Buxus microphylla, was able to provoke mitophagy in lung cancer cells. CVB-D-induced mitophagy potentiates apoptosis by promoting mitochondrial dysfunction. Mechanistically, CVB-D initiates mitophagy by enhancing the expression of the mitophagy receptor BNIP3 and strengthening its interaction with LC3 to provoke mitophagy. Our results further showed that p65, a transcriptional suppressor of BNIP3, is downregulated upon CVB-D treatment. The ectopic expression of p65 inhibits BNIP3 expression, while its knockdown significantly abolishes its transcriptional repression on BNIP3 upon CVB-D treatment. Importantly, nude mice bearing subcutaneous xenograft tumors presented retarded growth upon CVB-D treatment. Overall, we demonstrated that CVB-D treatment can provoke mitophagy and further revealed that the p65/BNIP3/LC3 axis is one potential mechanism involved in CVB-D-induced mitophagy in lung cancer cells, thus providing an effective antitumor therapeutic strategy for the treatment of lung cancer patientsMitophagy plays a pro-survival or pro-death role that is cellular-context- and stress-condition-dependent. In this study, we revealed that cyclovirobuxine D (CVB-D), a natural compound derived from  Lung cancer is one of the most common malignant tumors and a frequent cause of death worldwide ,2. SurgiBuxus microphylla, is primarily used to treat cardiovascular and cerebrovascular disease [Cyclovirobuxine D (CVB-D), a natural bioactive alkaloid component derived from  disease ,7. Recen disease ,9,10,11.Autophagy is defined as an evolutionally conserved process of recycling excess intracellular macromolecules, which are engulfed in autophagosomes, subsequently degraded in autolysosomes and ultimately, are broken down into their constituent parts ,13. AutoUnder certain conditions, the occurrence of mitophagy is of great importance for cells to preserve a population of normal-function mitochondria and protect cells from cellular damage ,26. TherIn this study, we demonstrated that CVB-D-induced lung-cancer cell death is closely associated with mitophagy activation. CVB-D treatment causes significant depletion of mitochondria, which is accompanied by the upregulation of the mitophagy receptors BNIP3 and BNIP3L. Our results further demonstrated that the upregulation of BNIP3 is due to the downregulation of p65, which relieves its inhibition on BNIP3 expression. Notably, mitophagy induced by CVB-D leads to a further increase of apoptotic death in lung cancer cells. These findings provide a cross link between mitophagy and apoptosis upon CVB-D treatment, which indicates that CVB-D is a potential candidate and is of particular clinical relevance for lung-cancer treatment.To evaluate whether CVB-D exhibits antitumor effects against lung cancer cells, the CCK8 (cell counting kit-8) assay was performed to evaluate the cytotoxic effects of CVB-D against lung cancer cells . As shown in Cell cycle regulation plays a key role in antitumor therapy, and we therefore tested whether CVB-D treatment can induce cell-cycle arrest in lung cancer cells. As expected, exposure to CVB-D treatment caused significant G2/M arrest in all lung cancer cells D,E. ConsRNA-seq was applied to investigate the underlying mechanism of apoptotic induction upon CVB-D treatment. Of these differentially expressed genes (DEGs) upon 30 μM CVB-D treatment, 1200 genes were upregulated (log2FC>2), and 1090 genes were downregulated (log2FC>2) A. For fuTranscriptome data showed that CVB-D treatment resulted in the downregulation of various mitochondria-related genes in A549 cells, which implied that mitochondria might be a potential factor involved in CVB-D-induced apoptosis. To test our hypothesis, we first evaluated the morphology change of mitochondria upon CVB-D treatment. In the control group, large number of normal mitochondria with tubular-shaped morphology can be seen in lung cancer cells, while CVB-D treatment led to a significant morphology change of mitochondria into an abnormal structure A, suggesMitophagy is an important mitochondria quality-control pathway that removes dysfunctional or damaged mitochondria to maintain normal cell growth. Given that CVB-D treatment causes mitochondrial dysfunction and decline, we then aimed to test whether CVB-D treatment provokes mitophagy. LC3I conversion to LC3II, which is regarded as a molecular marker of autophagy, was analyzed by Western blot. As expected, CVB-D treatment led to a significant conversion of LC3I to lipidated LC3II in a dose-dependent manner in all tested cells A. SQSTM1We next evaluated whether mitochondrial dysfunction and loss is ascribed to mitophagy activation upon CVB-D treatment. A shRNA against ATG5  was applied to explore the underlying mechanism in CVB-D-induced mitochondrial loss. As expected, the knockdown of ATG5 significantly inhibited the conversion of LC3I to lipidated LC3II upon CVB-D treatment in all lung cancer cells A. ImportThe role of CVB-D-induced mitophagy in disturbing the normal function of mitophagy in lung cancer cells and mitochondria is vitally important as an energy supplement to maintain the normal growth of cells; therefore, we presumed that this kind of mitophagy induced by CVB-D may contribute to cell apoptosis. Next, we aimed to investigate whether mitophagy induced by CVB-D was engaged in CVB-D-induced apoptotic death. Thus, the pharmacological and genetic inhibition of autophagy was applied to clarify the underlying connection between mitophagy and cytotoxicity upon CVB-D treatment. As expected, combined treatment with CVB-D and autophagy inhibitor  resulted in an alleviation of cytotoxicity in all lung cancer cells A, and a The parkin-Pink1 and BNIP3/BNIP3L pathways are known to mediate mitophagy activation in mammalian cells. To further explore the underlying mechanism involved in CVB-D-induced mitophagy, we first detected the expression of parkin/Pink1 and BNIP3/BNIP3L in lung cancer cells upon CVB-D treatment. The results showed that parkin exhibits decreased expression in all lung cancer cells, and Pink1 showed a decreased expression in 95-D cells, but no obvious change was detected in A549 and H446 cells . We concBNIP3 can be transcriptionally activated by HIF1α or FOXO3a or transcriptionally silenced by p65 ,18,19,20To evaluate the antitumor effects of CVB-D in vivo, nude mice bearing subcutaneous A549 and H446 xenografts were established to evaluate the antitumor ability of CVB-D. As shown in Buxus microphylla, can provoke apoptotic death in lung cancer cells. In addition to apoptotic death, CVB-D treatment can also significantly provoke mitophagy activation. Under certain conditions, mitophagy serves a protective role by removing dysfunction or damaged mitochondria to alleviate cellular stress. The extent and duration of mitophagy activation is a crucial impact factor for cell survival or death decisions, so we conducted a series of experiments to address the role of CVB-D-induced mitophagy in the apoptosis of lung cancer cells. Interestingly, we found that mitophagy induced by CVB-D serves a pro-death role in CVB-D-treated lung cancer cells by promoting mitophagy-mediated mitochondrial dysfunction. It is apparent that mitophagy induced by CVB-D is more likely to be overactivated and that this kind of mitophagy serves as a destroyer of mitochondria in lung cancer cells and ultimately potentiates CVB-D-induced apoptotic death. Our results further demonstrated that the p65/BNIP3/LC3 axis is a potential mediator for mitophagy activation upon CVB-D treatment. Treatment of lung cancer cells with CVB-D can significantly suppress the expression of p65, a transcriptional suppressor of BNIP3, and the downregulation of p65 can significantly relieve its inhibition on BNIP3 transcription upon CVB-D treatment and can cause enhanced expression of BNIP3, thus enhancing its interaction with LC3 and mediating mitophagy activation. We demonstrated that CVB-D exhibits a kind of favorable anti-lung-cancer activity in vitro and in vivo and revealed a novel antitumor mechanism of CVB-D in lung cancer cells, namely that mitophagy-mediated mitochondrial dysfunction is a contributor to CVB-D-induced apoptotic death through the targeting of the p65/BNIP3/LC3 axis.In this study, we revealed that CVB-D, a natural compound derived from In our preliminary experiments, we found that CVB-D exhibited favorable anti-lung-cancer potential both in vitro and in vivo. Next, we investigated the underlying mechanism by which CVB-D is involved in lung-cancer cell death. A RNA-seq analysis was conducted to detect the change in gene expression upon CVB-D treatment, and we found that numerous mitochondria-related genes were significantly downregulated, so it revealed that CVB-D treatment causes direct or indirect damage to mitochondria in lung cancer cells. In line with RNA-seq results, we also provided novel evidence that CVB-D treatment induces mitochondria dysfunction and loss by testing cellular ATP concentration, mitochondrial mass and mitochondrial DNA copy-numbers, as well as the degradation of mitochondrial proteins. Along with these findings, we also revealed that mitophagy was significantly activated upon CVB-D treatment. Subsequently a series of ATG5 knockdown experiments were conducted to confirm our results. These observations strongly supported the idea that CVB-D treatment promotes mitophagy activation and that mitophagy is a contributing factor involved in mitochondria loss in lung cancer cells.Although mitophagy is a specific autophagy that selectively removes dysfunctional or damaged mitochondria to protect cells from death , abnormaNotably, we found that mitophagy induced by CVB-D is not associated with the parkin-Pink1 pathway, a well-studied pathway involved in mitophagy activation. CVB-D treatment caused a significant decrease in parkin protein in all lung cancer cell lines upon CVB-D treatment in a dose-dependent manner for 24 h, suggesting that CVB-D-induced mitophagy in lung cancer cells is independent of parkin, at least at our time point. A previous study showed that chemical-induced mitophagy is not accompanied by the enhanced expression of parkin but with the enhanced expression of Pink1 recruited to outer membrane mitochondria . Next we125I seeds brachytherapy process, 125I seeds irradiation causes both mitochondrial damage and mitophagy activation, and the activated mitophagy contributes to the clearance of damaged mitochondria to maintain cellular homeostasis and survival [In physiological conditions, mitochondrial quality and mass are accurately controlled by undergoing mitochondrial biogenesis and mitophagy constantly . Generalsurvival . Contrarsurvival  and thatsurvival . BAY 87-survival . Ketoconsurvival . Similarsurvival  but trigsurvival .Based on our findings that mitophagy induced by CVB-D could significantly potentiate the apoptotic death of lung cancer cells, it is certain that mitophagy induced by CVB-D is excessive and toxic to lung cancer cells. Despite our experimental evidence proving that CVB-D induced mitophagy and promoted apoptotic cell death, the knockdown of ATG5 or BNIP3 