cDCs) play critical roles in DNA vaccine induced antibody and T cell responses. STING activation by double stranded (dsDNA) sensing proteins initiate the production of type I interferon (IFN),but the DC-intrinsic effect of STING signaling is still unclear. Here, we investigated the role of STING within cDCs on DNA vaccine induction of antibody and T cell responses. STING knockout (-/-STING) and conditional knockout mice that lack STING in cDCs (cDC STING cKO), were immunized intramuscularly with a DNA vaccine that expressed influenza A nucleoprotein (pNP). Both -/-STING and cDC STING cKO mice had significantly lower type I T helper (Th1) type antibody (anti-NP IgG2C) responses and lower frequencies of Th1 associated T cells (NP-specific IFN-γ+CD4+ T cells) post-immunization than wild type (WT) and cDC STING littermate control mice. In contrast, all mice had similar Th2-type NP-specific (IgG1) antibody titers. -/-STING mice developed significantly lower polyfunctional CD8+ T cells than WT, cDC STING cKO and cDC STING littermate control mice. These findings suggest that STING within cDCs mediates DNA vaccine induction of type I T helper responses including IFN-γ+CD4+ T cells, and Th1-type IgG2C antibody responses. The induction of CD8+ effector cell responses also require STING, but not within cDCs. These findings are the first to show that STING is required within cDCs to mediate DNA vaccine induced Th1 immune responses and provide new insight into the mechanism whereby DNA vaccines induce Th1 responses.DNA vaccines elicit antibody, T helper cell responses and CD8 However, these studies showed that TLR9, and its downstream adaptor protein, myeloid differentiation primary response gene 88 (MyD88) were not required to mediate DNA vaccine immunogenicity (DNA vaccines induce robust antibody (Ab) and T cell responses in small animals, but their immunogenicity in humans has generally been much lower and often below the threshold needed for protection –5. To imgenicity .cyclic GMP-AMP synthase (cGAS), binds to dsDNA within the cytoplasm and induces the production of type I interferon (IFN) responses via stimulator of interferon genes (STING). However, studies to determine if cGAS is a major regulator of DNA vaccine immunogenicity, showed that it is dispensable for DNA vaccine induced immune responses, whereas STING (stimulator of interferon genes) and other proteins downstream of STING including TANK-binding kinase 1 (TBK1) and interferon regulatory factor 7 (IRF7) were required (Double stranded DNA (dsDNA) sensing pathways have been hypothesized to be key mediators of DNA vaccine immunogenicity. Specifically, required .STING governs downstream DNA vaccine immunogenicity including the induction Th1 and Th2-biased Ab and polyfunctional CD8+ T cell responses. Previous studies showed that conventional dendritic cells (cDCs) play a central role in coordinating DNA vaccine induction of Ab and T cell responses . fl/flSTING mice were made by a commercial service  using a Cas9/sgRNA plasmid construct. In brief, a targeting vector was designed with a Neo cassette, flanked by Frt sites, and STING Exon 6 flanked by LoxP sites, was introduced into a B6-derived embryonic stem cells (ES). Targeted ES cells were introduced into host embryos, and cell embryos were surgically transferred into pseudo-pregnant (surrogate) mothers resulting in F0 heterozygous floxed mice on the B6 background. Chimeric mice were crossed to B6J (JAX #000664) for 6 generations. fl/flSTING mice were bred to zbtb46Cre mice (JAX #028538) to generate Crezbtb46 x f/fl/STINGmice (cDC STING cKO). The Crezbtb46 x f/flSTING genotyping was carried out using tail snips followed by PCR . Red blood cells were lysed using an RBC lysis buffer (Thermofisher cat. no. 00-4300-54). Splenocytes were stained with Live/Dead fixable aqua dead (ThermoFisher Scientic cat. no. L34957), PerCP-conjugated anti-mouse CD11c (Biolegend cat. no. 117326), BV605-conjugated anti-mouse CD19 (Biolegend cat. no. 115540), BV605-conjugated anti-mouse CD3 (Biolegend cat. no. 100237), APC-conjugated anti-mouse Ly-6G (Biolegend cat. no. 127614), PE-conjugated anti-mouse NK1.1 (Biolegend cat. no. 108708), APC-Cy7-conjugated anti-mouse CD11c, and Pacific blue-conjugated anti-mouse MHC-II (Biolegend cat. no. 107620). A BD Biosciences FACS ARIA III cell sorter was used to isolate cDC (CD11c+MHCIIhi), and B and T cells (CD19+CD3+). RNA was isolated (Qiagen RNeasy kit cat. no. 74004) and then converted to cDNA (ThermoFisher cat. no. 4368814). STING expression in each cell type was measured via qPCR using primer F: GGGAGCCGAAGACTGTACAT; primer R: CGCTGTTGGAAAAACCCGA. All mice were maintained by the department of animal welfare (OAW) according to institutional Animal Care and Use Committee (IACUC)- approved protocols.C57BL/6J (WT), toid DCs , and as A DNA vaccine that encodes a codon-optimized full-length nucleoprotein from A/Puerto Rico/8/1934 (pNP) was used to immunize mice. Construction of the plasmid, pNP, employed for these studies is previously described . Briefly1, and IgG2C antibody titers, and sera samples were stored at -80°C until the time of assay. Microtiter plates were coated at 1μg/ml of recombinant NP  overnight at 4°C. Anti-NP IgG, IgG1 and IgG2C concentrations (μg/ml) in sera were calculated using purified mouse IgG (Southern Biotech cat. no. 0107-01), IgG1 (Southern Biotech cat. no. 0102-01), or IgG2C (Southern Biotech cat. no. 1078-01) to produce a standard curve. Purified mouse antibodies were detected using 1μg/ml of goat anti-mouse IgG-HRP (Southern Biotech cat. no. 1030-05), IgG1-HRP (Southern Biotech cat. no. 1070-05) or IgG2C–HRP (Sothern Biotech cat. no. 1078-05), respectively. O-phenylenediamine dihydrochloride (OPD) substrate tablets (ThermoFisher cat. no. 34006) were used to observe HRP induced color changes. The HRP substrate reaction was terminated using 2N sulfuric acid after 2 minutes. Absorbance was measured at 450nm and concentrations of antibody were calculated using each plate’s internal standard curve. Sera dilutions were optimized for each experiment and timepoint to ensure absorbance reading fell within each plates internal standard curve. Data are representative of at least three independently preformed studies (n=3-9 mice/genotype/study).Sera were collected at 14-, 21- and 28-days post-vaccination to measure NP specific IgG, IgG+ (+ T cells (366-374 (H-2k(b)) and PE-conjugated- NP311-325 (I-A(b)) tetramers, respectively. Tetramers were obtained from the NIH tetramer core facility . Mice were sacrificed, spleens collected and single cell suspensions were generated for each mouse as outlined above. A total of 1x106 splenocytes were stained and analyzed via flow cytometry (BD bioscience LSRII). To measure the frequencies of Ag-specific CD4+ T cells, splenocytes were stained with the following antibodies: Live/Dead fixable aqua dead (ThermoFisher Scientic cat.no. L34957), BUV395-conjugated anti-mouse CD4 (BD cat. no. 565975), PerCP5.5-conjugated anti-mouse CD3 (Biolegend cat. no. 1002180), BV711-conjugated anti-mouse CD8 (Biolegend cat. no. 100748), PE-Cy7-conjugated anti-mouse CD44 (BD cat. no. 560569) and PE-conjugated- NP311-325. To measure the frequencies of Ag-specific CD8+ cells, splenocytes were stained as follows: Live/Dead fixable aqua dead (ThermoFisher Scientic cat. no. L34957), PerCP5.5-conjugated anti-mouse CD3 (Biolegend cat. no. 1002180), BUV395-conjugated anti-mouse CD4 (BD cat. no. 565975), BV711-conjugated anti-mouse CD8 (Biolegend cat. no. 100748) and PE-Cy7-conjugated anti-mouse CD44 (BD cat. no. 560569).Antigen (Ag)-specific CD86 cells were plated in duplicate wells in a 96 well plate and rested for 24 hours (hrs.) in full RPMI media (details above) at 37°C. To evaluate CD8+ T cell polyfunctionality cells were either stimulated with 1μg/ml NP366-374, or media only (unstimulated). After one hour, 1x brefeldin A (Biolegend cat. no. 420601) and FITC-conjugated CD107a/GranzymeB (Biolegned cat. no. 121606) were added to the wells and incubated overnight (~16hrs). The following