Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, w...Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant viruslike particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21(baby hamster kidney) cells. Collectively, the mutant virus-likeparticle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.展开更多
Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available ...Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available for the treatment of alphaviruses infection.For most highly pathogenic alphaviruses are classified as risk group-3 agents,the requirement of biosafety level 3(BSL-3)facilities limits the live virus-based antiviral study.To facilitate the antiviral development of alphaviruses,we developed a high throughput screening(HTS)platform based on a recombinant Semliki Forest virus(SFV)which can be manipulated in BSL-2 laboratory.Using the reverse genetics approach,the recombinant SFV and SFV reporter virus expressing eGFP(SFV-eGFP)were successfully rescued.The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 cells.Using a broad-spectrum alphavirus inhibitor ribavirin,we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study.The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z0 score.A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses.This assay provides a safe and convenient platform for antiviral study of alphaviruses.展开更多
Alphaviruses are a group of important viruses that cause significant diseases in humans.Among them,Semliki Forest vi-rus(SFV)not only causes symptoms such as joint pain but also infects neuron cells and induces enceph...Alphaviruses are a group of important viruses that cause significant diseases in humans.Among them,Semliki Forest vi-rus(SFV)not only causes symptoms such as joint pain but also infects neuron cells and induces encephalitis in rodents.Recently,the very-low-density lipoprotein receptor(VLDLR)was identified as the cellular receptor for SFV entry.In this study,we present the cryo-electron microscopy structure of SFV bound to human VLDLR.VLDLR targets E1-DIII region of SFV using its membrane-distal LDLR class A(LA)repeats.Structural and functional analyses emphasize the synergistic role of multiple VLDLR repeats in the SFV entry.Remarkably,VLDLR’s binding mode to SFV closely mirrors that of minor group human rhinoviruses but differs significantly from other alphaviruses’interactions with receptors in the canyon re-gion of the E protein.We also assessed SFV binding to VLDLR or apolipoprotein E receptor 2(ApoER2)proteins in horses and mosquitoes and revealed their use of multiple but different LA repeats for binding.Our findings illuminate SFV’s cross-species infectivity,offering insights into potential antiviral strategies against alphavirus infections.展开更多
目的:构建炭疽毒素保护性抗原第四结构域PA4基因的重组Sem lik i森林病毒(Sem lik i forest virus,SFV)复制子载体,并观察PA4抗原在重组SFV复制子载体中的表达。方法:将PA4基因克隆到基于RNA和DNA的SFV复制子表达载体中,获得的重组SFV...目的:构建炭疽毒素保护性抗原第四结构域PA4基因的重组Sem lik i森林病毒(Sem lik i forest virus,SFV)复制子载体,并观察PA4抗原在重组SFV复制子载体中的表达。方法:将PA4基因克隆到基于RNA和DNA的SFV复制子表达载体中,获得的重组SFV复制子载体直接转染BHK21细胞,通过间接免疫荧光和W estern印迹试验检测PA4在细胞中的表达;与辅助病毒载体共转染制备重组病毒颗粒,通过间接免疫荧光和W estern印迹检测PA4在重组病毒颗粒感染细胞中的表达。结果与结论:成功地构建了基于RNA和DNA的PA4重组SFV复制子载体,并且在体外基于RNA和DNA的重组SFV复制子表达载体能够在细胞中有效地表达非分泌型和分泌型的PA4抗原,制备了具有感染能力并能表达PA4抗原的重组病毒颗粒,为进一步以SFV复制子作疫苗载体观察炭疽新型复制子疫苗的免疫原性奠定了基础。展开更多
基金supported by the National Natural Science Foundation of China(31771197,31830035 and 91732304)the National Basic Research Development Program(973 Program)of China(2015CB755600)+2 种基金the Strategic Priority Research Program(B)Chinese Academy of Sciences,China(XDBS01030200)the Major Research Plan of the National Natural Science Foundation of China(91632303)
文摘Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant viruslike particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21(baby hamster kidney) cells. Collectively, the mutant virus-likeparticle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.
基金supported by the Creative Research Group Program of Natural Science Foundation of Hubei Province (2022CFA021)National Natural Science Foundation of China (81702005).
文摘Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available for the treatment of alphaviruses infection.For most highly pathogenic alphaviruses are classified as risk group-3 agents,the requirement of biosafety level 3(BSL-3)facilities limits the live virus-based antiviral study.To facilitate the antiviral development of alphaviruses,we developed a high throughput screening(HTS)platform based on a recombinant Semliki Forest virus(SFV)which can be manipulated in BSL-2 laboratory.Using the reverse genetics approach,the recombinant SFV and SFV reporter virus expressing eGFP(SFV-eGFP)were successfully rescued.The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 cells.Using a broad-spectrum alphavirus inhibitor ribavirin,we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study.The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z0 score.A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses.This assay provides a safe and convenient platform for antiviral study of alphaviruses.
基金supported by the National Natural Science Foundation of China (82072290,82122040,and 32100129)supported by CAS Project for Young Scientists in Basic Research (YSBR-010)the Youth Innovation Promotion Association CAS (Y2021033).
文摘Alphaviruses are a group of important viruses that cause significant diseases in humans.Among them,Semliki Forest vi-rus(SFV)not only causes symptoms such as joint pain but also infects neuron cells and induces encephalitis in rodents.Recently,the very-low-density lipoprotein receptor(VLDLR)was identified as the cellular receptor for SFV entry.In this study,we present the cryo-electron microscopy structure of SFV bound to human VLDLR.VLDLR targets E1-DIII region of SFV using its membrane-distal LDLR class A(LA)repeats.Structural and functional analyses emphasize the synergistic role of multiple VLDLR repeats in the SFV entry.Remarkably,VLDLR’s binding mode to SFV closely mirrors that of minor group human rhinoviruses but differs significantly from other alphaviruses’interactions with receptors in the canyon re-gion of the E protein.We also assessed SFV binding to VLDLR or apolipoprotein E receptor 2(ApoER2)proteins in horses and mosquitoes and revealed their use of multiple but different LA repeats for binding.Our findings illuminate SFV’s cross-species infectivity,offering insights into potential antiviral strategies against alphavirus infections.
文摘目的:构建炭疽毒素保护性抗原第四结构域PA4基因的重组Sem lik i森林病毒(Sem lik i forest virus,SFV)复制子载体,并观察PA4抗原在重组SFV复制子载体中的表达。方法:将PA4基因克隆到基于RNA和DNA的SFV复制子表达载体中,获得的重组SFV复制子载体直接转染BHK21细胞,通过间接免疫荧光和W estern印迹试验检测PA4在细胞中的表达;与辅助病毒载体共转染制备重组病毒颗粒,通过间接免疫荧光和W estern印迹检测PA4在重组病毒颗粒感染细胞中的表达。结果与结论:成功地构建了基于RNA和DNA的PA4重组SFV复制子载体,并且在体外基于RNA和DNA的重组SFV复制子表达载体能够在细胞中有效地表达非分泌型和分泌型的PA4抗原,制备了具有感染能力并能表达PA4抗原的重组病毒颗粒,为进一步以SFV复制子作疫苗载体观察炭疽新型复制子疫苗的免疫原性奠定了基础。