led to a partial alleviation of cytotoxicity. Therefore, it is undeniable that other factors may be involved in CVB-D-triggered cell death and need to be further elucidated.In this study, we showed that CVB-D demonstrated favorable anti-lung-cancer activity both in vitro and in vivo and that it has potential application in the treatment of lung cancer. Based on its autophagy/mitophagy induction activity, its potential application in the treatment of other diseases also deserves to be explored. Under certain conditions, the activation of autophagy may serve as a treatment strategy to defend against intracellular bacteria, parasites and virus infection by targeting intracellular microorganisms (xenophagy) and mediating the clearance of microorganisms or via modulating the antiviral immune response of organisms to defend against infection . In termHuman lung cancer cell lines  were purchased from the Cell Bank of the Chinese Academy of Sciences , and cultured in DMEM containing 10% fetal bovine serum (FBS) and a 100 U/mL mixture of penicillin and streptomycin. All cells were kept at 37 °C and 5% CO2. Cyclovirobuxine D (CVB-D) was purchased from the Chengdu Alpha Biotechnology Incorporation and dissolved in methanol to generate a 70 mM stock solution. The primary antibodies for Western blotting, immunoprecipitation and immunohistochemistry against target proteins are as follows: caspase3 , cleaved-caspase3 , PARP , cleaved-PARP , VDAC1 , TOMM20 , COX4I1 , LC3 , ATG5 , SQSTM1/p62 , BNIP3 , BNIP3L , β-actin , p65 , CDC2 , cyclinB1 , p-BNIP3L (phosphoS81)  and phosphoserine .3 cells were seeded in 35 mm cell culture dishes, cultured overnight, and then cells were pretreated with various concentrations of CVB-D for 6 h, followed by replacing the CVB-D-containing medium with a fresh medium to form colonies in 2–3 weeks. Colonies were fixed with glutaraldehyde (6.0% v/v) and stained with crystal violet (0.5% w/v) .Cell viability was assessed through a cell counting kit-8 (CCK8) assay . Briefly, cells were seeded in 96-well plates at a concentration of 5 × 103 per well, incubated overnight, then subjected to different concentrations of CVB-D treatments and cultured for 24, 48 or 72 h. The CVB-D-contained medium was replaced with 100 μL fresh DMEM; CCK8 (10 μL/well) was added, and then incubated at 37 °C for 2 h, and the absorbance value was measured at a 450 nm wavelength . For clonogenic assay, 1 × 10A549, H446 and 95-D cells were seeded into 6-well plates, cultured overnight, and then the cells were treated with CVB-D for 24 h. Next, cells were harvested and stained with an Annexin V-FITC/PI apoptosis detection kit  to perform a apoptosis assay or stained with 100 nM mitotracker green  to perform a mitochondria mass assay. Reactive oxygen species (ROS) levels were assessed by staining with 10 μM fluorescent dye 2′7′-dichlorfluorescein-diacetate (DCFH-DA)  for 20 min. For a cell-cycle analysis, cells were incubated with various concentrations of CVB-D for 24 h, rinsed in precooled PBS, followed by fixing with 75% alcohol overnight at 4 °C. Then cells were digested with 1% RNase A  and stained with 1mg/mL propidium iodide (PI)  for 30 min. All experiments were performed according to the corresponding manufacturer’s instructions. At least 10,000 live cells were collected and analyzed on the flow cytometer . Experimental data were analyzed using Flow Jo 7.6 and Modifit software.g, 10 min), total proteins were quantified with a BCA Protein Assay Kit , and 30 μL of protein A-agarose beads  and 2 μg BNIP3 primary antibody were added into equal amounts of lysates and then subjected to immunoprecipitation assay overnight at 4 °C. Subsequently, the precipitates were centrifuged , resuspended and washed with washing buffer, and this process was repeated three times. Then immune complexes were eluted with SDS-PAGE loading buffer , 50% (w/v) glycerol, 5% (v/v), β-mercaptoethanol, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue) at 100 °C for 10 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Subsequently it was transferred onto 0.45 μm PVDF membranes  and blocked with 5% (w/v) skimmed-milk dissolved in a TBST buffer  Tween 20, 20 mM Tris-HCL) for 1 h and then incubated with the corresponding primary antibodies overnight at 4 °C. The PVDF membranes were washed three times (10 min/time) with the TBST buffer. Secondary HRP-labeled goat anti-Mouse lgG (H+L)  or HRP-labeled goat anti-Rabbit lgG (H+L) antibodies were applied  at 1:5000 for 1 h at room temperature, then washed three times (10 min/time) with the TBST buffer. The immunoreactions were visualized with the Western BrightTM ECL reagent  using ChemiDocTM XRS+ . For the analysis of protein expression upon CVB-D treatment, cells were rinsed in precooled PBS, and total proteins were extracted using a RIPA lysis buffer containing protease inhibitors. After centrifugation , total proteins were quantified with a BCA Protein Assay Kit . Equal amounts of protein samples (30–80 μg) were subjected to SDS-PAGE analysis. For analysis of the phosphorylation level of BNIP3, briefly, lung cancer cells were treated with the indicated CVB-D concentration for 24 h, and total proteins were extracted using a RIPA lysis buffer containing a protease inhibitor cocktail and phosphorylase inhibitor . After centrifugation, total proteins were quantified, and 30 μL of protein A-agarose beads and 2 μg BNIP3 primary antibody were added into equal amounts of lysates and then subjected to immunoprecipitation assay overnight at 4 °C, and the subsequent steps were conducted as in the above description. The phosphorylation modification of BNIP3 was detected using a phosphoserine antibody.For the interaction analysis of BNIP3 and LC3, lung cancer cells were treated with or without 30 μM CVB-D for 24 h, rinsed in precooled PBS , and total proteins were extracted using a RIPA lysis buffer  containing a protease inhibitor cocktail (EDTA-free) . After centrifugation  according to the corresponding manufacturer’s standard instructions. Briefly, cells were seeded into 60 mm dishes, cultured overnight, and then cells were treated with CVB-D for 24 h. Total proteins were extracted using an ATP detection lysis buffer and quantified with a BCA Protein Assay Kit . A total of 100 μg protein for each sample was subjected to the detection of ATP concentration, and chemiluminescence intensity was detected using a multi-mode ELISA reader . ATP concentration was calculated according to the standard curve, and the relative ATP level for each sample was normalized to the control group. The full length of LC3 cDNA was cloned into pEGFP-C1 vector  to generate a recombinant pEGFP-LC3 plasmid. To generate knockdown cell lines, shRNA against ATG5, BNIP3 and P65 was constructed into the PLKO.1 vector. Lentivirus particles were produced using a three-plasmid packaging system. pMD2G, psPAX2 and pLKO.1 ; the 293T cells and the supernatant containing lentivirus particles were collected after transfection for 48 h. A549, H446 and 95-D cells were infected with lentivirus in the presence of 8 μg/mL polybrene  for 24 h and were selected with 2 μg/mL puromycin  for two weeks. Stable expression clones were validated by Western blotting and used in the following experiments. The target sequences were synthesized by the Sangon company  and are as follows: ATG5, forward: CCGGCCTGAACAGAATCATCCTTAACTCGAGTTAAGGATGATTCTGTTCAGGTTTTT, reverse: AATTAAAAACCTGAACAGAATCATCCTTAA CTCGAGTTAAGGATGATTCTGTTCAGG. BNIP3: forward: CCGGGCCTCGGTTTCTATTTATAATCTCGAGATTATAAATAGAAACCGAGGCTTTTT, reverse: AATTAAAAAGCCTCGGTTTCTATTTATAATCTCGAGATTATAAATAGAAACCGAGGC. P65 forward: CCGGCGGATTGAGGAGAAACGTAAACTCGAGTTTACGTTTCTCCTCAATCCGTTTTT, reverse: AATTAAAAACGGATTGAGGAGAAACGTAAACTCGAGTTTACGTTTCTCCTCAATCCG. To generate P65 overexpression cell lines, the full length of p65 cDNA was inserted into pCMV-Flag vector (Addgene Company) and transfected into A549, H446 and 95-D cells, then selected with 500 μg/mL G418  to generate p65 stable expression cell lines.−ΔΔCT method. For mitochondrial DNA quantitation, total DNA was isolated with a DNA Isolation kit , followed by a qPCR analysis using the mtDNA primers : MT-ND1 , forward: CCCTAAAACCCGCCACATCT, reverse: GAGCGATGGTGAGAGCTAAGGT. Nuclear target gene LPL (lipoprotein lipase), forward: ATGAGAGTTGGGTGCCAAAAC, reverse: AGTCCACCACAATGACATTGG. All experiments were performed with the standard amplification protocol according to the corresponding manufacturer’s instructions. Relative mitochondria DNA copy numbers were normalized to LPL using the 2−ΔΔCT method.Total RNA was extracted using the trizol reagent ; a total of 1 µg RNA for each sample was reverse-transcribed using SuperRT cDNA Synthesis Kit , and quantitative real-time PCR was performed using the SYBR-Green qPCR master mix . The qRT-PCR primers in this study were synthesized by the Sangon company  and used as follows: p62, forward: CCAGAGAGTTCCAGCACAGA, reverse: TGAGGAACAGATGGAGTCGG, β-actin, forward: TGACGTGGACATCCGCAAAG, reverse: CTGGAAGGTGGACAGCGAGG, FIS1 forward: AGCGGGATTACGTCTTCTACC, reverse: CATGCCCACGAGTCCATCTTT, MFF forward: ACTGAAGGCATTAGTCAGCGA, reverse: TCCTGCTACAACAATCCTCTCC, CKMT1B forward: CTGGTTGGACGCTAGATCAGT, reverse: GGTCAGCAAATACCTCATAGGTC, PMPCA forward: CTCCTGCGTACAGACGGTTTA, reverse: CGAAGCCCATTATCCAATGTGG, AIFM1 forward: CCCAATGCTATTGTGCAATCCG, reverse: GAAGCCACCAAAATCTGAGTCTA, OXA1L forward: GCCTCAGTACCTCTGCTATCT, reverse: TCCCACTGGGGTGTATGACC, FMC1 forward: TCCCCGTCGCACACTTTTC, reverse: TGAAGCTCATGTTGGGCTCTG, IMMT forward: CGATTCAGTCGGGTCCACTAA, reverse: AGCTGGAGTATCTCCCTTTTGT, TOMM22 forward: GGCCGGAGCCACTTTTGAT, reverse: AGGTCCTAGAAGTATCTGCCG, MSTO1 forward: ATCCCAAGAACCCTTATCTCCA, reverse: GGGAGTGGTGAGGAACCTTT. The relative gene level was normalized to β-actin using the 2Cells were cultured in confocal dishes  overnight and treated with 30 μM CVB-D for 24 h. The morphology of mitochondria was determined by staining 100 nM Mito-Tracker green  for 30 min at 37 °C. Cells were washed twice, and images were visualized under confocal microscopy . To evaluate autophagic flux, GFP-LC3-transfected cells were treated with 30 μM CVB-D for 24 h, followed by fixation with 4% paraformaldehyde, then cells were washed twice and visualized under confocal microscopy (Leica TCS SP8 microscope). To detect the endogenous LC3, cells were treated with 30 μM CVB-D for 24 h, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then this process was followed by blocking with 10% goat serum. Then cells were incubated with the anti-LC3 antibody and Alexa Fluor 555 goat anti-mouse IgG (H+L) antibody, and images were visualized under confocal microscopy (Leica TCS SP8 microscope). To evaluate the colocalization of mitochondria and lysosomes, cells were treated with 30 μM CVB-D for 24 h, labeled with 100 nM Mito-Tracker green  and 50 nM Lyso-Tracker Red  for 30 min at 37 °C and visualized under confocal microscopy (Leica TCS SP8 microscope) immediately. To evaluate the change of MMP , we treated cells with 30 μM CVB-D for 24 h. Then cells were labeled with a JC-1 probe using the mitochondrial membrane