Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exte...Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exten-sively;however,further genetic engineering of HSV-1 is hindered by its complex genome structure.In the present study,we designed and constructed a synthetic platform for HSV-1 based on H129-G4.The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination(TAR)in yeast,and was named H129-Syn-G2.The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus.According to growth curve assay and electron microscopy results,the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus.This synthetic platform will facilitate further manipulation of the HSV-1 genome for the devel-opment of neuronal circuit tracers,oncolytic viruses,and vaccines.展开更多
目的探讨曲古抑菌素A(TSA)联合Ⅰ型单纯疱疹病毒(HSV-1)对C6鼠胶质瘤细胞株细胞增殖、细胞凋亡的影响。方法体外培养C6胶质瘤细胞,设对照组(加入等体积培养基)、TSA组(加入0.5×10^-3μmol/L TSA处理)、HSV-1(加入10 MOI HSV-1处理)...目的探讨曲古抑菌素A(TSA)联合Ⅰ型单纯疱疹病毒(HSV-1)对C6鼠胶质瘤细胞株细胞增殖、细胞凋亡的影响。方法体外培养C6胶质瘤细胞,设对照组(加入等体积培养基)、TSA组(加入0.5×10^-3μmol/L TSA处理)、HSV-1(加入10 MOI HSV-1处理)及TSA+HSV-1组(加入0.5×10^-3μmol/L TSA和10 MOI HSV-1处理)共4组,每组设5个复孔。CCK-8法检测细胞增殖活性;流式细胞仪检测细胞凋亡率;RT-PCR、免疫印迹法检测血管内皮细胞生长因子(VEGF)的mRNA和蛋白表达水平。结果作用48、72 h,与对照组相比,TSA组、HSV-1组、TSA+HSV-1组C6细胞增殖活性显著降低(P<0.01),细胞凋亡率明显增高(P<0.05),VEGF mRNA和蛋白表达水平明显降低(P<0.05);而且,TSA+HSV-1组明显优于TSA组和HSV-1组(P<0.05)。结论TSA联合HSV-1对体外培养的C6细胞可产生协同或叠加杀伤作用,抑制VEGF表达可能是作用机制之一。展开更多
单纯疱疹病毒2型潜伏相关转录体(LAT)-RL1对放线菌素D诱导的凋亡作用的研究。构建重组pEGFP-RL1质粒,转染Vero细胞,RT-PCR及荧光鉴定重组质粒的表达。放线菌素D诱导Vero细胞凋亡,通过Hochest33342荧光染色观察细胞形态变化,流式细胞术...单纯疱疹病毒2型潜伏相关转录体(LAT)-RL1对放线菌素D诱导的凋亡作用的研究。构建重组pEGFP-RL1质粒,转染Vero细胞,RT-PCR及荧光鉴定重组质粒的表达。放线菌素D诱导Vero细胞凋亡,通过Hochest33342荧光染色观察细胞形态变化,流式细胞术检测细胞凋亡率,JC-1荧光观察膜电位变化,Caspase 3凋亡蛋白检测。RT-PCR和荧光观察表明该真核表达载体能在Vero细胞中高表达。Hochest33342染色转染了pEGFP-RL1的Vero细胞经放线菌素D凋亡诱导后,细胞形态正常。流式结果表明转染重组质粒pEGFP-RL1且经诱导凋亡组与正常对照组凋亡率无差异,而显著低于诱导凋亡组和转染空质粒pEGFP-C2诱导凋亡组。转染重组质粒pEGPF-RL1的细胞JC-1染色后,红色细胞的比例要远大于绿色细胞的比例,而转染空质粒pEGFP-C2被染成绿色细胞的数量较多。Caspase-3结果表明转染了空质粒pEGFP-C2的Vero细胞经诱导凋亡后,活性显著高于转染了pEGFP-RL1经放线菌素D诱导凋亡后的Vero细胞和正常Vero细胞。HSV-2 LAT RL1具有抗放线菌素D诱导的Vero细胞的凋亡作用。展开更多
Herpes simplex virus 1(HSV-1),a member of a herpesviruses,shows a high infectivity rate of 30%-60%in populations of various ages.Some herpes simplex(HSV)vaccine candidates evaluated during the past 20 years have not s...Herpes simplex virus 1(HSV-1),a member of a herpesviruses,shows a high infectivity rate of 30%-60%in populations of various ages.Some herpes simplex(HSV)vaccine candidates evaluated during the past 20 years have not shown protective efficacy against viral infection.An improved understanding of the immune profile of infected individuals and the associated mechanism is needed.HSV uses an immune evasion strategy during viral replication,and various virus-encoded proteins,such as ICP47 and Vhs,participate