day, cells were stained for IL-2 (Biolegend cat. no. 503808), IFN-γ (Biolegend cat. no. 505818), FITC-conjugated anti-human/mouse Granzyme B (Biolegend cat. no. 515403) and TNF-α (Biolegend cat.no 506308) using BD’s fixation/permeabilization solution kit (cat.no. 554714) and the manufacture’s protocol. T cells were first gated using BV605-conjugated anti-mouse CD3 (Biolegend cat. no. 100237). CD8+ T cells were gated using BV711-conjugated anti-mouse CD8 (Biolegend cat. no. 100748). The frequencies of CD8+ T cells expressing IL-2, TNF-α, IFN-γ and/or cytolytic markers CD107a/Granzyme B were then measured by flow cytometry (BD Biosciences LSRII) and Boolean gating  with 1μg/ml NP311-325. Supernatants were collected and analyzed for IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17a via flow cytometry (BD Bioscience LSRII) using BD’s mouse Th1/Th2/Th17 Cytometric Bead Array kit (cat. no. 560485) using the manufacture’s protocol.To measure Th1 and Th2 cytokine expression, 1x10+ T cells mouse splenocytes were isolated as outlined above. A total of 1x106 cells were pated in duplicate in a 96 well plate and rested for 24hrs. The cells were then stimulated with 1μg/ml NP311-325, the immunodominant NP peptide for C57BL/6 CD4+ T cells or remained in media only. After one hour, 1x brefeldin A (Biolegend cat. no. 420601) was added to wells and the cells were incubated overnight (~16hrs). The following day splenocytes were stained with PerCP5.5-conjugated anti-mouse CD3 (Biolegend cat. no. 1002180), BUV395-conjugated anti-mouse CD4 (BD cat. no. 565975) and IFN-γ (Biolegend cat. no. 505818) using BD’s fixation/permeabilization solution kit (cat. no. 557414) and the manufacture’s protocol. The frequencies of CD4+ T cells expressing IFN-γ was measured by flow cytometry (DB Bioscience LRSII).To measure IFN-γ expressing CD46 splenocytes in 100μl of media were plated in NP-coated plates in duplicate and then incubated overnight (~16hrs) at 37°C. The following day, the cells were discarded and the wells were washed 5 times with PBS. Following the final wash, 100μl of anti-mouse IgG-HRP, IgG1-HRP, or IgG2C–HRP diluted at 1:2000 in PBS were added to each well. Spots were developed using BD ELISPOT AEC substrate set (BD cat. no. 551951) and then visualized using a CTL imager (Cellular Technologies) and were enumerated using an ImageJ cell counter.Mouse splenocytes were used to enumerate NP-specific antibody secreting cells 21 days post-vaccination. ELISPOT plates (Millipore Sigma cat. no. S2EM004M99) were coated with 10μg/ml recombinant NP  in PBS and then incubated overnight at 4°C. Mouse splenocytes were processed as outlined above and then 3x10Cre x STINGfl/flzbtb46 (cDC STING cKO) and cDC STING LitC mice were diluted 1:5 while sera from -/-STING mice were diluted 1:2. Levels of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10 and IL-17a in diluted sera were then measured by flow cytometry (BD Bioscience LSRII) using BS’s mouse Th1/Th2/Th17 Cytometric Bead Array kit (cat. no. 560485) following the manufacture’s protocol. IFN beta (IFN-β) was measured in sera diluted 1:2 prior to vaccination and 6hrs post-vaccination using mouse IFN-β ELISA kit (PBL Assays cat. no. 42410-1).Sera were collected from mice prior to vaccination (baseline), and at 6hrs and 24hrs post-vaccination. Sera from WT, All data are represented as the mean of individual mice ± SD. Statistical analyses were performed using one-way ANOVA followed by a turkey post-test and Student’s t test using Graphpad Prism 7.cGAS and/or STING play a role in the ability of DNA vaccines to induce antibody (Ab) responses, WT, -/-cGAS and -/-STING mice were vaccinated by intramuscular delivery and electroporation (IM/EP) with 10μg of a codon optimized DNA vaccine expressing influenza A nucleoprotein (pNP). Sera were collected 14-, 21- and 28-days post-vaccination to analyze induction of NP-specific IgG Ab responses. -/-STING mice generated significantly lower anti-NP IgG Ab concentrations at 21- and 28-days post-vaccination compared to WT and -/-cGAS mice  and Th2 (IgG1) Ab responses were impacted by STING or cGAS. Analysis of IgG2C and IgG1 titers, and IgG2C/IgG1 ratios in vaccinated WT, -/-cGAS and -/-STING mice showed that -/-STING mice developed significantly lower Th1 associated anti-NP IgG2C  then flow cytometry , but not in plasmacytoid DCs  mice  were vaccinated by IM/EP delivery of 10μg of pNP. Mice were sacrificed 21 days post-vaccination and splenocytes were isolated to measure Ag-specific CD8+ T cell responses by NP366-374 tetramer staining  are important for the generation of DNA vaccine induced Ab and CD8esponses –14, thertoid DCs . WT, STI+ and CD8+ T cell effector functions , and at 14-, 21- and 28- days post-vaccination. -/-STING and cDC STING cKO mice developed significantly lower NP-specific IgG  than the control mice (STING within cDCs is required to induce Th1 (IgG2C) type Ab responses. To determine if the lower IgG2C Ab responses observed in -/-STING and cDC STING cKO mice was due to a reduction in the number of Ab-secreting B cells (ASCs), the number of NP-specific IgG  and Th2 (IL-4) cytokines were analyzed in the supernatants using a flow-based cytokine detection kit  Ab responses, but neither STING nor cGAS is required to induce Ag-specific IgG1 (Th2) Ab responses  vaccinated -/-STING mice had significantly lower IgG2C concentrations compared to control mice, but generated normal concentrations of IgG1. The lack of significant impact of STING on the total IgG responses following GG delivery of DNA vaccines is likely due to GG inducing predominantly IgG1 responses whereas IM induced primarily IgG2 antibody responses , our studies surprisingly showed that STING is not required within cDCs for DNA vaccine induction of polyfunctional CD8+ T cell responses. The discrepancy between our findings that show STING is required for the induction of polyfunctional CD8+ T cell responses, but not within cDCs suggest that STING within other cell types likely mediates the ability of DNA vaccines to induce polyfunctional CD8+ T cells. In support of this, we found that the production of pro-inflammatory cytokines  after DNA vaccination was significantly diminished in -/-STING mice but similar in control mice and mice lacking STING in cDCs  produced by cDCs could adversely impact Th1 T cell polarization  Ab responses and Th1 IFN-γ producing CD4+ T cells. Taken together, these results provide new insight into the mechanisms whereby DNA vaccines induce Th1 responses. We propose that upon DNA vaccination, STING within cells other than cDCs triggers the production of pro-inflammatory cytokines  that are critical for the induction of polyfunctional CD8+ T cell responses. Alternatively, STING may facilitate apoptosis in DNA transfected cell and cross-presentation to CD8+ T cells by cDCs. Indeed, cross-presentation has been shown to be a mechanism whereby intramuscular delivered DNA vaccines induce CD8+ T cell responses  grant R01AI442257 and Washington Research Fund “A diametric genetic adjuvant strategy to maximize immunogenicity of gene gun vaccines”.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The discovery of antineutrophil cytoplasmic antibodies (ANCAs) in ANCA-associated vasculitis (AAV) marked a breakthrough in diagnostics . Subsequp < 0.0001, Supplementary Table 2). In contrast, a less robust association was observed for cANCA IIF and PR3-ANCA titers . Among other laboratory parameters, cANCA IIF correlated with C-reactive protein (CRP) elevation in PR3-ANCA GN  present in 7/26 (26.9%) of patients with MPO-ANCA GN  were MPO-ANCA GN and 27/53 (50.9%) were PR3-ANCA GN (Supplementary Table 1). In the subgroup of MPO-ANCA GN, pANCA IIF and respective MPO-ANCA titers measured by immunoassays correlated among each other  in ANCA GN. Nevertheless, our finding that pANCA IIF specifically correlated with arteritis is especially relevant because arteritis determines renal prognosis in ANCA GN , 9. MoreSupplementary file1 (PDF 103 KB)Below is the link to the electronic supplementary material.


