The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this ...The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.展开更多
Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have be...Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins,the nonstructural protein 5(NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of h NPCs.Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.展开更多
Zika virus(ZIKV)poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage.However,deficiency of human testicular samples hinders the in-depth research ...Zika virus(ZIKV)poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage.However,deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury.Organoids are relatively simple in vitro models,which could mimic the pathological changes of corresponding organs.In this study,we constructed a 3D testicular organoid model using primary testicular cells from adult BALB/c mice.Similar to the testis,this organoid system has a blood-testis barrier(BTB)-like structure and could synthesize testosterone.ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis.Therefore,our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.展开更多
Owing to the widespread distribution of mosquitoes capable of transmitting Zika virus, lack of clinical vaccines and treatments, and poor immunity of populations to new infectious diseases, Zika virus has become a glo...Owing to the widespread distribution of mosquitoes capable of transmitting Zika virus, lack of clinical vaccines and treatments, and poor immunity of populations to new infectious diseases, Zika virus has become a global public health concern. Recent studies have found that Zika virus can continuously infect human brain microvascular endothelial cells.These cells are the primary components of the blood–brain barrier of the cerebral cortex, and further infection of brain tissue may cause severe damage such as encephalitis and fetal pituitary disease. The present study found that a biologically active base, piperlongumine(PL), inhibited Zika virus replication in human brain microvascular endothelial cells, Vero cells, and human umbilical vein endothelial cells. PL also significantly increased heme oxygenase-1(HO-1) gene expression, while silencing HO-1 expression and using the reactive oxygen species scavenger, N-acetylcysteine, attenuated the inhibitory effect of PL on Zika virus replication. These results suggest that PL induces oxidative stress in cells by increasing reactive oxygen species. This, in turn, induces an increase in HO-1 expression, thereby inhibiting Zika virus replication. These findings provide novel clues for drug research on the prevention and treatment of Zika virus.展开更多
In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–te...In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier(BTB),or Sertoli cell barrier(SCB).However,little is known about the underlying mechanism.In this study,interaction between actin,an important component of the SCB,and ZIKV envelope(E)protein domainⅢ(EDⅢ)was inferred from coimmunoprecipitation(Co-IP)liquid chromatography–tandem mass spectrometry(LC–MS/MS)analysis.Confocal microscopy confirmed the role of actin filaments(F-actin)in ZIKV infection,during which part of the stress fibers,the bundles that constituted by paralleled actin filaments,were disrupted and presented in the cell periphery.Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement.Perturbation of F-actin by cytochalasin D(CytoD)or Jasplakinolide(Jas)enhanced the infection of ZIKV.More importantly,the transepithelial electrical resistance(TEER)of an in vitro mouse SCB(mSCB)model declined with the progression of ZIKV infection or overexpression of E protein.Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression,highlighting the role of E protein in ZIKV-induced disruption of the BTB.We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network,thereby compromising BTB integrity.展开更多
Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitation...Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitations,such as a long and arduous cycle,complicated operation,and high expenses.A chicken lymphoma cell line,DT40,is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture.Here,the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro.Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID)gene,AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected.In this study,we generated a novel AID-inducible DT40 cell line(DT40-H7),in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system,and an inducible AID gene,based on the Tet-Off expression system,was stably transfected.AID expression was controlled in DT40-H7 cells in a simple and efficient manner;gene conversion and point mutations were observed only when AID was expressed.Using the antibody library generated from this cell line,we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.展开更多
文摘The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.
基金The work is supported by the National Natural Science Foundation of China[NSFC Grant#81620108020 and#32041002,to D.G.,Grant#31800151,to J.W.]Guangdong Zhujiang Talents Program(to D.G.)+2 种基金Shenzhen Science and Technology Program[Grant#KQTD20180411143323605 and#JSGG20200225150431472 to D.G.]National Ten-thousand Talents Program(to D.G.)Guangdong Province “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project(Grant #2019ZT08Y464 to Li,C.M)。
文摘Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins,the nonstructural protein 5(NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of h NPCs.Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.
基金funded by the National Key Research and Development Plan of China(2021YFC2300202)the National Natural Science Foundation of China(U1902210,81871641,81972979,82172266,81902048)+1 种基金the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan(IDHT20190510)the Beijing Key Laboratory of Emerging Infectious Diseases(NO.DTKF202103).