potential assay kit with JC-1  according to the corresponding manufacturer’s instructions, incubated for 20 min at 37 °C, washed twice and visualized under confocal microscopy (Leica TCS SP8 microscope) immediately.Briefly, cells were treated with 30 μM CVB-D for 24 h, fixed with 2.5% glutaraldehyde buffered in a 0.1 mM phosphate buffer (pH 7.4) overnight at 4 °C, washed with phosphate buffer, followed by being embedded in 1% osmium tetroxide and dehydrated in a series concentration of ethanol. Next, cell pellets were embedded in epon resin; ultrathin sections were cut and stained with 2% uranyl acetate, and images were captured using TEM (JEM-1400plus).A549 cells were treated with 30 μM CVB-D for 24 h. Total RNA was extracted using the TRIZOL reagent , and genome DNA was digested with DNase I (Takara). The RNA library was constructed by using a TruSeqTM RNA sample preparation kit (Illumina), according to the corresponding manufacturer’s instructions, using 1 μg of total RNA. Briefly, mRNA was isolated, fragmented, and double-stranded cDNA was synthesized with random hexamer primers  using a SuperScript double-stranded cDNA synthesis kit . Subsequently, the synthesized cDNA was subjected to phosphorylation, end-repair and ‘A’ base addition according to Illumina’s library construction instructions. cDNA target fragments of 300 bp were selected and amplified. The library was subjected to sequencing with the Illumina NovaSeq 6000 sequencer (2 × 150 bp read length).Tumors obtained from A549 and H446 xenograft mice were fixed in 4% paraformaldehyde, then were paraffin-embedded and sectioned. Tumor slides were incubated with primary antibodies at 4 °C overnight, incubated with a secondary antibody, and then the chromogenic reaction was conducted with 3, 3-diaminobenzidine  and counterstained with hematoxylin  according to the corresponding manufacturer’s instructions. Images were captured using the Nikon microscope.6 of A549 and H446 cells were suspended in 100 μL of PBS and subcutaneously implanted into the flank of nude mice . When the tumor volume reached ~50 mm3, mice were randomly divided into different groups and were intraperitoneally injected with CVB-D (20 mg/kg) or a vehicle every two days. Tumor volume was measured every five days. The mice were sacrificed after 20 days of drug treatment, and tumors were harvested and analyzed.All animal experiments were approved by the institutional animal care and use committee of Chongqing University. Briefly, 5 × 10p < 0.05 was considered statistically significant.The Student’s t test and a one-way ANOVA were used to compare the differences of two groups or multiple groups. Data were presented as means ± SD of three independent experiments. A value of In summary, we propose that treatment with CVB-D in lung cancer cells can provoke mitophagy through the p65/BNIP3/LC3 axis, and that this overactivated mitophagy is one of the contributing factors in apoptosis induction. Our results provided a novel insight into the mechanism of the antitumor activity of CVB-D, and its favorable anti-lung-cancer activity in vitro and in vivo indicated that CVB-D is a potential therapeutic candidate in lung-cancer treatment.


Drosophila and mammals. Yorkie acts as a transcriptional co-activator in the Hippo pathway and cross-talks with other essential pathways. In this study, a Yorkie gene and two Cactus isoforms  were isolated and characterized from oriental river prawns (Macrobrachium nipponense). Results showed that MnYorkie includes 1620 bp open reading frame and encodes a protein of 539 amino acids (aa). MnCactus-a (377 aa) and MnCactus-b (471 aa) were produced by alternative splicing. MnYorkie and MnCactus were continuously expressed in all selected tissues. Upon Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Vibrio parahaemolyticus stimulation, the mRNA levels of MnYorkie and MnCactus in hemocytes and intestines underwent time-dependent enhancement. RNA interference studies showed that MnYorkie silencing remarkably downregulated the transcription of MnCactus but upregulated the expression of seven immune-related genes. In addition, MnYorkie silencing in vivo decreased the susceptibility of prawns to bacterial challenge. After S. aureus and V. parahaemolyticus infection, the survival rate of prawns increased significantly from 2 to 6 days, which corresponded to the period of MnYorkie knockdown. All these findings suggested that MnYorkie in the Hippo pathway might exhibit remarkable biological roles in the immune defense of M. nipponense by negatively regulating the expression of immune-related genes and promoting the transcription of MnCactus.The Hippo signaling pathway controls organ size and immune system in  Invertebrates, such as insects and crustaceans, rely mainly on various innate defense reactions to combat pathogen infections . When foDrosophila , Sav, large tumor suppressor (Lats) 1/2, Mps one binder 1A and 1B (MOB), and Yes-associated protein (YAP), respectively  were identified from M. nipponense. Their tissue distributions and temporal response to bacterial stimulation were examined. The biological function of MnYorkie was investigated by detecting the expression of downstream immune-related genes, bacterial clearance activity, and organism survival rate. The findings will provide insights into the immune defense mechanisms of crustaceans.As a member of the Palaemonidae family of decapod crustaceans, ountries . Prawns M. nipponense specimens with a weight of 2.5–3.5 g were purchased from an aquatic product market in Nanjing, China and kept in an aerated water tank filled with freshwater. After 10 days of acclimatization, hemolymph was extracted from five prawns and placed in an equal volume of an anticoagulant solution . Hemocytes were then isolated through centrifugation at 2000 rpm for 10 min. Heart, hepatopancreas, gills, intestine, and stomach were dissected from the prawns and quickly stored at –80°C until RNA extraction.A total of 200 healthy S. aureus (3 × 107 cells) and V. parahaemolyticus (3 × 107 cells) in phosphate-buffered saline . Hemocytes and intestines were collected from five live individuals at each time point  for total RNA extraction.Seventy-five prawns were randomly selected for bacterial infections . For imm® 1st Strand cDNA Synthesis Kit  with an oligo-dT primer. The obtained cDNA was kept at –20°C.Total RNA was isolated from the collected samples using the TRIzol Reagent  in accordance with the manufacturer’s protocols. RNA quality was assessed by electrophoresis on 1.5% agarose gel, and RNA concentration was measured by a NanoDrop 2000 Spectrophotometer . In brief, 1 μg of RNA was used to synthesize first-strand cDNA using the PrimeScriptM. nipponense transcriptome data, four specific primers  were designed to clone the full-lengths of MnYorkie and MnCactus. A SMARTer™ RACE cDNA Amplification Kit  was used to synthesize the first-strand cDNA. The 5′- and 3′-rapid amplification of cDNA ends (RACE) was performed using an Advantage® 2 PCR Kit  under the following PCR procedures: 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min, followed by 20 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min. The PCR products were purified using a gel extraction kit  and sequenced  after insertion into the pEasy-T3 vector .On the basis of the unigenes obtained from http://blast.ncbi.nlm.nih.gov/Blast.cgi). The deduced amino acid sequence was obtained using an open reading frame (ORF) finder program (https://www.ncbi.nlm.nih.gov/orffinder/) and then analyzed with the ExPASy Translate tool (https://web.expasy.org/translate/). Putative domains and motifs were predicted by the SMART program (http://smart.embl-heidelberg.de/). Molecular weight (Mw) and theoretical isoelectric point (pI) were calculated with the ExPASy Compute pI/Mw tool (https://web.expasy.org/compute_pi/). Multiple sequence alignments were carried out with the Clustal Omega program  and GENEDOC software. A phylogenetic tree was constructed with MEGA 7.0 software using a neighbor-joining (NJ) method  were synthesized to examine the tissue distribution and expression profiles of MnYorkie and MnCactus by RT-qPCR using the TransStart® Top Green qPCR SuperMix Kit  in LightCycler® 96 real-time PCR Detection System  under the following cycling conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 20 s, and a melting curve analysis from 65°C to 95°C. β-actin gene from M. nipponense was used as an internal PCR and cDNA template control and was positively amplified from all samples with primers (β-actin-qF: 5′-TATGCACTTCCTCACGCCATC-3′ and β-actin-qR: 5′-AGGAGGCGGCAGTGGTCAT-3′). All the experiments were repeated three times, and the obtained data were calculated using comparative CT (2−ΔΔCt) method  under the following conditions: 94°C for 3 min, 30 cycles of 94°C for 30 s, 53°C for 45 s, and 72°C for 30 s, and 72°C for 5 min. β-actin was amplified as reference gene for internal standardization (β-actin-SqF: 5′-AATGTGTGACGACGAAGTAG-3′ and β-actin-SqR: 5′-GCCTCATCACCGACATAA-3′). The PCR products were separated on agarose gels and photographed over NV light using Quantity One software .The tissue distribution of MnYorkie (MnYorkie-dsRNA-F: 5′-GCGTAATACGACTCACTATAGGGGTAGTGACCTGGTGTTG-3′ and MnYorkie-dsRNA-R: 5′-GCGTAATACGACTCACTATAGGCGCAGGCAAATGTCTCTTC-3′) and green fluorescent protein (GFP-dsRNA-F: 5′-GCGTAATACGACTCACTATAGGTGGTCCCAATTCTCGTGGAAC-3′ and GFP-dsRNA-R: 5′-GCGTAATACGACTCACTATAGGCTTGAAGTTGACCTTGATGCC-3′) were designed to synthesize DNA template for the transcription of MnYorkie-dsRNA and GFP-dsRNA. Double stranded RNAs (dsRNA) were prepared in vitro by using the HiScribe™ T7 quick high-yield RNA synthesis kit . The prawns were initially injected with 50 μL of PBS containing 15 μg of MnYorkie-dsRNA or GFP-dsRNA (as control). After 12 h, 15 μg of MnYorkie-dsRNA or GFP-dsRNA was injected into the same prawn. At 36 h post-injection (hpi), the RNAi efficiency of MnYorkie in hemocytes and intestines was checked using RT-qPCR.Primers specific to MnYorkie-dsRNA or GFP-dsRNA injection, the prawns were further injected with S. aureus or V. parahaemolyticus (3 × 107 cells) for immune challenge experiments. At 24 h post S. aureus or V. parahaemolyticus infection, the hemocytes and intestines were sampled, and the expression levels of several immune-related genes, namely, MnCactus, anti-lipopolysaccharide factor 1 (MnALF1), MnALF2, MnALF3, crustin 4 (MnCrus4), MnCrus5, MnCrus6, and lysozyme (MnLyso1) were determined using RT-qPCR with primers . Three independent experiments were performed in triplicate. The results were subjected to one-way ANOVA using SPSS 19.0, and P value less than 0.05 was considered statistically significant.At 36 h post MnYorkie-dsRNA or GFP-dsRNA was injected into the prawns twice. At 24 h post second dsRNA injection, 50 μL of S. aureus or V. parahaemolyticus (3 × 107 cells) was injected into the prawns. The hemolymph from 10 prawns was extracted and mixed with anticoagulant buffer at 20 min post-bacterial injection. After dilution 1:100/1:1000 with PBS, 50 μL of the hemolymph was loaded on LB