In summary, CenH3 plays both pro-viral and restriction functions during tombusvirus replication. This is a surprising novel role for a nuclear histone variant in cytosolic RNA virus replication.Tombusviruses, similar to other (+)RNA viruses, exploit the host cells by co-opting numerous host components and rewiring cellular pathways to build extensive virus-induced replication organelles (VROs) in the cytosol of the infected cells. Most molecular resources are suboptimal in susceptible cells and therefore, tomato bushy stunt virus (TBSV) drives intensive remodeling and subversion of many cellular processes. The authors discovered that the nuclear centromeric CenH3 histone variant  plays a major role in tombusvirus replication in plants and in the yeast model host. We find that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. CenH3 binds to the viral RNA and acts as an RNA chaperone. Although these data support a restriction role of CenH3 in tombusvirus replication, we demonstrate that by partially sequestering CenH3 into VROs, TBSV indirectly alters selective gene expression of the host, leading to more abundant protein pool. This in turn helps TBSV to subvert pro-viral host factors into replication. We show this through the example of hypoxia factors, glycolytic and fermentation enzymes, which are exploited more efficiently by tombusviruses to produce abundant ATP locally within the VROs in infected cells. Altogether, we propose that subversion of CenH3/Cse4p from the nucleus into cytosolic VROs facilitates transcriptional changes in the cells, which ultimately leads to more efficient ATP generation  Biogenesis of viral replication organelles (VROs) containing membrane-bound viral replicase complexes is critical for replication of (+)RNA viruses. To achieve efficient VRO formation, RNA viruses reprogram host gene transcription through incompletely characterized processes. Using tombusviruses and the model host yeast, the authors discovered that the centromeric Histone 3 variant (CenH3 or CENP-A) is recruited into tombusvirus VROs. Tombusvirus replication protein and the viral RNA interact with CenH3 leading to partial sequestration of CenH3 into cytosolic VROs from the nucleus. The subversion of CenH3 leads to selective re-programming of gene expression of the host, resulting in increased production of glycolytic and fermentation enzymes. These enzymes are exploited by tombusviruses to produce ATP locally within the VROs to achieve robust replication. Positive-strand (+)RNA viruses use the abundant resources of the host cells to build large viral replication compartments/organelles (VROs), which support their replication in a membranous protective microenvironment –9. Tomatpol, which are essential for virus replication [TBSV codes for two viral replication proteins, termed p33 and p92lication ,14. Theslication ,15,16. TUsing a library of temperature-sensitive mutants of yeast (a model host for TBSV), we identified Cse4 centromeric H3 protein variant as a restriction factor for TBSV replication . Based oThe nucleus of a eukaryotic cell is full of nucleic acid binding proteins, which can potentially be used by the host to fight viral infections. Indeed, many well-characterized nuclear proteins are shuttling in and out of the nucleus, making possible that these cellular proteins could also function in the cytosol ,25–28. Tin vitro. Based on knockdown, mutation or over-expression experiments, we showed that CenH3/Cse4p acts as a cellular restriction factor against TBSV replication. CenH3/Cse4p was found to be partially re-targeted from the nucleus into the cytosolic VROs. In vitro works showed that CenH3/Cse4p binds to the viral RNA and acts as an RNA chaperone. Co-purification and pulldown experiments demonstrated interaction between CenH3/Cse4p and the viral p33 replication protein. However, subsequent analysis showed that TBSV hijacks CenH3/Cse4p into VROs to sequester this histone 3 variant away from the nucleus, which affects the expression of a set of host genes. These genes include pro-tombusviral host factors. We chose to further test the role of CenH3/Cse4p in regulating the glycolytic and fermentation pathways, which are co-opted by tombusviruses. These pathways are usurped by TBSV to provide plentiful ATP within VROs to fuel the activities of additional co-opted host proteins, such as Hsp70, the ESCRT-associated Vps4 AAA ATPase and DEAD-box helicases needed for robust viral replication [In this paper, we studied the role of CenH3 in TBSV replication in yeast, plants and lication –46. Altocse4-1 [CSE4 (To explore the possible role of Cse4p (CenH3) in tombusvirus replication, we used the temperature-sensitive haploid yeast strain with a mutated single copy of cse4-1 ,47. Partcse4-1 ,49 repli-1 [CSE4 . This mi-1 [CSE4 . We also-1 [CSE4 . These fArabidopsis CenH3 in yeast replicating TBSV. We observed ~3-fold inhibition of TBSV repRNA accumulation in comparison with the control yeast  approach to deplete CenH3 level in thamiana  renderedthamiana .N. benthamiana followed by inoculation of the same leaves with two peroxisome-associated tombusviruses  and the mitochondrial membrane-associated CIRV. Northern blot analysis revealed ~8-to-10-fold reduction in TBSV, CNV and CIRV gRNA accumulation in the inoculated leaves  nuclear marker protein , we co-expressed TBSV p33-BFP replication protein and the GFP-tagged  protein . Confocating CNV . Interesting CNV . In contting CNV . The expting CNV , albeit  protein . The exp protein . Based opol and the repRNA to induce VRO formation [We also performed subcellular localization experiments in WT yeast cells expressing YFP-Cse4 and co-expressing CFP-p33 together with p92ormation . Time poin vitro, we reconstituted the tombusvirus replicase by using (+)repRNA transcripts and purified recombinant TBSV p33 and p92pol replication proteins in cell-free extracts (CFE) prepared from WT yeast ssRNA products in CFE with the highest amount of Cse4p in comparison with the RNA replication supported by WT CFE in the presence of GST control RNA template in vitro . Templatlication –56 compelication , we testin vitro . The abiin vitro RNA binding experiments suggested that the N-terminal region in Cse4p is not required, whereas the C-terminal HFD domain is critical for Cse4p to bind to the TBSV repRNA  and the C-terminal Histone-fold domain (HFD) containing the centromere targeting domain (CATD) . ExpressV repRNA . RNA strV repRNA , whereasV repRNA . Based o6-tagged p33 replication protein. After detergent-solubilization of the membrane-fraction of yeast, the Flag-CenH3 was immobilized to the Flag-column. Western blot analysis of the eluted proteins from the column revealed the co-purified His6-p33  and TEF1 (eEF1A) genes has not changed in cse4-1 yeast at the semi-permissive temperature  DEAD-box helicase  and Pdc1 (VIGS-based knockdown of CenH3 level in helicase . Moreoveand Pdc1 . TBSV inand Pdc1 . CenH3 eand Pdc1 . On the and Pdc1 . Thus si2 level due to plant submersion in water) by ERF-VII transcription factors in Arabidopsis plants, we measured the expression levels of RAP2.12, HRE1 and HRE2, which are known hypoxia transcription factors [N. benthamiana led to the highest expression levels of RAP2.12, HRE1 and HRE2, which showed ~4-to-9-fold increase (Because the fermentation enzymes (Adh1 and Pdc1) are induced during hypoxia  by the noncanonical function of CenH3/Cse4.in situ within VROs, which depends on the availability of co-opted glycolytic and fermentation enzymes [N. benthamiana leaves. The ATeam domain of the fusion protein can measure ATP level due to a conformational change in the enhanced ε subunit of the bacterial F0F1-ATP synthase upon ATP binding [N. benthamiana plants RNA viruses, exploit the host cells by co-opting the cellular translation machinery, subverting host proteins, intracellular membranes, metabolites and energy to build virus-induced extensive VROs in infected cells. It seems, however, that the molecular resources available in susceptible cells to support robust TBSV replication are suboptimal at the start of viral replication. Therefore, TBSV drives intensive remodeling and subversion of many cellular processes ,69,70. TOur gene and protein interaction network studies based on a dozen genome- and proteome-wide screens, which previously identified host components affecting TBSV replication and recombination or interactions with host components, revealed that Cse4 H3 histone variant is one of the highest connected nodes in the network . Becausein vitro via its HFD domain. We also showed RNA chaperone activity for Cse4 in vitro, which activity might contribute to the inhibitory function of Cse4 via unwinding critical cis-acting elements in the viral (+)RNA. We also documented interaction of CenH3/Cse4 with the p33 replication protein, which completely overlaps with the N-terminal region of the p92 RdRp. All these results agree with a proposed antiviral activity of the CenH3/Cse4. Accordingly, we have shown in this paper that the purified recombinant Cse4p is indeed inhibitory to TBSV replication in an in vitro replicase reconstitution assay.How can a nuclear, DNA-binding histone variant be a restriction factor for a cytosolic RNA virus? Subcellular localization studies confirmed that CenH3/Cse4 is partially relocalized to the cytosol, namely into the large VROs, during TBSV or CIRV replication. Moreover, CenH3/Cse4 binds to the viral RNA in vitro were not consistent with the idea that the canonical function of CenH3/Cse4 is exploited by the host to fight off tombusvirus infection. Moreover, tombusviruses replicate in mature plant leaf and root cells, which are not going through cell division and chromosomal segregation. An interesting noncanonical function of Cse4 is to replace histones bound to the chromosome in many places, including transcription factor hotspots [In situ production of ATP within VROs is essential for robust TBSV replication as shown previously [N. benthamiana and yeast. Albeit CenH3 acts as a restriction factor of TBSV replication in N. benthamiana and yeast, one can imagine that CenH3 might act as pro-viral in other hosts. However, demonstration