in this process through limiting the ability of CD8+cytotoxic T lymphocytes to recognize target cells.Other proteins,e.g.,Us3 and Us5,also play a role in viral immune evasion via interfering with cellular apoptosis.In this work,to study the mechanism by which HSV-1 strain attenuation interferes with the viral immune evasion strategy,we constructed a mutant strain,M5,with deletions in the Us3 and Us5 genes.M5 was shown to induce higher neutralizing antibody titers and a stronger cellular immune response than our previously reported M3 strain,and to prevent virus infection more effectively than the M3 strain in an in vivo mouse challenge test.展开更多
MicroRNAs(miRNAs)encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2(HSV-2)infection.In this study,miRNA-H4-5p and miRNA-H4-3p were ectopically expressed...MicroRNAs(miRNAs)encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2(HSV-2)infection.In this study,miRNA-H4-5p and miRNA-H4-3p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions.The results showed that miRNA-H4-5p could reverse apoptosis induced by actinomycin D(Act-D)and promote cell cycle progression,but miRNA-H4-3p had no such obvious functions.Bioinformatics analysis,luciferase report assay,quantitative reverse transcription polymerase chain reaction(qRT-PCR),and Western blotting demonstrated that miRNA-H4-5p could bind to the 3-′untranslated region(UTR)of cyclin-dependent kinase inhibitor 2A(CDKN2A)and cyclin-dependent kinase-like 2(CDKL2)to negatively regulate their expression.We verified that these two targeted genes were associated with cell apoptosis and cell cycle.Furthermore,in HeLa cells infected with HSV-2,we detected significantly reduced expression of CDKN2A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5p on these two target genes.Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection,providing further information on the roles of encoded herpesvirus miRNAs in pathogen-host interaction.展开更多
基金Wuhan Institute of Virology for financial support for the research(grant no.EISA020201).
文摘Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exten-sively;however,further genetic engineering of HSV-1 is hindered by its complex genome structure.In the present study,we designed and constructed a synthetic platform for HSV-1 based on H129-G4.The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination(TAR)in yeast,and was named H129-Syn-G2.The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus.According to growth curve assay and electron microscopy results,the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus.This synthetic platform will facilitate further manipulation of the HSV-1 genome for the devel-opment of neuronal circuit tracers,oncolytic viruses,and vaccines.