agar plates and incubated at 37°C overnight. Colony forming unit (cfu) in the plate was counted. The number of S. aureus or V. parahaemolyticus in prawn hemolymphs was calculated and expressed as cfu mL−1 hemolymph. The assay was repeated in triplicate for all samples.In brief, 30 μg of MnYorkie-dsRNA plus S. aureus group, GFP-dsRNA plus S. aureus group, MnYorkie-dsRNA plus V. parahaemolyticus group, and GFP-dsRNA plus V. parahaemolyticus group) to determine whether MnYorkie is involved in its host’s antibacterial immune defense. Twenty prawns per each experimental group were injected with 30 μg of MnYorkie-dsRNA or GFP-dsRNA twice and then with 50 μL of S. aureus (3 × 107 cells) or V. parahaemolyticus (3 × 107 cells) at 24 h after the second injection. The number of dead prawns in these four treatment groups was monitored, and the cumulative survival rate of prawns was calculated and recorded up to 144 h. The experiment was repeated three times.Eighty prawns were divided into four groups (MnYorkie mRNA (GenBank Accession No. OL901207) is 2558 bp long consisting of a 75 bp 5′-untranslated region (UTR), an 863 bp 3′-UTR, and a 1620 bp ORF encoding a predicted polypeptide of 539 amino acids with a calculated Mw of 58.3 kDa and a pI of 5.29  and MnCactus-b (GenBank Accession No. OL901209) based on their nucleotide sequences. MnCactus-a transcript is 1614 bp long and consists of a 275 bp 5′-UTR, a 205 bp 3′-UTR, and a 1134 bp ORF encoding a 377 amino acid protein  in the prawns with effective MnYorkie knockdown (MnALF1 (MnALF3 (MnCrus5 (MnLyso1 (P < 0.05) in the MnYorkie-dsRNA injection group compared with those in the GFP-dsRNA group. No change was observed in the expression of MnALF2, MnCrus4, and MnCrus6 (data not shown). Following MnYorkie knockdown and exposure to V. parahaemolyticus for 24 h (MnCactus in hemocytes (P < 0.05), and those of four AMP genes, MnALF2 (MnALF3 (MnCrus4 (MnCrus6 (P < 0.05). These results suggested that MnYorkie negatively regulated the expression of several immune-related genes in M. nipponense.After the stimulations with nockdown 8A. On tMnYorkie towards S. aureus  was identified and functionally studied. Sequence structure and homologous phylogenetic analysis revealed that the predicted MnYorkie protein exhibits a classical Yorkie structure. Two 33 amino acid WW domains were found in MnYorkie. Given that one of the outstanding features of this sequence motif is the presence of two conserved tryptophans (W), it was named as the WW domain  were cloned and identified from M. nipponense through transcriptomic analysis and amplification by PCR. The deduced MnCactus-a and MnCactus-b amino acid sequences contained the typical characteristics of IκB members: an N-terminal serine-rich region with conserved lysine and serine residues crucial for signal-dependent degradation , V. parahaemolyticus and S. aureus injections . By inhibiting the activity of the Notch pathway, Yorkie acts as an important repressor in specifying polar cells during oogenesis in Drosophila  were identified from M. nipponense. MnCactus-a and MnCactus-b were formed by alternative splicing. Upon immune challenge with S. aureus and V. parahaemolyticus, the expression levels of MnYorkie and MnCactus mRNA were upregulated in the hemocytes and intestines of M. nipponense. RNAi experiments showed that MnYorkie knockdown affected the transcription of MnCactus and seven immune-related genes, the susceptibility of prawns to bacterial infections, and the survival rate of prawns. These results suggested that MnYorkie in the Hippo pathway might have remarkable biological roles in the immune defense of M. nipponense by negatively regulating the expression of several immune-related genes and promoting the transcription of MnCactus. Further studies are required to understand the exact mechanism of MnYorkie in crustaceans.In conclusion, a The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.YH, data curation, formal analysis, funding acquisition, investigation, methodology, software, supervision, validation, visualization, writing - original draft, and writing - review and editing. QS, formal analysis, software, and visualization. JD, data curation, funding acquisition, and validation. QR, conceptualization, project administration, funding acquisition, resources, and writing - review and editing. All authors contributed to the article and approved the submitted version.The current study was supported by the National Natural Science Foundation of China (32002423), the Natural Science Foundation of Jiangsu Province (BK20180501), the Fundamental Research Funds for the Central Universities (B200202142), the Natural Science Fund of Colleges and universities in Jiangsu Province (14KJA240002), and Youth Support Project of Jiangsu Vocational College of Agriculture and Forestry (No. 2020kj011).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. 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Escherichia coli is a leading cause of community-acquired and health care-associated bloodstream infections (BSIs) worldwide. Limited information is available regarding the changes in population dynamics of human E. coli over extended time periods, especially among nonbiased E. coli isolates in large well-defined geographical regions. Coque and colleagues  conducted a longitudinal study of E. coli BSIs in a Madrid hospital over a 21-year period (1996 to 2016). Certain E. coli B2 phylogroups  dominated the community E. coli population in Madrid. These community clones were often introduced into the hospital setting. This study and other longitudinal surveys from England and Canada showed that ST131 subclades C1 and C2 were mainly responsible for the increase in fluoroquinolone and cephalosporin resistance among E. coli during the mid- to late 2000s. Escherichia coli is the most common cause of community-acquired and health care-associated bloodstream infections (BSIs) worldwide and is responsible for substantial health care costs and patient morbidity and mortality  (The population structure of human es (STs) : ST131, es (STs) .E. coli isolates. It causes millions of human infections and deaths annually .ST131 is the most widespread clone among human annually . ST131 bannually . ST131-CE. coli isolates over extended time periods, especially among nonbiased E. coli isolates in large well-defined geographical regions. Such information will aid with designing superior treatment and prevention strategies for E. coli BSIs.Limited information is available regarding the changes in population structure of human E. coli BSIs in a Madrid hospital over a 21-year period (1996 to 2016) . Isolates (n = 45) representing highly related pulsotypes also underwent short-read whole-genome sequencing (WGS).To address this issue, Coque and colleagues conducted a longitudinal study of to 2016) . They usE. coli BSI incidence increased from 5.5 (1996) to 10.8 (2016) BSI episodes/1,000 hospital admissions. E. coli phylogroup B2 was common and mainly obtained from community patients. Other E. coli phylogroups  were rare.The E. coli population). This ST often caused infections in the elderly (>80 years). The fluoroquinolone-resistant ST131 C subclades (C1 and C2) were dominant (71%), while ST131-A (9%) and ST131-B (20%) were rare. ST131-B was present among E. coli isolates causing BSIs during 1996. ST131-C0 was first detected in 2000, while ST131-C1 and ST131-A appeared during 2004. This was followed by ST131-C2 (with blaCTX-M-15) in 2006 and ST131-C1_M27 (with blaCTX-M-27) in 2016. These interesting longitudinal data support the current understanding of ST131 evolution and expansion (E. coli population for long time periods (up to 17 years).STc131 (phylogroup B2-I) was the most common and MDR clone in the Madrid study  dominated the Madrid community E. coli population and that these clones are often introduced into the hospital setting. Community “outbreaks” often predated the hospital dissemination. Such clones were seldom the cause of large hospital-acquired outbreaks.Coque and colleagues showed that the increased incidence of hospital pidemics . Their dE. coli BSI longitudinal studies were undertaken in England and Calgary, Canada. The England study undertook an 11-year (2001 to 2011) longitudinal hospital-based survey , ST131 (14%), ST95 (11%), and ST69 (6%). ST131 was first detected in 2003. The ST131 population was also dominated by MDR C subclades. The prevalence of ST131 clades remained stable from 2006 to 2011. Therefore, the expansion of ST131 was not driven by sequential introduction of increasingly resistant subclades (Comparable d survey . They inubclades . The proE. coli isolates causing BSIs in a region of 1.4 million people (blaCTX-M-14 and ST131-C1_M27 with blaCTX-M-27). ST131 was relatively rare among E. coli BSIs during the early 2000s but increased significantly toward the latter part of the 2000s (E. coli isolates from blood increased from 53/481 (11%) in 2006 to 150/621 (24.2%) in 2012 and 141/684 (20.6%) in 2016  included all community and hospital n people . The mosn people  , especially among long-term-care residents  causing BSIs. Eliminating ST131  would substantially decrease the overall global incidence rate and MDR burden within E. coli isolates causing hospital BSIs. This will lead to considerable worldwide public health benefits.The Madrid study provided fascinating information regarding longitudinal population dynamics of


Homologous recombination-mediated genome editing, also called gene targeting (GT), is an essential technique that allows precise modification of a target sequence, including introduction of point mutations, knock-in of a reporter gene, and/or swapping of a functional domain. However, due to its low frequency, it has been difficult to establish GT approaches that can be applied widely to a large number of plant species. We have developed a simple and universal clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated DNA double-strand break (DSB)-induced GT system using an all-in-one vector comprising a CRISPR/Cas9 expression construct, selectable marker, and GT donor template. This system enabled introduction of targeted point mutations with non-selectable traits into several target genes in both rice and tobacco. Since it was possible to evaluate the GT frequency on endogenous target genes precisely using this system, we investigated the effect of treatment with Rad51-stimulatory compound 1 (RS-1) on the frequency of DSB-induced GT. GT frequency was slightly, but consistently, improved by RS-1 treatment in both target plants. On the other hand, homologous recombination (HR)-mediated gene targeting (GT) allows precise genome engineering  of endogenous target genes via “copy and paste” of sequences from a repair template. This approach is essential to create gain-of-function-type mutants. However, establishing a universal GT method that can be applied to a large number of plant species remains a challenge.Genome editing techniques have come to be required for both the development of basic research and plant molecular breeding in recent years. Targeted