of this scenario requires future studies.Our initial results in yeast, plants and hotspots –36. In thotspots –35. Becaeviously ,67. On tHijacking and regulating CenH3 function might be conducted by other viruses as well. For example, Hepatitis B virus x protein (HBx) induces hepatocellular carcinoma (HCC) by inducing the over-expression of CENP-A (CenH3) protein . The hepCenH3/Cse4 histone variant plays a major role in tombusvirus replication in plants and in yeast model host. The emerging theme from our current studies is that sequestration of CenH3/Cse4 from the nucleus into the cytosolic VROs by tombusviruses requires a balancing act between restriction and pro-viral activities. Robust TBSV replication depends on the virus’ ability to reprogram host gene transcription, in which CenH3/Cse4 plays a mostly unexplored role. Subversion of CenH3/Cse4 is a “double-edge sword”: possibly advantageous for TBSV under given conditions, but disadvantageous under over-expression conditions, when CenH3/Cse4 acts as a strong restriction factor. Our data indicate that the nuclear CenH3 histone variant plays surprising novel pro-viral and restriction functions during tombusvirus replication. Nevertheless, the results point at a new frontier in cytosolic tombusvirus—host interactions.Saccharomyces cerevisiae strains BY4741 and R1158 (wt) [- (synthetic complete medium without leucine and histidine) medium containing 2% glucose and 100 μM BCS (bathocuproinedisulfonic acid). Then, cells were centrifuged and washed thoroughly with clean SC-LH- supplemented with 2% glucose medium, to remove BCS, and pellets were re-suspended in the same medium containing 50 μM CuSO4 to induce viral protein expression and repRNA replication. Then, a set of cultures was placed at 32°C and the other set remained at 23°C and grown for additional 24 h, time after which, total RNA and protein was extracted. For CIRV repRNA accumulation cells were co-transformed with plasmids HpESC-GAL1-Hisp36/GAL10-DI-72 and LpESC-CUP1-Flag-CIRVp95 and grown as above with the exception that pellets were thoroughly washed with SC-LH- medium  and re-suspended in SC-LH- medium containing 2% galactose and 50 μM CuSO4. Total RNA and protein was extracted after 30 h of viral induction.For measuring TBSV repRNA accumulation, yeast strains BY4741 and cse4-1 were transformed with HpGBK-CUP1-Hisp-33/ADH1-DI-72 and LpGAD-CUP1-His-p92. Two sets of cultures per strain were grown at 23°C 12 h overnight in SC-LH- medium supplemented with 2% galactose and 100 μM BCS. Cells then were centrifuged and washed thoroughly with clean SC-ULH- medium supplemented with 2% galactose and resuspended in the same medium containing 50 μM CuSO4 to induce viral repRNA replication. Total RNA and protein were isolated after 24 h. The same method was used when expressing the Cse4 truncation mutants, but BY471 yeast cells were transformed with HpGBK-CUP1-Hisp-33/ADH1-DI-72, LpGAD-CUP1-His-p92 and either UpYES empty vector, UpYES-His-CSE4, UpYES-His-cse4ΔN50, UpYES-His-cse4ΔN80, UpYES-His-cse4ΔN129, UpYES-His-cse4ΔC60, UpYES-His-cse4ΔC100 or UpYES-His-Histone H3.For over-expression analysis, BY4741 cells were transformed with plasmids HpGBK-CUP1-Hisp-33/ADH1-DI-72, LpGAD-CUP1-His-p92 and either UpESC empty vector, UpESC-HisAtCENH3, or pGAL-myc-CSE4. Transformed cells were grown for 24 h in SC-ULHN. benthamiana was done as described previously [The virus-induced gene silencing (VIGS) in eviously ,75. AfteN. benthamiana leaves were co-infiltrated with Agrobacterium cultures containing pGD-p19 and either pGD empty vector, pGD-NbCENH3 or pGD-AtCENH3. In the experiment with CNV, plants were also infiltrated with Agrobacterium carrying pGD-CNV20KSTOP 24 h after the first agroinfiltration. In the experiment with TBSV and CIRV, plants were inoculated with crude sap inoculum 48 h after agroinfiltration. Samples were collected from CNV infiltrated leaves about 84 h after the second agroinfiltration. For TBSV and CIRV infection, samples were taken from inoculated leaves 48 h and 72 h post-virus inoculation, respectively. All samples were used for total RNA extraction and northern blot as described, to analyze the accumulation levels of these viruses [To over-express CenH3,  viruses .N. benthamiana epidermal cells, transgenic N. benthamiana (constitutively expressing H2B fused to RFP) leaves were agroinfiltrated with plasmids pGD-T33-BFP or pGD-C36-BFP (OD600 0.3), pGD-p19 (OD600 0.3) and pGD-GFP-NbCENH3 (OD600 0.3). Likewise, wild-type N. benthamiana leaves were agroinfiltrated with plasmids pGD-T33-BFP or pGD-C36-BFP (OD600 0.25), pGD-p19 (OD600 0.25), pGD-GFP-NbCENH3 (OD600 0.25) and either pGD-RFP-SKL (OD600 0.25) or pGD-Cox4-RFP (OD600 0.25). In the experiment with CNV, plants were also infiltrated with Agrobacterium carrying pGD-CNV20KSTOP (OD600 0.2). Plants were additionally inoculated with TBSV or CIRV virions. Live confocal images were obtained with an Olympus FV1000 microscope (Olympus America) 48 h for CNV and TBSV-infection and 72 h for CIRV- infection. BFP/Alexa 405, GFP/Alexa 488, and RFP were excited using 405 nm, 488 nm, or 543 nm lasers, respectively. Images were obtained sequentially and merged using Olympus FLUOVIEW 1.5 software [To observe the subcellular localization of CenH3 in software .- supplemented with 2% galactose and 100 μM BCS. Next morning, cells were centrifuged and washed (with sterile water) and pellets were re-suspended in the same medium supplemented with 2% galactose and 50 μM CuSO4 to induce the expression of the fluorescently-tagged proteins. Samples were collected at the time points given in the main figure and analyzed by confocal microscopy as previously described [For confocal microscopy assays in yeast, BY4741 cells were co-transformed with plasmids UpYES-GAL-YFP-CSE4, LpGAD and HpESC empty vectors or UpYES-GAL-YFP-CSE4, LpGAD-CUP1-His-p92 and HpESC-GAL1-CFP-p33/GAL1-DI72. Transformed yeast cell cultures were grown 12 h overnight in the proper SC-ULHescribed .For the mRNA detection in yeast, BY4741 and cse4-1 cells were grown for 12 h at 32°C. Total RNA was isolated and analyzed by gel electrophoresis in a 1.5% agarose gel to normalize total RNA levels in the samples. Then, the same amounts of total RNA were used for semi-quantitative RT-PCR reactions. cDNA was first obtained using MMLV reverse transcriptase (Lucigen) and oligo dT. The cDNA was then used to perform PCR to detect the expression levels of the following host factors with the following primer sets: #5992/#7136 for Cdc19; #7123/#7137 for Eno2; #6275/#6367 for Pgk1; #5621/#5604 for Pdc1; #4308/#7140 for Ded1; #2030/#7138 for Ssa1 and #7141/#7142 for Tef1.N. benthamiana leaf samples. VIGS was performed as described using the pTRV2-3’CENH3-3’ construct only. After 12 d of VIGS treatment, total RNA was isolated and used for semi-quantitative RT-PCR using the following primers: #6380/#6381 for CenH3; #7293/#7294 for Eno2; #7291/#7292 for Pgk1; #7289/#7290 for Pdc1; #7295/#7296 for RH20 (DDX3-like); #2534/#2535 for Hsp70-1 and #7297/#7298 for eEF1A.For mRNA detection in plant, a similar approach was performed by using wild-type and CenH3 knockdown N. benthamiana gene expression as follows: VIGS was performed as described using the pTRV2-3’CENH3-3’ construct only. After 11 d of VIGS treatment, plants were inoculated with TBSV virions or mock inoculated. Samples were collected 2 d post-inoculation from infected leaves and 4 d post-inoculation from systemically-infected leaves. Total RNA was isolated and used for qRT-PCR. First, primers were designed using Real Time qPCR Tool from Integrated DNA Technologies website . Second, MMLV reverse transcriptase (Lucigen) and Oligo-dT were used to obtain cDNA. Finally, the qPCR reactions were prepared using Applied Biosystem Power UP SYBR green master mix (Thermo Fisher Scientific) in a 96 well plate and the Eppendorf’s Mastercycler ep realplex instrument and primers #8217/#8218 for GapC1; #8219/#8220 for Pgk1; #8221/#8222 for CenH3; #8174/#8175 for Pdc1; #8176/#8177 for PK1 and #8178/#8179 for Tubulin β2 as the housekeeping gene control. qPCR conditions were selected following the Power Up SYBR green master mix user manual recommendations.Real-Time quantitative qRT-PCR was also used for the detection of N. benthamiana plants, leaves were co-agroinfiltrated with plasmids pGD-p33-ATeamYEMK, pGD-p92, pGD-DI-72 and pGD-p19 for CNV infection or plasmids pGD-p36-ATeamYEMK, pGD-p95, pGD-DI-72 and pGD-p19 for CIRV infection. Samples were analyzed in a confocal microscope 2 d post-agroinfiltration. FRET images were obtained by exciting cells with a 405-nm laser diode, and CFP and YFP (Venus) signals were detected at 480–500 nm and 515–615 nm wavelength ranges, respectively [N. benthamiana leaves were co-infiltrated with the above plasmid combinations for CNV and CIRV with the addition of pGD-NbCENH3 plasmid as well. Confocal FRET images were obtained as above, 2.5 d post-agroinfiltration.Intracellular ATP levels within VROs were visualized using the ATeam-based biosensor  by usingS1 Text(DOCX)Click here for additional data file.S1 Figpol replication proteins and the (+)repRNA show partial co-localization of YFP-Cse4 with CFP-p33 at 12 h, 16 h and 24 h after induction of protein expression. Images on the right show the nuclear distribution of YFP-Cse4 in the absence of viral components in WT yeast cells at the same time points.Confocal laser microscopy analyses in WT yeast cells co-expressing YFP-Cse4 together with CFP-p33, p92(TIF)Click here for additional data file.S2 Fig6-p33 replication protein and His6-Cse4 mutants with anti-His antibody. Note that levels of ΔN129 are very low, suggesting that the N-terminal region of Cse4 has a role in protein stability.  RNA gel mobility shift analysis shows that GST-Cse4, GST-ΔN50 and GST-ΔN80 efficiently bind to 32P-labeled (+)repRNA (C) or (-)repRNA (D) in vitro, whereas GST-ΔC100 show defective binding capability to both repRNAs. Purified GST-Cse4, GST-ΔN50, GST-ΔN80, GST-ΔC100 and GST were added in increasing concentrations  to the assays. The 32P-labeled ssRNA—protein complexes were visualized on nondenaturing 5% polyacrylamide gels.  