文摘目的探讨曲古抑菌素A(TSA)联合Ⅰ型单纯疱疹病毒(HSV-1)对C6鼠胶质瘤细胞株细胞增殖、细胞凋亡的影响。方法体外培养C6胶质瘤细胞,设对照组(加入等体积培养基)、TSA组(加入0.5×10^-3μmol/L TSA处理)、HSV-1(加入10 MOI HSV-1处理)及TSA+HSV-1组(加入0.5×10^-3μmol/L TSA和10 MOI HSV-1处理)共4组,每组设5个复孔。CCK-8法检测细胞增殖活性;流式细胞仪检测细胞凋亡率;RT-PCR、免疫印迹法检测血管内皮细胞生长因子(VEGF)的mRNA和蛋白表达水平。结果作用48、72 h,与对照组相比,TSA组、HSV-1组、TSA+HSV-1组C6细胞增殖活性显著降低(P<0.01),细胞凋亡率明显增高(P<0.05),VEGF mRNA和蛋白表达水平明显降低(P<0.05);而且,TSA+HSV-1组明显优于TSA组和HSV-1组(P<0.05)。结论TSA联合HSV-1对体外培养的C6细胞可产生协同或叠加杀伤作用,抑制VEGF表达可能是作用机制之一。
文摘单纯疱疹病毒2型潜伏相关转录体(LAT)-RL1对放线菌素D诱导的凋亡作用的研究。构建重组pEGFP-RL1质粒,转染Vero细胞,RT-PCR及荧光鉴定重组质粒的表达。放线菌素D诱导Vero细胞凋亡,通过Hochest33342荧光染色观察细胞形态变化,流式细胞术检测细胞凋亡率,JC-1荧光观察膜电位变化,Caspase 3凋亡蛋白检测。RT-PCR和荧光观察表明该真核表达载体能在Vero细胞中高表达。Hochest33342染色转染了pEGFP-RL1的Vero细胞经放线菌素D凋亡诱导后,细胞形态正常。流式结果表明转染重组质粒pEGFP-RL1且经诱导凋亡组与正常对照组凋亡率无差异,而显著低于诱导凋亡组和转染空质粒pEGFP-C2诱导凋亡组。转染重组质粒pEGPF-RL1的细胞JC-1染色后,红色细胞的比例要远大于绿色细胞的比例,而转染空质粒pEGFP-C2被染成绿色细胞的数量较多。Caspase-3结果表明转染了空质粒pEGFP-C2的Vero细胞经诱导凋亡后,活性显著高于转染了pEGFP-RL1经放线菌素D诱导凋亡后的Vero细胞和正常Vero细胞。HSV-2 LAT RL1具有抗放线菌素D诱导的Vero细胞的凋亡作用。
基金supported by the National Natural Science Foundation of China (81802868 and 31670173)the Fundamental Research Funds for the Central Universities (3332018129, 3332018197)+1 种基金the CAMS Initiative for Innovative Medicine (2016I2M-1-019)the Science and Technology Major Project of Yunnan Province (2017ZF006 and 2017ZF020)
文摘Herpes simplex virus 1(HSV-1),a member of a herpesviruses,shows a high infectivity rate of 30%-60%in populations of various ages.Some herpes simplex(HSV)vaccine candidates evaluated during the past 20 years have not shown protective efficacy against viral infection.An improved understanding of the immune profile of infected individuals and the associated mechanism is needed.HSV uses an immune evasion strategy during viral replication,and various virus-encoded proteins,such as ICP47 and Vhs,participate in this process through limiting the ability of CD8+cytotoxic T lymphocytes to recognize target cells.Other proteins,e.g.,Us3 and Us5,also play a role in viral immune evasion via interfering with cellular apoptosis.In this work,to study the mechanism by which HSV-1 strain attenuation interferes with the viral immune evasion strategy,we constructed a mutant strain,M5,with deletions in the Us3 and Us5 genes.M5 was shown to induce higher neutralizing antibody titers and a stronger cellular immune response than our previously reported M3 strain,and to prevent virus infection more effectively than the M3 strain in an in vivo mouse challenge test.
基金supported by the National Natural Science Foundation of China (81371749) and (81171511)
文摘MicroRNAs(miRNAs)encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2(HSV-2)infection.In this study,miRNA-H4-5p and miRNA-H4-3p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions.The results showed that miRNA-H4-5p could reverse apoptosis induced by actinomycin D(Act-D)and promote cell cycle progression,but miRNA-H4-3p had no such obvious functions.Bioinformatics analysis,luciferase report assay,quantitative reverse transcription polymerase chain reaction(qRT-PCR),and Western blotting demonstrated that miRNA-H4-5p could bind to the 3-′untranslated region(UTR)of cyclin-dependent kinase inhibitor 2A(CDKN2A)and cyclin-dependent kinase-like 2(CDKL2)to negatively regulate their expression.We verified that these two targeted genes were associated with cell apoptosis and cell cycle.Furthermore,in HeLa cells infected with HSV-2,we detected significantly reduced expression of CDKN2A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5p on these two target genes.Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection,providing further information on the roles of encoded herpesvirus miRNAs in pathogen-host interaction.