mutagenesis using sequence-specific nucleases (SSNs) such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is one of the genome editing techniques that has become available for several plant species and crops Voytas, . HoweverpiggyBac transposon, which transposes without leaving a footprint at the excised site, to allow precise marker elimination from a GT locus in rice  have been utilized to improve the frequency of CRISPR/Cas9-mediated GT in mammalian cells  calli were induced from mature seeds as described previously  plants were grown in soil in a greenhouse (16 h light/8 h dark) at 21°C, and mature leaves of 3- or 4-week-old plants were used for Agrobacterium-mediated transformation.Rice (BbsI site of the single guide RNA (sgRNA) expression vector described as pU6gRNA-oligo  containing a partial OsALS coding sequence with W548L/S627I mutations was amplified by PCR with the vector used in our previous study . An all-in-one GT vector for the modification of OsCly1 gene was constructed as follows. The sgRNA expression cassette (OsU6::sgCly1) was digested and ligated into the AscI/PacI site in pZH_OsU6gRNA_MMCas9. To insert the GT donor template into the vector, an artificially synthesized DNA fragment  containing a partial OsCly1 coding sequence with mutations in the miR172 targeting site was cloned into the AscI site in pZH_OsU6sgOsCly1_MMCas9. For construction of an all-in-one GT vector in tobacco, the sgRNA expression cassettes  were generated via a cut-ligation reaction with BbsI as described above. Single (for NtALS) or double sgRNA modules (for NtEPSPS) were combined with pDe_Cas9_KAN using a Gateway LR reaction (Thermo Fisher Scientific). GT donor templates for the modification of NtALS  or NtEPSPS  were synthesized artificially and introduced into the PacI site of pDe_Cas9_AtU6sgNtALS or pDe_Cas9_AtU6sgNtEPSPSx2, respectively.An all-in-one GT vector for the introduction of point mutations into the Agrobacterium-mediated transformation as described previously , and 25 μM RS-1 or 0.05% DMSO for 2 weeks at 33°C. Hygromycin-resistant calli were transferred to N6D containing 50 mg/L hygromycin and 25 mg/L meropenem without RS-1 or DMSO and cultured for 2 more weeks. To evaluate GT frequency targeting the OsALS locus without CRISPR/Cas9-mediated DSB induction, a vector harboring an 8-kb fragment of genomic DNA encoding the 5′ truncated OsALS gene carrying W548L and S627I mutations was transformed into rice calli by Agrobacterium-mediated transformation  and treated with 25 μM RS-1 or 0.05% DMSO as a control for 30 min in Murashige and Skoog (MS) medium . A portion of these compounds as used in this study was synthesized as described previously .Genomic DNA extracted from small pieces of clonally propagated hygromycin-resistant rice calli or kanamycin-resistant tobacco calli using Agencourt chloropure (Beckman Coulter) according to the manufacturer's protocol was subjected to cleaved amplified polymorphic sequence (CAPS) analysis. PCR amplifications were performed with KOD FX neo or KOD ONE (TOYOBO) using the primer sets shown in PCR fragments derived from CAPS-positive calli or plants were cloned into pCR-BluntII-TOPO (Invitrogen) and subjected to sequence analysis using an ABI3130 sequencer (Applied Biosystems).hpt, nptII, and OsCly1 locus were synthesized with a PCR DIG probe synthesis kit (Sigma-Aldrich) according to the manufacturer's protocol using the primers shown in Genomic DNA was extracted from leaves of seedlings using the Nucleon Phytopure extraction kit (Cytiva) according to the manufacturer's protocol. Two micrograms of rice genomic DNA or 15 μg of tobacco genomic DNA was digested with the same restriction enzyme as that used in CAPS analysis and fractionated in a 0.8% agarose gel. Southern blot analysis was performed according to the digoxigenin (DIG) Application Manual (Sigma-Aldrich). Specific DNA probes for the 1 progeny of OsALS-GT RS-1_B were sown on 1/2 MS medium with or without 1.5 μM BS and were grown in a growth chamber at 27°C under a 16 h photoperiod. Seeds of wild-type tobacco (SR-1) and T1 progeny of NtALS_GT RS-1_B#1 were sown on 1/2 MS medium with or without 500 nM chlorsulfuron (CS) and grown in a growth chamber (16 h light/8 h dark) at 27°C.Seeds of wild-type rice (Nipponbare) and Tacetolactate synthase (OsALS) gene  gene expression cassettes, and a GT donor template (ca. 700 bp) carrying W548L and S627I, in which these point mutations generate recognition sites for the restriction enzyme MfeI, CAATTG, was constructed and transformed into rice calli  and 25 μM RS-1 treatment groups. CAPS analysis, i.e., PCR analysis coupled with MfeI digestion, revealed that there were no GT positive callus lines from among 960 independent transgenic calli harboring all-in-one GT vector treated with DMSO  under treatment with RS-1  and 37.5% (9/24), respectively  from OsALS-GT RS-1_B analyzed  had an identical mutation pattern, which was the desired point mutation introduced via GT and the long deletion (243 bp) between the two target sequences of CRISPR/Cas9 in each OsALS allele, respectively (1 plants from self-pollinating regenerated plants (OsALS-GT RS-1_B#1 and #2) of the OsALS-GT RS-1_B line and conducted genotyping of the OsALS gene and the BS-sensitivity test. We found that all progeny plants from OsALS-GT RS-1_B#1 T0 plants carried homozygous point mutations via GT in the OsALS gene  was a heterozygous mutant carrying GT-mediated point mutations and the CRISPR/Cas9-mediated deletion in each allele of OsALS  conferring tolerance to the herbicide bispyribac sodium (BS) into the rice ce calli . The desce calli . Therefoith DMSO , while Mith RS-1 . Sequencectively . These rectively . FurtherALS gene . In anotof OsALS . These rd plants . These Te plants . To provectively . These dcleistogamy 1 (OsCly1) gene , with no change to amino acid sequences  and 12 (50%), respectively, were true GT plants containing the desired GT-mediated mutations and CRISPR/Cas9-mediated indels in each allele of the OsCly1 gene, while all regenerated plants from OsCly1-GT DSMO_B1 and B2 callus lines lacked GT-mediated point mutations in the OsCly1 locus (0/24) (via GT/4 bp deletion) in the OsCly1 gene   . All GT ly1 gene , while dsertion) . Furtherregation .NtALS-B) using an all-in-one vector. A vector carrying an sgRNA targeting region proximal to S647 in the NtALS-B gene, Cas9, nptII expression cassettes, and GT donor template 1.0 kb NtALS-B coding region with mutations (W568L/MfeI site and S647I) was introduced into tobacco leaf discs with or without RS-1 by Agrobacterium-mediated transformation  and 0.22%  of transgenic calli treated with DMSO and RS-1, respectively, harbored a GT-modified NtALS-B gene  and 7.8%  of T0 plants regenerated from NtALS_GT RS-1_B and DMSO_C callus lines, respectively, but not from NtALS_GT RS-1_A1 and A2. Sequencing analysis of PCR fragments from CAPS-positive plants (NtALS_GT RS-1_B#1-B#4 and NtALS_GT_DMSO_C#1-C#4) showed that W568L (MfeI recognition site) and S647I mutations had been introduced into the targeted NtALS-B gene in the biallelic state in NtALS_GT_RS-1_B#1-B#4 or in the chimeric state in DMSO_C#1–C#4, respectively. However, in plants NtALS_GT_RS-1_B#2–B#4 and DMSO_C#1–C#4, unexpected mutations (insertion or deletion) also occurred at the CRISPR/Cas9 target region, suggesting that CRISPR/Cas9 recognized and digested again at the target sequence even after the introduction of S647I mutations via GT  conferring tolerance to the herbicide CS in tobacco ALS-B ; however, additional mutations were also generated by CRISPR/Cas9 in 37.5% of T1 progenies , a well-known target of the herbicide glyphosate that catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis . Thus, we attempted to introduce TIPS (T176I and P180S) amino acid substitutions into tobacco EPSPS-B (NtEPSPS-B) using a CRISPR/Cas9-mediated GT approach  with or without RS-1 treatment. We isolated GT callus lines by CAPS analysis (P180S mutations carry a new HindIII restriction enzyme recognition site). CAPS-positive bands were detected in one (NtEPSPS_GT DMSO_1) and two (NtEPSPS_GT RS-1_1 and 2) independent lines from 384 transgenic calli treated with DMSO and RS-1, indicating that GT frequency was 0.26% and 0.52% in the treatment of DMSO and RS-1, respectively . Structural optimization of RS-1 to plant Rad51, or structural modification of RS-1 to enhance delivery efficiency into plant cells, is expected to lead the development of more effective approaches to establish a universal GT system for various types of crops.Positive–negative selection to enrich GT cells has been developed and applied successfully to the modification of several endogenous target genes in rice 1 progenies with the all-in-one vector, but not in siblings in which the all-in-one vector was segregated out (via GT within the seed region of the target sequence. To completely abolish induction of these de novo mutations in T1 tobacco plants, it is essential that multiple mismatches, or a single mismatch, are introduced into the sgRNA target site or PAM sequence via GT, at least in dicot plants.In tobacco, we found that CRISPR/Cas9-mediated additional mutations were also generated at the target site in Tated out . These rin planta GT strategy in which DSBs were induced by SSNs at both ends of a GT donor template on a GT vector integrated into the host genome and at an endogenous target gene, resulting in repair of the target gene via HR using a released linear GT donor from a GT vector in Arabidopsis. Using these strategies, although GT frequency was still low in higher plants, GT cells were enriched by counter selection derived from the introduction of targeted point mutations conferring resistance to herbicide, knock-in of antibiotic resistance or a reporter gene into the target gene via GT. Miki et al. (Arabidopsis. This tool provides a powerful, but thus far Arabidopsis-specific, approach.CRISPR/Cas9-mediated GT strategies have been applied for precise gene modification in plants by several research groups. Geminivirus-based replicons have been used for transient expression of SSNs and delivery of DNA donor template, resulting in successful knock-in of reporter or antibiotic-resistance genes, etc., into an endogenous target gene in tobacco (Baltes et al., i et al.  revealedHere, we have established a CRISPR/Cas9-mediated GT strategy using an all-in-one vector in combination with RS-1 treatment in rice and tobacco. Although removal of an all-in-one vector remains challenging in vegetative propagation of plants, we are hopeful that our approach will become widely applicable for precise genome modification in a variety of crops. Therefore, we are currently screening several chemicals with the aim of improving GT efficiency in plants.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AN-Y designed the research and wrote the manuscript. AN-Y and MM conducted the experiments. ST commented on the research and edited the manuscript. All authors contributed to the article and approved the submitted version.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.