RNA-strand separation assays. Left: Schematic representation of the RNA/RNA duplexes used in the assays. See details in 32P-labeled RNA products after the in vitro RNA-strand separation assay were analyzed on nondenaturing 5% polyacrylamide gels. Full length Cse4 and all mutants were unable to unwind a fully duplexed dsRNA (E), whereas WT Cse4 and the N-terminal deletion mutants unwound the partial dsRNA (F). Three independent repeats were performed for each experiment.(A) Schematic diagram showing the endpoints of the Cse4 deletion mutants used in this study. The proteins were named by the number of the last amino acid deleted. (B) Top: northern blot analysis shows a reduction in the accumulation of repRNA in cells expressing full length Cse4 (lanes 3–4) and the N-terminal deletion mutants ΔN50 (lanes 5–6) and ΔN80 (lanes 7–8) compared to the control samples (lanes 1–2). Expression of ΔN129, ΔC60, ΔC100 and Histone H3 did not affect TBSV repRNA accumulation (lanes 9–16). Middle: Northern blot with 18S ribosomal RNA specific probe was used as a loading control. Bottom: Western blot analyses of the level of His(TIF)Click here for additional data file.S3 Fig6-p33 with Flag-affinity purified A. thaliana Flag-CenH3 from membrane fraction of WT yeast. His6-p33 was detected with anti-His antibody. The negative control was His6-tagged AtCenH3 which was not co-purified from yeast extracts when using a Flag-affinity column (lane 2). Second panel: Western blot of purified Flag-AtCenH3 and Flag-p33 detected with anti-Flag antibody. Bottom panels: Western blot of His6- or Flag-tagged proteins in total yeast extracts. (B) Co-purification of yeast Cse4p with the viral replicase complex. First panels: western blot analysis of co-purified Myc-tagged Cse4 (lane 2) and Cse4 C-terminal domain  with Flag-affinity purified Flag-p33 from WT yeast membrane fraction. Myc-Cse4 and Myc-Cse4-CTD were detected with anti-Myc antibody. The negative control was His6-tagged p33 (lane 1). Second panels: Western blots of purified Flag-p33 detected with anti-Flag antibody. Bottom panels: Western blot of Myc-Cse4, Myc-Cse4-CTD, Myc-Cse4 N terminal domain (Myc-Cse4-N) and Flag-tagged p33 in the total yeast extracts. Note that after affinity-purification, Myc-Cse4-N was not co-purified with p33 (lane 3). (C) Pull-down assay including TBSV GST-p33 replication protein and the MBP-tagged Cse4 or Cse4 deletion mutants. The C-terminal region of TBSV p33 replication protein was used instead of the full-length protein, which includes the non-soluble N-terminal region with the trans-membrane domains. Top: Western blot analysis of the captured GST-p33C with MBP purified WT GST-Cse4 or ΔN50, ΔN80, ΔN129 Cse4 deletion mutants. GST was used as a control. Note that similar to Myc-Cse4-N, the C-terminal deletion mutants ΔC60 and ΔC100 were not pulled-down with GST-p33C suggesting that the HFD of Cse4 is also important for the interaction between p33 replication protein and Cse4p. Bottom: Coomassie-blue stained SDS-PAGE of the purified recombinant proteins. All experiments were performed three times.(A) Co-purification of viral p33 replication protein with plant CenH3. First panel: western blot analysis of co-purified His(TIF)Click here for additional data file.S4 FigYEMK–p92pol. See further details in Comparison of the ATP level within the tombusvirus replication compartment in WT and cse4-1 yeasts grown at 23°C using ATeam(TIF)Click here for additional data file.


Beauveria species, mitochondrial genome (mitogenome) used to examine genetic diversity within fungal populations. Complete mitogenomes of Beauveria species can be easily sequenced and assembled using various sequencing techniques. However, since mitogenome annotations are mainly derived from similar species comparison and software prediction, and are not supported by RNA-seq transcripts data, it leads to problems with the accuracy of mitochondrial annotations and the inability to understand RNA processing. In this study, we assembled and annotated the mitogenome of eight Beauveria strains using Illumina DNA and RNA sequencing data. The circular mitogenome of eight Beauveria strains ranged from 26,850 bp  to 35,999 bp (B. brongniartii strain GYU-BMZ03), with the intronic insertions accounting for most of the size variation, thus contributing to a total mitochondrial genome (mitogenome) size of 7.01% and 28.95%, respectively. Intron number variations were not directly related to the evolutionary relationship distance. Besides ribosomal protein S3 (rps3), most introns are lost too quickly and lack the stability of protein-coding genes. The short RNA-seq reads from next-generation sequencing can improve the mitochondrial annotation accuracy and help study polycistronic transcripts and RNA processing. The transcription initiation sites may be located in the control region. Most introns do not serve as taxonomic markers and also lack open reading frames (ORFs). We assumed that the poly A tail was added to the polycistronic transcript before splicing and one polycistronic transcript (trnM(1)-trnL(1)-trnA-trnF-trnK-trnL(2)-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1-trnR(2)-nad1-nad4-atp8-atp6-rns-trnY-trnD-trnS-trnN-cox3-trnG-nad6-trnV-trnI-trnS-trnW-trnP-rnl(rps3)-trnT-trnE-trnM(3)) was first processed from the mitogenome and was subsequently processed into smaller mono-, di-, or tricistronic RNAs.Despite the significant progress that has been made in the genome sequencing of  Beauveria belonging to the family Cordycipitaceae, subclass Hypocreomycetidae, has a wide host range of insect species  (GenBank accession number: EU371503.2/NC_010652.2) was sequenced by Sanger sequencing and published in 2009 in the NCBI nucleotide database  provided insights into the mitogenome of the Beauveria genus (B. bassiana isolate k4), whereas the maximum length was nearly double the minimum length, i.e., 59,014 bp (B. lii strain RCEF5500). Complete mitogenomes of Beauveria species are haploid, maternally inherited, and have fewer introns, which can be easily sequenced and assembled using various sequencing techniques , and PacBio sequencing)  . However studies . PacBio osporium . The Pacosporium . HoweverBeauveria strains using the Illumina sequencing platform in this study, and downloaded the SRA data (genome and transcriptome) of one Beauveria strain from the NCBI SRA database, which was sequenced using the Illumina sequencing platform in previous study , B. amorpha strain GYU-BMZ02 (BMZ072690), B. brongniartii strain GYU-BMZ03 (BMZ101344), B. bassiana strain GYU-BMZ04 (BMZ104186), B. bassiana strain YMM (CGMCC No. 11464), B. caledonica strain ATCC 64970, and B. pseudobassiana strain ATCC 90518 were selected for mitogenome sequencing. The B. amorpha strain GYU-BMZ01, B. amorpha strain GYU-BMZ02, B. brongniartii strain GYU-BMZ03, and B. bassiana strain GYU-BMZ04 (BMZ104186) were obtained from Ningbo Mingzhou Biotechnology Co., Ltd . B. bassiana strain YMM was isolated from Alissonotum impressicolle Arrow obtained from the Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Institute of Plant Protection, Guangxi Academy of Agricultural Sciences. The B. bassiana strain YMM was isolated from the odonatan larva of Guizhou University. B. caledonica strain ATCC 64970 and B. pseudobassiana strain ATCC 90518 were obtained from the American Type Culture Collection (ATCC). B. bassiana was cultivated on potato dextrose agar (PDA) for genomic DNA (gDNA) extraction. The surface of the PDA medium was scraped with a scalpel after 20 days of cultivation. We downloaded the SRA data (genome (SRR3269141) and transcriptome (SRR3269778–SRR3269780)) of B. bassiana strain ARSEF 8028 from the NCBI SRA database, which was sequenced using the Illumina sequencing platform in previous study  following the kit handbook. The purity and concentration of the obtained gDNA were tested using a NanoPhotometer® spectrophotometer  and a Qubit® 2.0 fluorometer , respectively. Sequencing libraries for the quality-checked gDNA were generated using a TrueLib DNA Library Rapid Prep Kit for Illumina sequencing . The libraries were subjected to size distribution analysis using an Agilent 2100 bioanalyzer , followed by real-time PCR quantitative test. The successfully generated libraries were sequenced using an Illumina NovaSeq 6000 platform , and 150-bp paired-end reads with an insert of approximately 350 bp were generated.Samples were collected from seven Beauveria were extracted using Trizol reagent  following the reagent handbook. The obtained RNA samples were subjected to RNase-free DNase I treatment to remove residual DNA. The purity, concentration, and integrity of the RNA were tested using a NanoDrop 2000 spectrophotometer , Aglient 2100 bioanalyzer , and Qubit 2.0 fluorometer , respectively. The mRNAs were purified from the total RNAs using poly-T oligo-attached magnetic beads and fragmented. Subsequently, cDNA libraries were generated for the quality-checked samples using a Tru Seq RNA Sample Prep Kit following the kit handbook. The successfully generated cDNA libraries were sequenced using using an Illumina NovaSeq 6000 platform .Total RNAs from seven https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (https://github.com/OpenGene/fastp)  , and file/fastp) . The quaThe QC standards for RNA reads were as follows: (1) Removing reads with >50% bases with Phred quality < Q10, (2) Removing reads with an average quality score of < 20, (3) trimming reads longer than 150 bases at their tail region to modify them to ≤ 150 bases, (4) trimming the poly-G tails of the reads, (5) filtering low-complexity reads (complexity is defined as the percentage of the base that differs from its next base); the threshold for the low-complexity filter is 10, which means that 10% complexity is required, and (6) Removing reads with < 80 bases.the Beauveria strains were assembled using NOVOPlasty version 4.3.1 (https://github.com/ndierckx/NOVOPlasty) (B. bassiana (GenBank accession number: EU371503.2/NC_010652.2)  The value of K-mer (33) was the default parameter.The mitogenomes of OPlasty) . The mai10652.2)  was selede novo into transcripts using Trinity version 2.13.2 (https://github.com/trinityrnaseq/trinityrnaseq/) (https://github.com/DaehwanKimLab/hisat2) (https://github.com/samtools/samtools)   with defrnaseq/) . The res/hisat2)  with def/hisat2) . Alignedamtools)  and assen 2.13.2 , with deCordyceps bassiana isolate Bb147 (EU100742.1) (B. pseudobassiana strain C1010 (KF297618.1/NC_022708.1)  (B. bassiana isolate k4 (KT201148.1) (Cordyceps brongniartii isolate IMBST95031 (EU100743.1/NC_011194.1) (B. bassiana (NC_010652.2) (https://chlorobox.mpimp-golm.mpg.de/geseq.html) (https://github.com/PASApipeline/PASApipeline) (http://research-pub.gene.com/gmap/) (https://github.com/icebert/pblat) (https://github.com/lh3/minimap2)  , B. pseu22708.1) , B. cale30636.1) , B. bass01148.1) , Cordyce11194.1) , and B. 10652.2)  as refereq.html) , includieq.html) , ARAGORNeq.html) , and BLAeq.html) . To impripeline) , includim/gmap/) , PBLAT vt/pblat)  (includit/pblat) ), and miinimap2) . Comparainimap2)  and mVISinimap2)  with LAGinimap2) . The seqinimap2)  with Meginimap2)  on the NBeauveria were deduced using MEGA 11 software  scores were considered to best describe the substitution pattern. Based on BIC = 50395.840, the general reversible chloroplast (cpREV) (Phylogenetic relationships of the genus software  with 15  (cpREV)  + gamma  (cpREV) . There wRNA was reversed to cDNA using the Kit PrimeScript™ RT Reagent Kit with gDNA Eraser  . According to the target region, primers were designed using Primer3 version 2.6.1 , and PCRB. caledonica strain ATCC 64970 to 3151 times for B. bassiana strain ARSEF 8028), comprising a typical single circular DNA molecule with a total length ranging from 26,850  to 35,999 bp (B. brongniartii strain GYU-BMZ03)  to 73.05% (B. amorpha strain GYU-BMZ01 and GYU-BMZ02)  to 0.0124 (B. brongniartii strain GYU-BMZ03) (B. bassiana strain YMM to 0.1101 for B. brongniartii strain GYU-BMZ03)  . BeauverU-BMZ02) , and theU-BMZ03) . HoweverU-BMZ03) .B. caledonica strain ATCC 64970) to 5.47% (B. bassiana strain ARSEF 8028), those from the RNA accounted for <0.18% of the total short reads, especially the mitochondrial RNA read proportion of B. caledonica strain ATCC 64970 being almost zero  (rps3), seven NADH dehydrogenase subunits (nad1-6 and nad4L genes), three cytochrome oxidase subunits (cox1-3), apocytochrome b (cob), three subunits of ATP synthase , small and large subunit ribosomal RNA genes (rnl and rns), 25 tRNAs that include three tRNAs for methionine, two for arginine, two for leucine, two for serine, and only one tRNA for other amino acids, and one control region located between trnM(3) and trnM(1). Transcriptome annotation revealed that rnl was longer than the reference mitogenome (NC_010652.2), extending 30 bp forward and 10 bp backward. These regions have multiple AT repeats, possibly circularizing the rnl gene. We also observed some reads of the B. amorpha strain GYU-BMZ01 and B. pseudobassiana strain ATCC 90518 from RNA-seq with the half bases aligned at the head of rnl and half bases aligned at its tail, thus causing errors in the de novo assembly of the transcriptome. We found a read in the B. amorpha strain GYU-BMZ02 showing that the rnl gene has a polyA tail at the 3′ end.The conserved genes of the 10652.2) , which eBeauveria mitogenome sequences (Beauveria mitogenomes for coding DNA sequence (CDS), with some structural variations existing in the mitogenomes. We observed mitogenome length differences mostly in the introns (B. pseudobassiana strain ATCC 90518) to eight (B. brongniartii strain GYU-BMZ03), and were located in rnl, cob, cox1, cox2, and nad1 (B. pseudobassiana strain ATCC 90518) to 10,422 bp (B. brongniartii strain GYU-BMZ03), with their contribution to the respective total mitogenome size ranging from 7.01% (B. pseudobassiana strain ATCC 90518) to 28.95% (B. brongniartii strain GYU-BMZ03). The intron of rnl (the second intron of rnl for B. brongniartii strain GYU-BMZ03) was conserved and contained an open reading frame (ORF) for rps3. Introns in other genes are thought to contain ORFs . The cox2 gene of B. brongniartii has one intron, including a putative GIY-YIG homing endonuclease  to 6092 bp (B. bassiana strain GYU-BMZ04), thus contributing to a total mitogenome size ranging from 15.76% (B. brongniartii strain GYU-BMZ03) to 20.33% (B. bassiana strain GYU-BMZ04). The A + T content was similar for these regions in the mitogenomes (∼74.5%), with the largest intergenic region being between cox1 and trnR-UCU. Overlapping regions of 1 bp were located between both nad2 and nad3, and between nad4L and nad5.The mitogenomes contained 39 intergenic and two overlapping regions. The total length of the intergenic regions ranged from 5198 bp , we constructed a phylogenetic tree using the maximum likelihood (ML) method and a general reversible chloroplast  we performed the de novo assemble and mapping of the transcripts to the mitochondrial genome. There are differences between mitochondrial transcripts and nuclear transcripts. While nuclear transcripts consist of a very high proportion of the entire transcriptome, we reduced the computational resources used during de novo assembly by filtering reads of nuclear transcripts. Although the first process was much more efficient than the second, it filtered out reads during their alignment, i.e., reads of B. amorpha strain GYU-BMZ01 and B. pseudobassiana strain ATCC 90518 from RNA-seq with the half bases aligned the head of rnl, and half bases aligned the tail. Although NGS short reads resulted in incompletely assembled transcripts that lack some vital information  (We adopted two annotation processes : (1) we rmation)  and do nde novo assembled transcripts into (1) intron-free and (2) intron-containing transcripts. mRNA was purified from the total RNA using poly T oligo-attached magnetic beads. Mature mRNAs with poly A tails were intron-free. Although some introns may contain ORFs, the origin and termination sites of genes located within introns are hundreds of bases away from the exon region. There should not be any second type of reads. Owing to the “efficiency of splicing”, , and with the addition of some tRNA regions that are not reads, the coverage will be even higher. Based on the transcript alignment results (rnl (rps3)-trnT-trnE-trnM(3). We found contig A that exists in B. bassiana strain ARSEF 8028 (rnl (rps3)-trnT-trnE-trnM(3) is present. (2) The second region is trnM(1)-trnL(1)-trnA-trnF-trnK-trnL(2)-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1-trnR(2)-nad1-nad4-atp8-atp6-rns-trnY-trnD-trnS-trnN-cox3-trnG. Owing to defects in the Trinity software assembly, some overlapping contigs such as contigs B3 and B4, were not assembled together (trnL(1)-trnA-trnF-trnK-trnL(2)-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1, nad1-nad4-atp8-atp6-rns-trnY, and trnD-trnS-trnN-cox3 were formed. Some tRNAs did not contain reads, such as trnM(1), trnR(2), trnG, and trnP. The tRNAs preceding mRNAs or rRNAs in the polycistronic transcripts were removed by 30–50 cleavages (trnM(1) is 2 bp apart from trnL(1), therefore, it might be trnM(1)-trnL(1)-trnA-trnF-trnK-trnL(2)-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1. trnG began at base 25,199 on the mitogenome sequence of B. pseudobassiana strain ATCC 90518, with contig B12 ending at base 25,172, which is 26 bp apart from trnG  ranged from 17,359–17,429 in the mitogenome sequence of B. pseudobassiana strain ATCC 90518, located between cox1 and nad1. Since the distance between contig B6 and contig B7 was exactly the length of one trnR(2) (cox1-trnR(2)-nad1 can be shown using cDNA and PCR in mitogenome of B. bassiana strain GYU-BMZ04 (trnM(1)-trnL-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1 and nad1-nad4-atp8-atp6-rns-trnY-trnD-trnS-trnN-cox3-trnG into a polycistronic transcript, trnM(1)-trnL(1)-trnA-trnF-trnK-trnL(2)-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1-trnR(2)-nad1-nad4-atp8-atp6-rns-trnY-trnD-trnS-trnN-cox3-trnG. (3) The third region is nad6-trnV-trnI-trnS-trnW-trnP. According to contigs B13, C8, and C9  . For verYU-BMZ04  and S4. , and C9 , trnV antrnG is 53 bp apart from nad6. For verification, cox3-trnG-nad6 can be shown using cDNA and PCR in mitogenome of B. bassiana strain GYU-BMZ04 -trnL(1)-trnA-trnF-trnK-trnL(2)-trnQ-trnH-trnM(2)-nad2-nad3-atp9-cox2-trnR(1)-nad4L-nad5-cob-trnC-cox1-trnR(2)-nad1-nad4-atp8-atp6-rns-trnY-trnD-trnS-trnN-cox3-trnG-nad6-trnV-trnI-trnS-trnW-trnP-rnl (rps3)-trnT-trnE-trnM(3)) was processed from the mitogenome. For B. bassiana strain ARSEF 8028 (trnM(3) ended at base 5,543 and contig A ended at base 5,664 located in the control region. Therefore, transcription initiation sites may be located in the control region.Polycistronic transcripts are subsequently processed into smaller mono-, di-, or tricistronic RNAs . trnG isYU-BMZ04  and S5. CC 90518  and S6. SEF 8028 , trnM(3)Beauveria species. The short RNA-seq reads from next-generation sequencing can improve the mitochondrial annotation accuracy and help study polycistronic transcripts and RNA processing. The pipeline of this study offers a reference for mitochondrial transcriptome. Beauveria species tend to process the growing polycistronic transcript first, and the poly A tail was added to the polycistronic transcript before splicing. The small proportion of mitochondrial transcripts among the total transcripts for Beauveria species, B. bassiana transcriptome sequenced by PacBio sequencing only have 11 full-length transcripts from mitogenome in previous study (In the present study, we utilized Illumina DNA and RNA sequencing data to assemble and annotate mitogenome of us study , the Oxf10.7717/peerj.14067/supp-1Table S1Beauveria.Base composition, AT%, AT-skew and GC-skew mitogenomes of 8Click here for additional data file.10.7717/peerj.14067/supp-2File S1Click here for additional data file.10.7717/peerj.14067/supp-3File S2Click here for additional data file.10.7717/peerj.14067/supp-4File S3Click here for additional data file.10.7717/peerj.14067/supp-5File S4Click here for additional data file.10.7717/peerj.14067/supp-6File S5Click here for additional data file.10.7717/peerj.14067/supp-7File S6Click here for additional data file.