文摘Zika virus(ZIKV)poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage.However,deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury.Organoids are relatively simple in vitro models,which could mimic the pathological changes of corresponding organs.In this study,we constructed a 3D testicular organoid model using primary testicular cells from adult BALB/c mice.Similar to the testis,this organoid system has a blood-testis barrier(BTB)-like structure and could synthesize testosterone.ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis.Therefore,our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.
基金supported by the National Natural Science Foundation (Nos. 31670168, 31470271 and 81730110)National Key R&D Program of China (Grant No. 2018YFC1602206)Guangdong Provincial Science and Technology (No. 2018B020207006)。
文摘Owing to the widespread distribution of mosquitoes capable of transmitting Zika virus, lack of clinical vaccines and treatments, and poor immunity of populations to new infectious diseases, Zika virus has become a global public health concern. Recent studies have found that Zika virus can continuously infect human brain microvascular endothelial cells.These cells are the primary components of the blood–brain barrier of the cerebral cortex, and further infection of brain tissue may cause severe damage such as encephalitis and fetal pituitary disease. The present study found that a biologically active base, piperlongumine(PL), inhibited Zika virus replication in human brain microvascular endothelial cells, Vero cells, and human umbilical vein endothelial cells. PL also significantly increased heme oxygenase-1(HO-1) gene expression, while silencing HO-1 expression and using the reactive oxygen species scavenger, N-acetylcysteine, attenuated the inhibitory effect of PL on Zika virus replication. These results suggest that PL induces oxidative stress in cells by increasing reactive oxygen species. This, in turn, induces an increase in HO-1 expression, thereby inhibiting Zika virus replication. These findings provide novel clues for drug research on the prevention and treatment of Zika virus.
基金We graciously acknowledge Dr.George F.Gao of Institute of Microbiology,Chinese Academy of Sciences,Beijing,China for the gifts of ZIKV(ZIKA-SMGC-1,GenBank accession number:KX266255)ZIKV antibody FITC-Z6.This work was supported by grants from the National Science and Technology Major Project(Grant No.2018ZX10733403)。
文摘In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier(BTB),or Sertoli cell barrier(SCB).However,little is known about the underlying mechanism.In this study,interaction between actin,an important component of the SCB,and ZIKV envelope(E)protein domainⅢ(EDⅢ)was inferred from coimmunoprecipitation(Co-IP)liquid chromatography–tandem mass spectrometry(LC–MS/MS)analysis.Confocal microscopy confirmed the role of actin filaments(F-actin)in ZIKV infection,during which part of the stress fibers,the bundles that constituted by paralleled actin filaments,were disrupted and presented in the cell periphery.Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement.Perturbation of F-actin by cytochalasin D(CytoD)or Jasplakinolide(Jas)enhanced the infection of ZIKV.More importantly,the transepithelial electrical resistance(TEER)of an in vitro mouse SCB(mSCB)model declined with the progression of ZIKV infection or overexpression of E protein.Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression,highlighting the role of E protein in ZIKV-induced disruption of the BTB.We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network,thereby compromising BTB integrity.
基金sponsored by the Project of the National Defense Science and Technology Innovation Special Zone (to HH, 17-163-12-ZT-005-013-01)the CAMS Innovation Fund for Medical Sciences (to HH, CIFMS 2016-12 M-1-013 and to ZZ, CIFMS 2016-12 M-1-014 and 2016-12 M-3-020)+1 种基金the National Key Research and Development Program (to ZZ, 2016YFD0500300)the Fundamental Research Funds for the Central Universities (to HH, 2018PT31032)
文摘Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitations,such as a long and arduous cycle,complicated operation,and high expenses.A chicken lymphoma cell line,DT40,is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture.Here,the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro.Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID)gene,AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected.In this study,we generated a novel AID-inducible DT40 cell line(DT40-H7),in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system,and an inducible AID gene,based on the Tet-Off expression system,was stably transfected.AID expression was controlled in DT40-H7 cells in a simple and efficient manner;gene conversion and point mutations were observed only when AID was expressed.Using the antibody library generated from this cell line,we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.