Mitochondria have been shown to play vital roles during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) development. Currently, it is unclear whether mitochondrial DNA (mtDNA) variants, which define mtDNA haplogroups and determine oxidative phosphorylation performance and reactive oxygen species production, are associated with COVID-19 risk.n = 615) and COVID-19 patients (n = 536). COVID-19 patients were diagnosed based on molecular diagnostics of the viral genome by qPCR and chest X-ray or computed tomography scanning. The exclusion criteria for the healthy controls were any history of disease in the month preceding the study assessment. MtDNA variants defining mtDNA haplogroups were identified by PCR-RFLPs and HVS-I sequencing and determined based on mtDNA phylogenetic analysis using Mitomap Phylogeny. Student’s t-test was used for continuous variables, and Pearson’s chi-squared test or Fisher’s exact test was used for categorical variables. To assess the independent effect of each mtDNA variant defining mtDNA haplogroups, multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) with adjustments for possible confounding factors of age, sex, smoking and diseases  as determined through clinical and radiographic examinations.A population-based case–control study was conducted to compare the distribution of mtDNA variations defining mtDNA haplogroups between healthy controls (ND2) and A249d /T6392C /G10310A (in ND3) were associated with a reduced risk of severe COVID-19 , while A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) were related to an increased risk of severe COVID-19 .Multivariate logistic regression analyses revealed that the most common investigated mtDNA variations (> 10% in the control population) at C5178a (in NADH dehydrogenase subunit 2 gene, This is the first study to explore the association of mtDNA variants with individual’s risk of developing severe COVID-19. Based on the case–control study, we concluded that the common mtDNA variants at C5178a and A249d/T6392C/G10310A might contribute to an individual’s resistance to developing severe COVID-19, whereas A4833G, A4715G, T3394C and G5417A/C16257a/C16261T might increase an individual’s risk of developing severe COVID-19.The online version contains supplementary material available at 10.1186/s40779-021-00351-2. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a worldwide crisis of formidable morbidity and mortality. The epidemiology, diagnosis, risk factors and treatments of COVID-19 have been explored intensively since the outbreak in Wuhan  in December 2019. Many studies have shown that the clinical features of COVID-19 range from an asymptomatic state to acute respiratory distress syndrome (ARDS) and multiorgan dysfunction. Most COVID-19 patients develop a respiratory tract infection with common symptoms of cough, fever and shortness of breath. Other reported symptoms are weakness, malaise, respiratory distress, muscle pain, sore throat and loss of taste and/or smell. A number of patients develop severe fatal consequences resulting from a surge of inflammatory events  ; a genomRecently, mitochondria have been inferred to be interrelated and interacted with oxidative stress and inflammation during SARS-CoV-2 infection and COVID-19 progression , 9. FurtAs the hub of cellular oxidative homeostasis, mitochondria generate approximately 85% reactive oxygen species (ROS) when they produce usable energy through oxidative phosphorylation (OXPHOS). In contrast to other cellular organelles, mitochondria have their own DNA (mtDNA). Interestingly, the common “nonpathological” mtDNA variation defining mtDNA haplogroups determines OXPHOS performance and ROS production in humans and mice . Addition = 536) were recruited from Taihe Hospital  and the People’s Hospital of Hubei Province  from February 2020 to March 2020. COVID-19 patients were diagnosed based on molecular diagnostics of the viral genome by qPCR and chest X-ray or computed tomography scanning, and stratified into cases with moderate (non-ICU) and severe (ICU) disease . Age- and sex-matched healthy volunteers (n = 615) were individuals who underwent physical examinations at the two hospitals. The exclusion criterion for the healthy controls was any history of disease in the one-month preceding the study assessment. All subjects were unrelated for at least three generations. After explaining the purpose and procedures of the study, all the participants signed a written informed consent form and completed a detailed questionnaire on their smoking habits.This population-based case–control study was approved by the Ethics Committee of Hubei University of Medicine  (2020-TH-063 and 2020-TH-064). COVID-19 patients .AluI, AvaII, BamHI, BstNI, DdeI, HaeII, HaeIII, HhaI, HincII and HinfI were used in this study . The primers for PCR-RFLPs and HVS-I sequencing were synthesized by Sangon Biotech Co., Ltd. , and the primer sequences are presented in Additional file MtDNA variants defining mtDNA haplogroups were identified using PCR-restriction fragment length polymorphism (PCR–RFLP) and replenished by hypervariable segment I (HVS-I) sequencing as previously described , 18, 19.To explore whether the mtDNA variants defining mtDNA haplogroups were associated with individual’s susceptibility to COVID-19, the distribution of mtDNA variants defining mtDNA haplogroups were compared between the pooled cases and controls after the mtDNA variants defining mtDNA haplogroups were identified using PCR–RFLP replenished by HVS-I sequencing for all of the subjects. Additionally, the distribution of mtDNA variants defining mtDNA haplogroups were analysed between controls and moderate cases or severe cases.t-test was used for continuous variables, and Pearson’s chi-squared test or Fisher’s exact test was used for categorical variables. For multiple comparisons of mtDNA variants defining mtDNA haplogroups, Bonferroni correction was applied (the required significance level was P = 0.05/number of comparisons). To assess the independent effect of each mtDNA variant defining mtDNA haplogroups, multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) with adjustments for the possible confounding factors of age, sex, smoking and diseases  as determined through clinical and radiographic examinations. All statistical analyses were performed using SPSS Statistics 25 for Mac .Student’s P < 0.001). After the cases were stratified into moderate and severe cases, the severe patients were found to be older than controls (P < 0.001). MtDNA variants defining mtDNA haplogroups were detected for all subjects. Pearson’s chi-squared test or Fisher’s exact test showed that mtDNA variants A4833G , A4715G , T3394C , and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) (defining mtDNA haplogroup N9a) were significantly higher , while mtDNA variants C5178a  and A249d (D-loop)/T6392C /G10310A (ND3)  were significantly lower in COVID-19 patients than in controls . When Bonferroni correction was applied, A4715G, G5417A/C16257a/C16261T and C5178a reached the required P value of < 0.0033 (0.05/15). Multivariate logistic regression analyses with adjustments for age, sex, smoking and diseases revealed that, based on a P value of < 0.05, A4833G, A4715G, T3394C and G5417A/C16257a/C16261T were associated with an increased risk of COVID-19 . In contrast, C5178a and A249d (D-loop)/T6392C (CO1)/G10310A (ND3) variants were associated with a reduced risk of COVID-19  , while the 8281–8289d variant  was significantly lower in moderate COVID-19 patients than in controls (P < 0.001). All of these mtDNA variants  reached the required P value of < 0.0033 when Bonferroni correction was applied. Multivariate logistic regression analyses with adjustments for age, sex, smoking and diseases showed that 8281–8289d was associated with a reduced risk of moderate COVID-19 , whereas A663G and G5417A/C16257a/C16261T variants were related to an increased risk of moderate COVID-19  (Table When COVID-19 patients were stratified into cases with moderate (non-ICU) and severe (ICU) disease , Pearson’s chi-squared test or Fisher’s exact test showed that the mtDNA variants A663G  and G5417A/C16257a/C16261T were significantly higher , while C5178a and A249d/T6392C/G10310A were significantly lower in severe COVID-19 patients than in controls . When Bonferroni correction was applied, A4833G, A4715G and C5178a remained significant. Multivariate logistic regression analyses with adjustments for covariates showed that C5178a and A249d/T6392C/G10310A variants were associated with a reduced risk of severe COVID-19 , while A4833G, A4715G, T3394C and G5417A/C16257a/C16261T variants were related to an increased risk of severe COVID-19   and A249d (D-loop)/T6392C (CO1)/G10310A (ND3) contributed to an individual’s resistance to developing severe COVID-19, whereas A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) polymorphisms increased the risk of severe COVID-19 in this population. Additionally, the mtDNA variants A663G (12S rRNA) and G5417A/C16257a/C16261T increased an individual’s risk of developing moderate COVID-19, while the 8281–8289d variant (located in a non-coding region) decreased an individual’s risk of developing moderate COVID-19. The mtDNA variants G5417A/C16257a/C16261T were risk factors for both moderate and severe COVID-19.To provide insight into COVID-19 risk in terms of mtDNA variation, we conducted a case–control study in a Han Chinese population from Central China. Our data demonstrated that the most common investigated mtDNA variants (> 10% in the control population) at C5178a  and G10310A  variants are common variants of mtDNA sub-haplogroups F1–4 [ND5, defining mtDNA sub-haplogroup F1) and T16298C/C16304T/T16362C  were detected, and both had significantly lower frequencies in COVID-19 patients . As expected, the combined sub-haplogroups F1 and F3 (representing haplogroup F) were associated with a decreased risk of COVID-19 and severe COVID-19. In Asian populations, haplogroup F is a positive factor associated with a long life-span [CO1)/G10310A (ND3) might have protective functions against COVID-19 by regulating the replication and transcription of mtDNA and/or the translation speed through codon optimization, protein folding or other mechanisms.The A249d (D-loop), T6392C , A4715G (synonymous mutation in ND2), T3394C (Tyr30His in ND1) and G5417A (synonymous mutation in ND2)/C16257a (D-loop)/C16261T (D-loop) were found to increase an individual’s risk of developing severe COVID-19 in our study. Of note, A4715G, T3394C and G5417A/C16257a/C16261T are reported to be associated with an increased risk of type II diabetes mellitus (T2DM) in the Chinese population [P = 0.0001) [The mtDNA variants A4833G  , COPD [1ND2  confers a higher risk of MetS development in HIV-infected patients [The mtDNA variant G5417A in patients . In the patients , which mInterestingly, certain variants in human mtDNA significantly change specific OXPHOS enzyme activity and response to OXPHOS targeting agents in yeast models , indicatOf note, a mtDNA deletion of approximately 800 bp was detected during the PCR–RFLP analysis in this study. In our previous studies, an 822 bp mtDNA deletion was identified and demonstrated to be positively associated with cigarette smoking and mtDNA haplogroups , 19. BecThis study had some limitations. First, during the extreme clinical circumstances of the pandemic, especially at the beginning of the epidemic, we were unable to recruit asymptomatic patients and collect detailed clinical data  in a very short period of time, which will be important to investigate in follow-up studies. Second, PCR–RFLP and HVS-I sequencing approaches were used to identify mtDNA variants in the study. Although they can detect known variants defining mtDNA haplogroups, these methods are unable to detect other mtDNA variants and heteroplasmy information on all variants, as other novel techniques such as the next generation sequencing (NGS) of mtDNA do. In future projects, NGS and other novel methods will be utilized to provide more comprehensive information on mtDNA variation. Third, two mtDNA macrohaplogroups M and N, emerging from African-specific mtDNA L3 in Northeast Africa, left Africa successfully and colonized the rest of the world. The mtDNA macrohaplogroup N gave rise to multiple European, Asian and Native American mtDNA lineages . Of all the Asian mtDNA lineages, only haplogroups A, C, D and X became enriched in Northeast Siberia and crossed the Bering Land Bridge into the Americas and haplogroup B colonized the Pacific Islands, demonstrating that haplogroups A, C, D and X have been subjected to much greater cold stress than haplogroup B or the African macro-haplogroup L , 44. TheND2) and A249d (D-loop)/T6392C (CO1)/G10310A (ND3) contributed to resistance to COVID-19 development, whereas A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) variants may be risk factors, providing evidence that the gene-environment interactions exist in COVID-19 progression.Our findings revealed for the first time that the common mtDNA variants at C5178a  validation of mtDNA variations mentioned in the study