The convergence of transcriptional and epigenetic changes in the peripheral nervous system (PNS) reshapes the spatiotemporal gene expression landscape in response to nerve transection. The control of these molecular programs exhibits sexually dimorphic characteristics that remain not sufficiently characterized. In the present study, we recorded genome-wide and sex-dependent early-phase transcriptional changes in regenerating  sciatic nerve 24 h after axotomy. Male nerves exhibited more extensive transcriptional changes with male-dominant upregulation of cytoskeletal binding and structural protein genes. Regulation of mRNAs encoding ion and ionotropic neurotransmitter channels displayed prominent sexual dimorphism consistent with sex-specific mRNA axonal transport in an early-phase regenerative response. Protein kinases and axonal transport genes showed sexually dimorphic regulation. Genes encoding components of synaptic vesicles were at high baseline expression in females and showed post-injury induction selectively in males. Predictive bioinformatic analyses established patterns of sexually dimorphic regulation of neurotrophic and immune genes, including activation of glial cell line-derived neurotrophic factor Gfra1 receptor and immune checkpoint cyclin D1 (Ccnd1) potentially linked to X-chromosome encoded tissue inhibitor of matrix metallo proteinases 1 (Timp1). Regulatory networks involving Olig1, Pou3f3/Oct6, Myrf, and Myt1l transcription factors were linked to sex-dependent reprogramming in regenerating nerves. Differential expression patterns of non-coding RNAs motivate a model of sexually dimorphic nerve regenerative responses to injury determined by epigenetic factors. Combined with our findings in the corresponding dorsal root ganglia (DRG), unique early-phase sex-specific molecular triggers could enrich the mechanistic understanding of peripheral neuropathies. The peripheral nervous system (PNS) displays strong regenerative potential compared to the brain and spinal cord. Substantial knowledge of mechanisms involved in PNS injury has been acquired using rodent models of sciatic nerve axotomy originally described by Augustus Waller in 1850 . The sucAxonal regrowth in the damaged PNS depends on trophic, immune, metabolic, and structural support of Schwann cells , formingSex is emerging as a key biological variable in models of PNS injury, as certain neuropathic states exhibit sex-specific prevalence, incidence, mechanisms, and clinical presentation . Sexual Using high-depth RNA-seq, the present study identified sex-specific early-phase transcriptional changes in protein-coding and ncRNAs in regenerating  nerves at 24 h after sciatic nerve axotomy in male and female mice. Applying predictive bioinformatics, we determined unique signaling events related to regenerative, trophic, metabolic, and sex chromosome-linked systems.To determine the sex specificity of an early-phase PNS regenerative transcriptional program, sciatic nerve axotomy or sham operation was conducted in female and male mice. At 24 h post-axotomy, regenerating  tissues  were subadj) < 0.1) and the expression fold change (FC) filter  were identified in males relative to females as presented on MA and scatter plots , leucine-rich repeat-containing protein 15 (Lrcc15), neuronal pentraxin-2 (Nptx2), heterodimeric neurotropic cytokine receptor-like factor 1 (Crlf1), cardiotrophin-like cytokine factor 1 (Clcf1), and sonic hedgehog protein (Shh). In males, DEGs encoding the potassium channel subfamily T member 1 (Kcnt1), apoptosis-promoting dendrin (Ddn), inactive serine/threonine-protein kinase (Plk5), Toll-like receptor 9, and myelin-associated oligodendrocyte basic protein (Mobp) were the most significant drivers of variance. These and other DEGs identified by PCA  enrichment analysis was conducted. Molecular-level biochemical characteristics of DEGs were used to identify the most relevant GO clusters of molecular function . GO clus2+ ion pump , CaM kinase-like vesicle-associated protein (Camkv), ephrin type-A receptor 10 (Epha10), and N-acetylaspartylglutamate synthase A (Rimkla) were significantly upregulated in male nerves. In females, cyclin-dependent kinase 18 (Cdk18) and cGMP-dependent protein kinase 2 (Prkg2) were upregulated. Notably, most proteins that bind charged ions (cluster 2) were upregulated only in males, including the protein kinase C-binding protein Nell1, and Ca2+ sensors regulating vesicular release, the double C2-like domain-containing protein α (Doc2a), and Ca2+-binding protein 7 (Cabp7), the hippocalcin-like protein 4 , and β-synuclein (Sncb) involved in neuronal plasticity.Heterocyclic compound binding proteins interact with ATP, GTP, nucleobases, and their derivatives (cluster 1). Subunits of the ATP-driven CaNell2), inactive heparanase-2 (Hpse2), phospholipase A2 (Pla2g5), matrix-assembly related SPARC-related modular Ca2+-binding protein (Smoc2), and a cysteine-rich secretory protein LCCL (Crispld2). In males, these proteins exhibited high levels of expression. In addition, genes encoding Nell1, neuronal adhesion neurocan core protein (Ncan), brevican core protein (Bcan), and β-amyloid precursor protein (Aplp1) were upregulated and expressed in males.Several carbohydrate-binding proteins (cluster 4) were downregulated in female nerves, including the protein kinase C (PKC)-binding protein (Dcx), the actinin-interacting Cdk5 activator Cdk5r2, Gap junction β6 protein (Gjb6), GAR domain-containing protein (BC024139), the microtubule-stabilizing MAP6 domain-containing protein (Map6d1), cytoplasmic dynein intermediate (Dync1i1), and the microtubule-associated serine/threonine-protein kinase (Mast1) (cluster 8). Oppositely, mRNAs encoding kinesin-like motor enzymes Kif15/Kif22/Kif23, nucleolar and spindle associated protein (Nusap1), spindle/kinetochore-associated protein (Ska1), and a centromere-associated protein (Cenpe) were reduced in males. Microtubule-associated oxygen-regulated protein Rp1 and neuron navigator Nav3 demonstrated more robust upregulation in female nerves.Male nerves displayed higher up-regulation of the microtubule-associated neuronal migration protein doublecortin , collagen , and glial fibrillary acidic protein (Gfap) genes. In contrast, β-adducin (Add2), light and heavy chains of neurofilament , spectrin β chain (Sptbn2), and Laminin subunit α5 (Lama5) showed male-dominant upregulation.Genes encoding structural proteins demonstrated strong upregulation in both sexes (cluster cl24) of tubulin , chemokine receptors , and other receptors . Despite the decrease, the absolute expression levels of many cytokine receptor genes remained high. Only cytokine receptor-like factor 1 (Crlf1) and corticotrophin-like cytokine factor 1 (Clcf1) demonstrated significant upregulation in both sexes.Reduced expression of many cytokine receptors was observed in both sexes (cluster 9), including interleukin receptors (Ucn2), nerve growth factor inducible (Vgf), endothelin (Edn2), calcitonin , glial cell differentiation regulator meteorin (Metrn), leukemia inhibitory factor (Lif), glial-derived neurotrophic factor (Gdnf), epidermal growth factor β-cellulin (Btc), epidermal growth factor-like (Egfl8), and fibroblast growth factor (Fgf5). Male-specific upregulation of somatostatin (Sst), pituitary adenylate cyclase-activating polypeptide (Adciap1), cholecystokinin (Cck), neuropeptide galanin , ghrelin (Ghrl), and pro-opiomelanocortin (Pomc). Proglucagon (Gcg) and stanniocalcin-1 (Stc1) exhibited upregulation in females.Both males and females exhibited significant upregulation of ligand-encoding DEGs (cluster 10), including urocortin 2 .Phosphotransferases (cluster 15), including non-receptor tyrosine kinases encoded by the adj < 0.001 and log2FC > 1 or log2FC < –1). Significant canonical pathways were further ranked based on Fisher’s exact test (p < 0.05). Positive or negative z-scores determined pathways’ activation and deactivation.Signaling pathways, upstream regulation, and interactive networks were predicted by Ingenuity Pathway Analysis (IPA) software using a highly stringent subset of DEGs , any gene or small molecule that, with high probability, could affect the