Background:The immune system of insects exerts fundamentally different antiviral mechanisms than mammals.MicroRNAs(miRNAs)play vital roles in developing insect antiviral immunity.MiRNAs expression profiles of insects ...Background:The immune system of insects exerts fundamentally different antiviral mechanisms than mammals.MicroRNAs(miRNAs)play vital roles in developing insect antiviral immunity.MiRNAs expression profiles of insects changed significantly during baculovirus infection.Methods:Differential expression profiles of miRNAs in Spodoptera frugiperda were monitored by next-generation sequencing(NGS)and RT-qPCR during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.The transcription levels of genes were detected by RT-qPCR.The 50%tissue culture infective dose(TCID_(50))endpoint dilution assay was used to determine the proliferation of progeny virus.Results:NGS revealed that 49 miRNAs were differentially expressed in Sf9 cells,and 10 of them were significantly up-or down-regulated.Though RT-qPCR analysis,we observed the similar trends for the expression patterns of significantly differentially expressed miRNAs from NGS.Moreover,the transcription levels of core genes,Exportin5,Dicer1,and Argonaute1,in miRNA biogenesis pathways were significantly increased after AcMNPV infection.For five selected miRNAs,miR-34-5p could regulate the proliferation of baculovirus progeny virus and energy metabolism.Conclusion:The miRNAs biogenesis pathway in Sf9 cells plays an important role and may be stimulated to resist AcMNPV infection.This work provides evidence for the molecular mechanism of baculovirus-insect interaction and offers novel ideas and directions for green pest control technology.展开更多
The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been perform...The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.展开更多
Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabac...Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus(Plxy GV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.展开更多
Baculoviruses have been widely used as a vector for expressing foreign genes.Among numerous baculoviruses,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the most frequently used and it encodes 155 open...Baculoviruses have been widely used as a vector for expressing foreign genes.Among numerous baculoviruses,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the most frequently used and it encodes 155 open reading frames(ORFs).Here,we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses(BVs)by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays.The results showed that among the 39 functionally unverified genes and 3 recently reported genes,36 are dispensable for infectious BV production,as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus.Three genes(ac62,ac82 and ac106/107)are essential for infectious BV production,as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus.In addition,three genes(ac13,ac51 and ac120)are important but not essential for infectious BV production,as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses.We then grouped the 155 AcMNPV genes into three categories(Dispensable,Essential,or Important for infectious BV production).Based on our results and previous publications,we constructed a schematic diagram of a potential mini-genome of AcMNPV,which contains only essential and important genes.The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.展开更多
Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ...Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ac13 was a late gene and that the encoded protein,bearing a putative nuclear localization signal motif,colocalized with the nuclear lamina.Deletion of ac13 did not affect viral genome replication,nucleocapsid assembly or occlusion body(OB)formation,but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus.Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm,while the OB morphogenesis was unaffected.Taken together,our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding,but was dispensable for OB formation.展开更多
GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy an...GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.展开更多
基金funded by the Natural Science Foundation of Shanxi Province,Grant No.201801D121193the Central Government Guiding Local Science and Technology Development Fund Project,Grant No.YDZJSX2022A001.
文摘Background:The immune system of insects exerts fundamentally different antiviral mechanisms than mammals.MicroRNAs(miRNAs)play vital roles in developing insect antiviral immunity.MiRNAs expression profiles of insects changed significantly during baculovirus infection.Methods:Differential expression profiles of miRNAs in Spodoptera frugiperda were monitored by next-generation sequencing(NGS)and RT-qPCR during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.The transcription levels of genes were detected by RT-qPCR.The 50%tissue culture infective dose(TCID_(50))endpoint dilution assay was used to determine the proliferation of progeny virus.Results:NGS revealed that 49 miRNAs were differentially expressed in Sf9 cells,and 10 of them were significantly up-or down-regulated.Though RT-qPCR analysis,we observed the similar trends for the expression patterns of significantly differentially expressed miRNAs from NGS.Moreover,the transcription levels of core genes,Exportin5,Dicer1,and Argonaute1,in miRNA biogenesis pathways were significantly increased after AcMNPV infection.For five selected miRNAs,miR-34-5p could regulate the proliferation of baculovirus progeny virus and energy metabolism.Conclusion:The miRNAs biogenesis pathway in Sf9 cells plays an important role and may be stimulated to resist AcMNPV infection.This work provides evidence for the molecular mechanism of baculovirus-insect interaction and offers novel ideas and directions for green pest control technology.
基金The project BACULOGENES of the European Union (LSHB-CT-2006-037541)The Netherlands Scientific Organisation (NWO) MEERVOUD program
文摘The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.
基金supported by the fund of Hubei Key Laboratory of Genetic Regulation and Integrative Biology
文摘Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus(Plxy GV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.
基金This research was supported by the grants from the National Natural Science Foundation of China(No.31872640)the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences(grant no.QYZDJ-SSW-SMC021)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11030400).
文摘Baculoviruses have been widely used as a vector for expressing foreign genes.Among numerous baculoviruses,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the most frequently used and it encodes 155 open reading frames(ORFs).Here,we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses(BVs)by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays.The results showed that among the 39 functionally unverified genes and 3 recently reported genes,36 are dispensable for infectious BV production,as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus.Three genes(ac62,ac82 and ac106/107)are essential for infectious BV production,as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus.In addition,three genes(ac13,ac51 and ac120)are important but not essential for infectious BV production,as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses.We then grouped the 155 AcMNPV genes into three categories(Dispensable,Essential,or Important for infectious BV production).Based on our results and previous publications,we constructed a schematic diagram of a potential mini-genome of AcMNPV,which contains only essential and important genes.The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.
基金supported by the National Key Research and Development Program of China(2017YFD0201206)the WIV “One-Three-Five”strategic program(Y602111SA1 to XS)。
文摘Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)orf13(ac13)is a conserved gene in all sequenced alphabaculoviruses.However,its function in the viral life cycle remains unknown.In this study,we found that ac13 was a late gene and that the encoded protein,bearing a putative nuclear localization signal motif,colocalized with the nuclear lamina.Deletion of ac13 did not affect viral genome replication,nucleocapsid assembly or occlusion body(OB)formation,but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus.Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm,while the OB morphogenesis was unaffected.Taken together,our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding,but was dispensable for OB formation.
基金supported by aBBSRC grant (LAK, RDP)a BBSRC-funded PhDstudentship (FJH)
文摘GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.
基金supported by grants from the National Natural Science Foundation of China(No.31272100,31372199)the Natural Science Foundation of Shanxi Province,China(No.2014011038-1)+1 种基金the National High Technology Research and Development Program of China(863 Program,No.2012AA020809)the Program for the Top Young Academic Leaders of Higher Learning Institutions of Shanxi Province,China