C. elegans was the first animal to have its genome completely sequenced. In the decades since, the genome continues to be actively curated, annotated, and improved. Here we report the discovery of a new gene in a region of the genome that is currently not associated with any annotated gene or feature. We present RNA-seq and RT-PCR evidence that this gene is expressed at detectable levels, and that it is alternatively spliced. The new gene (Y97E10C.2) shares an operon with two upstream genes. We provide RNA-seq and RT-PCR evidence for a missing exon in the upstream gene T05B11.7, as well as an alternatively-spliced exon. C. elegans genome sequence was completed , ongoing curation efforts continue to further improve and annotate the genome . In the course of searching for alternatively-spliced genes using RNA-seq data, we happened upon an alternative splicing event in a region of the C. elegans genome with no associated gene or feature annotation. Upon closer inspection, we identified RNA-seq support for an unannotated four-exon gene, with the third exon being alternatively spliced . The putative coding sequences of this gene encode proteins of either 86 or 122 amino acids, depending on the splicing choice. The longer isoform (exon three included) is the major isoform according to our RNA-seq data , and this isoform encodes four predicted transmembrane domains . RT-PCR of wild-type L4 stage worms further confirms that the gene is expressed at detectable levels, and that exon three is alternatively spliced . We have submitted this information to WormBase, and the new gene is set to appear as WBGene00306123 (transcripts Y97E10C.2a and Y97E10C.2b) in release WS283.In the decades since the T05B11.7) that deviates from its annotated gene model. The RNA-seq data indicate that prior to reaching the annotated stop codon in exon three, the mRNA is spliced to an unannotated downstream exon . Therefore, the annotated stop codon is in fact located within an intron, and the T05B11.7 gene contains four exons. This increases the coding sequence of T05B11.7 from 852 to 924 nucleotides. We also found evidence for alternative splicing in the T05B11.7 gene. Exon one harbors an alternative 5’ splice site which is used in a small fraction of the transcripts detected by RNA-seq  and which is also detectable via RT-PCR . These alternative isoforms (including the new addition of exon four) will appear in WS283 as transcripts T05B11.7a and T05B11.7b.While inspecting this locus, we also noted RNA-seq evidence for the gene immediately upstream (T05B11.7 is currently annotated as part of an operon (CEOP5584) along with its upstream gene clic-1 . Our newly-described gene Y97E10C.2, which is located immediately downstream of T05B11.7, is now included as the final gene in this operon, making CEOP5584 a 3-gene operon .Finally, the gene Analysis of RNA-seq data: Wild-type RNA-seq data used in this manuscript were previously published  using polyA-selected RNA obtained from either L3-stage or L4-stage wild-type (N2) hermaphrodites. In our initial search for alternative splicing which led to the identification of gene Y97E10C.2, we performed differential splicing analysis using JUM  on STAR-mapped  short read (150 bp paired end) sequencing, stipulating that to be considered an alternative splicing event, both alternative junctions must be represented by at least 5 unique junction-spanning reads in at least two out of the three biological wild-type replicates tested.RT-PCR: RT-PCR was performed on total RNA extracted from L4-stage wild-type (N2) hermaphrodites. Primers for Y97E10C.2: CGAGTCAAACTGAGCATGTG & AACACTCCACCAACAAGTAGAC. Primers for T05B11.7: TGTGCAACGGCAGCAAGAAG & CCAACAGTTTCCAGCTCCGAATC.


African swine fever virus (ASFV) is responsible for enormous economic losses in the global swine industry. The ASFV genome encodes approximate 160 proteins, most of whose functions remain largely unknown. In this study, we examined the roles of ASFV K205R in endoplasmic reticulum (ER) stress, autophagy, and inflammation. We observed that K205R was located in both the cytosolic and membrane fractions, and formed stress granules in cells. Furthermore, K205R triggered ER stress and activated the unfolded protein response through activating the transcription factor 6, ER to nucleus signaling 1, and eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3/PERK) signaling pathways. Moreover, K205R inhibited the serine/threonine kinase 1 and the mechanistic target of the rapamycin kinase signaling pathway, thereby activating unc-51 like autophagy activating kinase 1, and hence autophagy. In addition, K205R stimulated the translocation of P65 into the nucleus and the subsequent activation of the nuclear factor kappa B (NF-κB) signaling pathway. Inhibition of ER stress with a PERK inhibitor attenuated K205R-induced autophagy and NF-κB activation. Our data demonstrated a previously uncharacterized role of ASFV K205R in ER stress, autophagy, and the NF-κB signaling pathway. African swine fever virus (ASF), the only known DNA arbovirus, is prevalent in Africa, Europe, and Asia . It causVarious viruses induce endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) . Three sAutophagy is a conserved self-degradative process, which is important for cellular homeostasis and host-pathogen interactions . ASFV E1ASFV K205R has been found to be expressed in the early stages of infection, from 4 h post-infection, and is located in viral factories. Therefore, the K205R gene has received extensive research attention . Adenovi2. The 3D4/21 P65−/−, PK-15 ATG5−/−, and Beclin-1−/− cells were used and cultivated as previously described , Atf4, Gadd34, and Chop were all up-regulated in response to K205R expression (Cellular stress, such as ER stress, is a strong inducer of SG formation . Therefopression C–G. Treapression H. TogethWe next attempted to examine whether K205R activates autophagy, given that ER stress is a potent trigger of autophagy . We tranATG5−/− and Beclin-1−/− cells. K205R failed to induce autophagy in the ATG5−/− and Beclin-1−/− cells, as indicated by immunoblotting analysis of LC3-II, ATG5, ATG12, and Beclin-1 (Given that ATG5 and Beclin-1 are essential for the formation of autophagosomes , we veriBeclin-1 E. InhibiBeclin-1 F. The AKBeclin-1 G. This rBeclin-1 G. CollecIt is known that ER stress can elicit proinflammation , therefoIl-6, Il-18, and Tnfa by qRT-PCR analysis. K205R increased the mRNA levels of Il-6, Il-18, and Tnfa in a K205R dose-dependent manner (P65−/− cells. Neither LPS nor K205R stimulated the transcription of Il-6 and Il-18 in the 3D4/21 P65−/− cells (Il-6, Il-18, and Tnfa, as well as the secretion of IL-18 when K205R-HA was expressed in cells (Given that NF-κB activation triggers the expression of proinflammatory cytokines, we next examined the transcription of t manner D. K205R t manner E. We fur/− cells F,G. We f/− cells H. GSK trin cells I,J. TogeASFV causes an acute and fatal disease affecting domestic pigs. Its genome encodes more than 160 proteins. Better understanding of the roles of ASFV proteins would be valuable in supporting vaccine development, given that no vaccine is available for the prevention and control of ASFV. In the present study, we examined the roles of ASFV K205R in ER stress, autophagy, and inflammation. K205R induced ER stress, thereby activating the UPR via the ATF6, IRE1, and PERK signaling pathways. Subsequently, K205R stimulated autophagy and NF-κB activation, which were prevented by inhibition of ER stress. Our results suggested that K205R induced ER stress and consequently activated autophagy and the NF-κB signaling pathway.K205R has been shown to have high antigenicity and can be used for the detection of ASFV-specific antibodies . AdenoviSeveral lines of evidence indicate that ASFV modulated ER stress. Xia and colleagues have shown that ASFV P17 inhibited cell proliferation through ER stress and ROS-mediated cell cycle arrest . Our datInnate immunity is the front-line defense against viral infections . Cyclic


Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping.The unconventional mRNA capping enzyme  domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal  Vesicular stomatitis virus (VSV), an animal rhabdovirus closely related to rabies virus, has served as a paradigm for understanding the basic molecular mechanisms of transcription and replication by rhabdoviruses  and other non-segmented negative strand (NNS) RNA viruses, such as measles and Ebola. NNS RNA viral polymerases sequentially synthesize the non-coding leader RNA and monocistronic mRNAs from the 3′-terminal leader region and internal genes, respectively, on their genomes by the stop-start transcription mechanism. A hallmark of NNS RNA viral polymerases is the presence of a unique enzymatic domain, called GDP polyribonucleotidyltransferase (PRNTase), which catalyzes pre-mRNA 5′-capping, one of the essential mRNA modifications. Our recent study revealed that the VSV PRNTase domain directs transcription initiation at the 3′-end of the genome as well as pre-mRNA capping with the dual functional priming-capping loop during stop-start transcription. Here, we further show that a helix structure flanked by the priming-capping loop regulates not only transcription elongation at an early phase of leader RNA synthesis but also polyadenylation-coupled transcription termination at gene junctions. These findings indicate that the PRNTase domain acts as a key regulatory domain for stop-start transcription as well as a catalytic domain for pre-mRNA capping. Rhabdoviridae family in the order Mononegavirales, has been used as a prototypic virus to study the molecular mechanisms of transcription and replication in this order of viruses  into monocistronic mRNAs with a 5′-cap 1 structure  and 3′-poly(A) tail via a stop-start transcription mechanism GDP as a pRNA acceptor substrate . Relative activities of these mutants to produce GpppA with GDP  were roughly consistent with their total cap synthesis activities with GTP . The K1172A , R1178A (lane 5), and R1178K (lane 6) mutations enhanced the GpppA formation with GDP to approximately 230, 310, and 160% of the WT level, respectively. It was also confirmed that the R1181A mutant retains approximately 12% of the WT activity . Although some mutations of these basic amino acid residues in the helix structure were found to modulate the cap synthesis activities, none of these residues were critical for both the GTPase and PRNTase reactions.To analyze which step(s) of the capping reaction was affected by these mutations, we performed the GTPase and PRNTase assays separately. The GTPase reaction was monitored using [α-Fig 3A). As reported [N [1.3 kilo nucleotides (knt)], P (0.8 knt), M (0.8 knt), and G (1.6 knt) mRNAs and some minor transcripts  . It should be noted that monocistronic P and M mRNAs co-migrated in the denaturing gel (labeled by P/M). The K1172A (lane 3), K1177A (lane 4), and R1181A (lane 7) mutations had small or moderate effects on mRNA synthesis, whereas the R1183A (lane 8) and R1183K (lane 9) mutations almost completely abolished mRNA synthesis. Unexpectedly, the R1178A (lane 5) and R1178K (lane 6) mutations induced increased production of unknown transcripts  significantly longer than the monocistronic mRNAs produced by the WT L protein. These mutations reduced their