expression of their target DEGs. The activation or inhibition efficiency of each upstream regulator on target DEGs could be defined by positive or negative z-scores, respectively, as illustrated in via the neuron-specific adaptor Shc3, the Gdnf receptor α1 (Gfra1), and neural cell adhesion proteins Chl1 and Cadm1 to regulate cell-cell adhesion and neuronal plasticity , artemin (Artn), and persephin (Pspn). Notably, the Gdnf gene was highly upregulated in both sexes in the proximal stumps  were rapidly induced by axotomy in the corresponding DRG . Fos, Jue nerves . Cebpd, Both male and female nerves upregulated matrix metalloproteinase (MMP) Mmp17 (MT4-MMP) expression . Mmp24 mWfdc5), the cysteine protease inhibitor (Fetub), and serine protease inhibitor Kazal-type 1 (Spink1). Timp1 increased in both sexes, in agreement with our previous reports . It is important to note that SV-related genes show high levels of expression in sham and axotomized female nerves; therefore, male-prevalent gene activation could level up pre-synaptic activity to match the activity in females.Synaptogenesis Signaling Pathway was distinctly regulated in both sexes. Genes encoding components of synaptic vesicles (SV) showed greater activation in males . ConsequAxonal transport is essential for neuronal survival and function restoration after damage. Many kinesins involved in anterograde axonal transport decreased in males , except Ermn), α-internexin (Ina), myelin oligodendrocyte glycoprotein (Mog), opalin , contactin 2 (Cntn2) (Mobp) demonstrated significant sexually dimorphic regulation by upregulating in males and downregulating in females.Male nerves upregulated myelinogenesis- and neuron morphogenesis-related DEGs (according to GO term GO:0043209), including ermin ( (Cntn2) . The expGabra1, Gabra5, gabrg2, Gabrb1, Gabrb2, Gabrb3, and Gabrg1), members of ionotropic glutamate receptor N-methyl-D-aspartic acid (NMDA) receptor subunits , α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits Gria1 and Gria2, kainic acid receptor subunit Grik1. Notably, mRNAs encoding 5-hydroxytryptamine 2C (serotonin) receptors Htr2c and Htr3a also increased. Male nerves exhibited increased potassium, calcium, and sodium voltage-gated ion channel-related mRNAs. Remarkably, levels of these mRNAs diminished in the respective DRGs measured in the same animal cohorts , neurogenesis-related TFs , and nuclear protein Nupr1 were male-specific. Also, males upregulated Fox-family TFs , Hox-family TFs , and Sox-family TF genes (Sox2 and Sox10), and the RNA-binding proteins ZFP36 known to restrain T cell activation and antiviral immunity , and POU domain-containing Pou3f3 . Upregulimmunity . Sox2 an2+-transporting ATPase subunits Atp2b2 and Atp2b3  and ribonuclease P RNA component H1 (Rpph1) were upregulated in females.Levels of  females . In addivia cis-acting mechanisms. In females, genes encoding the axonemal dynein (Dnah2), tetraspanin 32 (Tspan32), pseudopodium-enriched atypical kinase 1 (Peak1), SAP30 binding protein (Sap30bp), and H3.3 histone (H3f3a) could be targeted by the respective asRNA (Dios3), MAP2 kinase (Map2k3), RAB26 oncogene (Rab26), homeobox B2/B3 , dynamin 3 (Dnm3), the transcriptional repressor CCCTC-binding factor (Ctcf), de novo DNA methyltransferase 3a (Dnmt3a), and insulin-like growth factor 1 (Igf1).Antisense lncRNAs (asRNA) can potentially target co-localized PNS injury-related genes ve asRNA . In maleMagix, Tmem255a, Tmem28, Map7d2, Timp1, Erdr1, 2010204K13Rik, Gjb1, Pcsk1n, Bex2, and Pwwp4c, were elevated in both sexes. Male-specific Y-linked transcript Gm47283 was upregulated post-axotomy in males.As reported in the DRGs of the same animal cohort , genomicWe obtained novel evidence of sexually monomorphic and dimorphic regulation of the early-phase transcriptional programs in regenerating segments of injured peripheral nerves . The conFos, Smad3, Creb1, Rac2, Klf6, and Atf3, that function as RAGs , known survival factors for neurons, bind to GFRA receptors, trigger the phosphorylation of RET tyrosine kinase receptor  that is ogenesis  and, somogenesis . Furtherogenesis . GDNF faogenesis  to the iThe outcome of nerve regeneration after sciatic nerve injury is partly dependent on the integrity of ECM-rich Schwann cell basal lamina and endoneurial tubes . ProteolIntriguingly, a group of MMPs co-localized on the mouse chromosome 9  have decreased in both sexes. Because MMPs in the orthologous human chromosome 11q22.3 region could carry different histone modification marks , epigeneNotably, protease expression levels were high in intact and axotomized nerves. Processes of post-translational protease activation and binding to intrinsic proteinase inhibitors control potentially cytotoxic proteolytic activities. Aberrant cleavage of mediators of nociceptive signaling during neurogenesis potentially causes neuropathic pain . PreviouAn intact connection to DRG allows for dynamic axonal transport of protein and mRNA content between neuronal soma and the proximal segment of the axotomized nerve . The antKinesins facilitate anterograde axonal transport of neurotransmitter receptors, mitochondria [reviewed in ], and alRetrograde transport of transcription factors from the site of nerve injury to DRG has been shown to facilitate the neuronal survival  and axonTimp1, known to promote regeneration  were housed in a temperature-controlled room (22°C) on a 12 h light/dark cycle with ad libitum access to food and water. The mice were randomly assigned to axotomy and sham groups by sex (n = 6/group). Under isoflurane anesthesia, the left sciatic nerve was exposed at the mid-thigh level, followed by a complete transection using sterile microsurgery scissors. In the sham group, anatomically equivalent nerves were subjected to exposure without transection. The muscle was sutured, and the skin stapled. At 24 h after surgery, proximal segments of the sciatic nerve, corresponding to previously investigated lumbar (L)4 and L5 DRG tissues , 3 sample/group) with RIN ≥ 7.0 were used for RNA-seq.Nerve tissues were transferred in Trizol solution (Invitrogen) and disrupted by mechanical homogenization. Total RNAs were purified using RNeasy RNA purification reagents. RNA concentrations and quality were determined using Nanodrop absorbance ratios at 260/280 nm and 260/230 nm. RNA integrity indices were determined using the Agilent Bioanalyzer Nano RNA chip. 500 ng of total RNA samples (mRNA libraries were generated following the TruSeq Stranded mRNA library preparation protocol (Illumina). In brief, the Poly-A enriched mRNAs were purified using poly-T oligo coupled magnetic beads, followed by mRNA fragmentation, first and second strands synthesis, cleaning on AMPure XP magnetic beads, and 3’-adenylation. Ligation of TruSeq dual-index adapters was used for barcoding. The quality of RNA-seq libraries was validated using qPCR. Libraries were sized on Agilent Bioanalyzer DNA high sensitivity chip and normalized. RNA-seq was performed using the paired-end 100 cycle program on the NovaSeq 6000 system at the Genomics High Throughput Facility . Base calls were recorded and converted to FASTQ files containing sequencing reads and the corresponding quality scores using Illumina software. Sequencing was conducted until we acquired at least 50 million paired-end reads per sample.Trimmomatic  cutoff (Padj < 0.1)  [Amazon Web Services (AWS)] running Ubuntu Server 20.04 LTS (64-bit ARM). Data analysis steps are summarized in mmomatic . Transcrmmomatic  and the (MultiQC . Transcr(Tximeta . Gene co(Tximeta . Log2FC m method . Signifij < 0.1) . Padj < ect tool  and RUVs .The datasets presented in this study can be found in the Gene Expression Omnibus (GEO) public repository (accession numbers: The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the VA San Diego Healthcare System.AC: conceptualization, methodology, software, formal analysis, investigation, data curation, writing – original draft, review and editing, and visualization. VS: conceptualization, resources, project administration, funding acquisition, and writing – original draft, review and editing. All authors contributed to the article and approved the submitted version.