LeRNA synthesis activities mildly  or severely . The R1183A (lane 8) and R1183K (lane 9) mutants produced smaller amounts of full-length LeRNA, while making larger amounts of a short RNA , compared to the WT L protein . This suggests that the R1183A and R1183K mutant L proteins tend to prematurely terminate LeRNA synthesis during chain elongation more frequently than the WT L protein. In contrast, the R1178A mutant did not produce detectable amounts of such short RNAs (lane 5), suggesting that this mutation may affect a very early stage of LeRNA synthesis. Consistent with these observations, de novo initiation of LeRNA synthesis (first-phosphodiester bond formation) at the 3′-terminal of the genomic RNA was significantly diminished by the R1178A mutation . Thus, this R1178A mutant has multiple abnormalities in RNA synthesis and capping. In contrast, the R1178K mutant retained about 70% of the WT initiation activity (lane 6), suggesting that a basic amino acid residue at this position is required for efficient transcription initiation as well as GpppA-cap formation. On the other hand, the R1183A (lane 8) and R1183K (lane 9) mutants exhibited transcription initiation activities comparable to that of the WT L protein. In addition, all these mutant L proteins were able to interact with the essential RdRp co-factor P protein  as in the case of the WT L protein (lane 3), suggesting that these mutations do not perturb the folding of the L protein or disrupt this essential interaction.In order to investigate effects of the mutations of the basic amino acid residues on transcription, we used our transcription system reconstituted with the N-RNA template and recombinant L and P proteins . As reported [32P]GMP-labeled LeRNA with residues 1–18 (LeRNA18), some transcripts longer than expected (19–21 nt), and abortive transcripts (3–12 nt) . By adding excess concentrations of GTP and UTP after the pulse reaction, the WT L protein elongated [α-32P]GMP-labeled LeRNA18 into full-length LeRNA (47 nt) (lane 3). Similarly, the R1178K mutant produced LeRNA18 (lane 4) and elongated it to full-length LeRNA (lane 5) during the pulse and chase periods, respectively, although its LeRNA synthesis activity was slightly lower than that of the WT L protein. As in the case of the WT L protein, the R1183K mutant produced abortive transcripts with 3–12 nt during the pulse period (lane 6). However, the R1183K mutant produced lower and higher amounts of LeRNA18 and LeRNA12, respectively (lane 6), by comparison to the WT L protein . Interestingly, once LeRNA18 was formed , the R1183K mutant was found to elongate it to full-length LeRNA during the chase period (lane 7). Since the R1183K mutation did not affect terminal de novo initiation as shown in Le promoter followed by the formation of a stable LeRNA elongation complex.We further analyzed LeRNA chain elongation activities of selected mutants, R1178K and R1183K, using our pulse-chase LeRNA synthesis assay  reported , during 32P-labeled oligo-DNA probes complementary to N, P, M, and G mRNAs . As reported previously  . Although a positive control RNA containing the Le and N regions synthesized by T7 RNA polymerase (Le-NΔ) was detected with the Le(−) probe, any transcripts synthesized by the R1178 mutants (lanes 2 and 3) as well as the WT L protein (lane 1) were not reacted with this probe. In contrast, when the same samples were re-probed with the N(−) probe , transcripts containing the N region were detected (lanes 1–3) as shown in Fig 5A. These results indicate that the R1178 mutants frequently read through the gene-junctions, but not the Le-N junction . We analyzed sequences of the N-P junction in 12 cDNA clones derived from polyadenylated polycistronic mRNAs synthesized by the R1178K mutant L protein and confirmed that all the clones contain no extra A residues introduced in the junction , indicating that the polycistronic mRNAs are bona fide read-through products .In order to identify the long transcripts synthesized by the R1178A and R1178K mutants, Northern blotting was performed with eviously ,34, thesL gene of the VSV genome on generation and growth of recombinant mutant VSVs in host cells. However, VSV harboring the R1183K mutation could not be recovered, although we tried multiple times, suggesting that the R1183K mutation is lethal to VSV. On the other hand, VSV with L mutation R1178K was successfully generated, but exhibited a small-plaque phenotype  when compared to the WT virus (left upper). We confirmed that the L gene from plaque-purified R1183K mutant VSV has the R1183K mutation , but not any other nucleotide changes in the N, P, and L genes. Single-step growth curve experiments showed that the R1178K mutant is grown more slowly than the WT virus . Unexpectedly, the R1178K mutant produced 5–10-fold higher levels of N, P, M, and G monocistronic mRNAs than the WT virus at 6-h, 9-h, and 12-h post-infection . On the other hand, levels of the N, P, and G proteins expressed in the R1178K mutant virus-infected cells were slightly higher than those in the WT virus-infected cells, while levels of the M protein in the R1178K mutant virus-infected cells were equivalent or lower than those in the WT virus-infected cells . Although, it is, at present, not clear whether all these unique phenotypes of the mutant virus are caused by the R1178K mutation alone and/or another unidentified complementation mutation(s) in the genome, we observed that the mutant virus produced large amounts of polycistronic mRNAs in addition to the monocistronic mRNAs throughout the course of infection  as expected. To identify these polycistoronic mRNAs, we performed Northern blotting with the probes against N , P , M , and G  mRNAs. Since the amount of the monocistronic N mRNA accumulated in R1178K mutant VSV-infected cells at 9-h post-infection were approximately 10-fold higher than that in WT VSV-infected cells , a 10-fold lower amount of the total RNAs from R1178K mutant-infected cells than those from WT virus-infected cells were analyzed. The WT virus mainly generated monocistronic mRNAs , whereas R1178K mutant VSV was found to synthesize polycistronic read-through mRNAs, such as N-P, N-P-M, and P-M, in addition to monocistronic mRNAs (lane 3) as observed in in vitro transcription. The percentages of read-through at the N-P and P-M junctions by the R1178K mutant virus were 27% and 48%, respectively, whereas those by the WT virus were 0.3% and 2%, respectively , indicating that the R1178K mutation significantly increases read-through at the gene junctions in infected cells.Using the VSV reverse genetics system , we examde novo initiation at the 3′-terminal Le promoter to generate the AC dinucleotide , [α-32P]GTP (PerkinElmer), and pppAACAG oligo-RNA for 2 h, as described in [32P]GDP was used as a substrate instead of [α-32P]GTP, capping reaction mixtures were analyzed by 20% polyacrylamide gels containing 7 M urea (urea-PAGE) followed by autoradiography [32P]GTP as a substrate GTP and the other three NTPs for 2 h as detailed previously CTP for 1 h GTP during the 3-min pulse period, and then chased into LeRNA47 by incubating with excess concentrations of GTP and UTP for 1 min. Transcripts were analyzed along with RNA size markers (synthesized by T7 RNA polymerase) by 20% urea-PAGE as described in  as described in  containing Halt Protease Inhibitor Cocktail (Thermo Scientific). After centrifugation of the cell lysates at 15,000 × g for 5 min at 4°C, the resulting supernatants were collected. Protein concentrations of the lysates were determined by a Bradford protein assay (Bio-Rad) using bovine γ-globulin as a standard. Proteins (1 μg) in the lysates were resolved by 10% SDS-PAGE (acrylamide-bisacrylamide ratio of 75:1) and electroblotted onto polyvinylidene difluoride membranes . The membranes were probed with rabbit anti-VSV , -P (GMP-labeled LeRNA47 and LeRNA12  synthesized by the WT or mutant L protein during 2-h transcription were estimated. The amount of LeRNA47 synthesized by the WT L protein (12 synthesis by the mutant L proteins compared to the WT L protein (open column 2) was examined by one-way ANOVA . (C) Relative molar amounts of LeRNA18 (gray columns) and LeRNA12 (open columns) synthesized by the WT or mutant L protein during 3-min transcription in the absence of UTP (18 synthesized by the WT L protein (12 synthesis by the mutant L proteins compared to the WT L protein (open column 2) was examined by one-way ANOVA.Click here for additional data file.S2 FigN-P junction in 12 cDNA clones derived from polyadenylated polycistronic mRNAs synthesized by the R1178K mutant L protein in vitro were sequenced. Electropherograms are shown with the N-P junction sequence (top). The N gene-end and P gene-start sequences are boxed. IG indicates the intergenic sequence.The (TIF)Click here for additional data file.S3 FigN (A), P (B), M (C), and G (D) mRNAs as in E) Cellular 18S rRNA on the same membrane was detected with an antisense probe.BHK-21 cells were mock-infected or infected with the WT or R1178K mutant rVSV at a multiplicity of infection of 5 and cultured at 37°C. Total RNAs were extracted from the cells at 6-h, 9-h, and 12-h post-infection and treated with RNase H in the presence of oligo(dT). The RNase H-treated RNAs (0.1 μg) were analyzed by Northern blotting sequentially with the probes against (TIF)Click here for additional data file.S4 FigA) The cell lysates (1 μg protein) were analyzed by Western blotting with rabbit anti-VSV , -P, or -GAPDH polyclonal antibody. The graphs show relative amounts of the N, P, M, and G proteins synthesized in cells infected with the WT (closed circles) or R1178K mutant (open triangles) rVSV. The amounts of the viral proteins in the lysates from the WT virus-infected cells at 12-h post-infection was set to 1. Symbols and error bars represent the means and standard deviations, respectively, of three independent experiments (n = 3). (B) The cell lysates (10 μg protein) were analyzed by 10% SDS-PAGE followed by staining with Coomassie Brilliant Blue.BHK-21 cells were mock-infected or infected with the WT or R1178K mutant rVSV as in (TIF)Click here for additional data file.S5 FigThe model of a putative elongation complex of the VSV L protein is shown with colors as in (TIF)Click here for additional data file.S6 FigThe modeled structure of the VSV L elongation complex shown in (TIF)Click here for additional data file.S7 FigA), a panned out view of the L protein in absence of RNA, noted as the apo-state in the text, is shown in the context of R1178. Secondary structure elements and key residues (shown as sticks) surrounding R1178 are shown with colors as in B) shows a close-up view of the direct environment of R1178, with local and key functional residues noted. (C) shows an alternate stick model for a subset of residues in the vicinity of R1178. Residue interactions with guanidinium group of the R1178 side chain are noted with dashed lines. Carbon-backbone colors, in (C), correspond to colors as in (A) and (B). Models in this figure were generated from coordinates in PDB id: 6U1X. For clarity, the N-terminal, fingers (excluding residues 457–62), palm, and thumb subdomain are not shown.In ((TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


The descriptors for panels D-F were mislabeled as E-G. The caption now appears as “Preoperative magnetic resonance imaging. A, Axial T1-weighted images demonstrated a well-circumscribed solid-cystic mass measuring 3 cm in the right parietal lobe. The mass displayed hypointensity for the most part with a cystic component showing very low intensity. Prominent peritumoral edema and midline shift were seen. B, On axial T2-weighted images, the mass showed heterogeneous high intermediate intensity. C, On axial T2flair-weighted images, the mass showed heterogeneous low intermediate intensity. D, On diffusion-weighted images, the mass showed low and slightly high intensity. E,F, Axial and coronal T1-weighted images with administration of the contrast medium demonstrated a well-demarcated lobular mass with heterogeneous enhancement. A cystic component with no enhancement was seen in the mass.”In the article, “Relapsing cerebral atypical teratoid/rhabdoid tumor after trimodality therapy: A case report”