The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor（F_0） and then cleaved in...The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor（F_0） and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies（mAbs） against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus（HearNPV）（Ha F） were generated. Two kinds of mAbs were obtained according to their different recognition epitopes： one kind of mAbs, as represented by 38F10,recognizes amino acid（aa） 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process. 基金展开更多
The Autographa californica nucleopolyhedrovirus(AcMNPV) contains three apoptosis suppressor genes:p35,iap1 and iap2.AcMNPV P35 functions as a pancaspase inhibitor,but the function of IAP1 and IAP2 has not been entirel...The Autographa californica nucleopolyhedrovirus(AcMNPV) contains three apoptosis suppressor genes:p35,iap1 and iap2.AcMNPV P35 functions as a pancaspase inhibitor,but the function of IAP1 and IAP2 has not been entirely resolved.In this paper,we analyze the function of IAP1 and IAP2 in detail.AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4(Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV(HearNPV) infection and rescued the replication of HearNPV and BV production in these cells.Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection.Recombinant HearNPVs expressing AcMNPV iap1,iap2 and p35,respectively,not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells.However,the iap1,iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production.These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.展开更多
A recombinant Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ex- pressing the insect-selective neurotoxin （RjAalTjO from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP-glucosyltransfera...A recombinant Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ex- pressing the insect-selective neurotoxin （RjAalTjO from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP-glucosyltransferase gene （egt） using ）^- red homologous recombination system. Another egt deleted control HearNPV was con- structed in a similar way by inserting egfp gene into the egt locus. One-step viral growth curve and viral DNA replication curve analysis confirmed that the recombination did not affect the viral growth and DNA replication in host cells. There is no discernable differ- ence in occlusion-body morphogenesis between RjAalTf-HearNPV,, Egfp-HearNPV and HZ8-HearNPV, which was confirmed by transmission electron microscopy analysis. How- ever, the insecticidal activity of RjAal 7f-HearNPV is enhanced against the third instar H. armigera larvae according to the bioassay on virulence comparison. There is a dramatic reduction （56.9%） in median lethal dose （LD50） and also a reduction （13.4%） in median sur- vival time （ST50） for the recombinant RjAalTf-HearNPV compared to the HZ8-HearNPV, but only a 27.5% reduction in LD50 and 10.1% reduction in ST50 value when Egfp- HearNPV is compared with HZ8-HearNPV. The daily diet consumption analysis showed that the RjAal 7f-HearNPV was able to inhibit the infected larvae feeding compared with the egt minus HearNPV. These results demonstrated that this novel recombinant RjAa17f- HearNPV could improve the insecticidal effect against its host insects and RjAa17fcould be a considerable candidate for other recombinant baculovirus constructions.展开更多
Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the la...Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.展开更多
Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (...Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group II NPV. The HearNPV vfgf transcripts were detected from 18 to 96 h post-infection (hpi) of Hz-AM1 cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.展开更多
Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV...Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Thr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.展开更多
P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (Hear...P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.展开更多
Fibroblast growth factor(FGF) is found throughout multicellular organisms; however, fgf homologs(vfgf) have only been identified among viruses in lepidopteran baculoviruses. The function of v FGFs from Group I alphaba...Fibroblast growth factor(FGF) is found throughout multicellular organisms; however, fgf homologs(vfgf) have only been identified among viruses in lepidopteran baculoviruses. The function of v FGFs from Group I alphabaculoviruses, including Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) and Bombyx mori nucleopolyhedrovirus(Bm NPV), involves accelerated killing of infected larvae by both viruses. The v FGF of Group II alphabaculovirus is structurally different from that of Group I alphabaculovirus, with a larger C-terminal region and additional N-linked glycosylation sites. In this study, we characterized the Group II alphabaculovirus v FGF of Helicoverpa armigera single nucleopolyhedrovirus(Hear NPV). The transcription and expression of vfgf was detected at 3 h and 16 h post-infection in Hear NPV-infected cells. To further study v FGF function, we constructed vfgf-knockout and-repaired Hear NPV bacmids and investigated their affect in both cultured cells and insects. Deletion of vfgf had no effect on budded-virus production or viral DNA replication in cultured Hz AM1 cells. However, bioassays showed that Hear NPV vfgf deletion significantly increased the median lethal dose and delayed the median lethal time by ~12 h in the host insect when the virus was delivered orally. These results suggested that v FGF is an important virulent factor for HearN PV infection and propagation in vivo.展开更多
基金supported by the grants from the National Science Foundation of China (No. 31370191 and 31621061)the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB11030400)Open Research Fund Program of the Key Laboratory of Agricultural and Environmental Microbiology, Chinese Academy of Sciences
文摘The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor（F_0） and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies（mAbs） against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus（HearNPV）（Ha F） were generated. Two kinds of mAbs were obtained according to their different recognition epitopes： one kind of mAbs, as represented by 38F10,recognizes amino acid（aa） 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process. 基金
基金Supported by the National Basic Research and Development Program of China (Grant No. 2003CB1140)the National Nature Science Foundation of China (Grant Nos. 30325002 and 30670077)in part by a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW) (Program Strategic Scientific Alliances Project,Grant No. 04-PSA-BD-02)
文摘The Autographa californica nucleopolyhedrovirus(AcMNPV) contains three apoptosis suppressor genes:p35,iap1 and iap2.AcMNPV P35 functions as a pancaspase inhibitor,but the function of IAP1 and IAP2 has not been entirely resolved.In this paper,we analyze the function of IAP1 and IAP2 in detail.AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4(Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV(HearNPV) infection and rescued the replication of HearNPV and BV production in these cells.Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection.Recombinant HearNPVs expressing AcMNPV iap1,iap2 and p35,respectively,not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells.However,the iap1,iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production.These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.
文摘A recombinant Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ex- pressing the insect-selective neurotoxin （RjAalTjO from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP-glucosyltransferase gene （egt） using ）^- red homologous recombination system. Another egt deleted control HearNPV was con- structed in a similar way by inserting egfp gene into the egt locus. One-step viral growth curve and viral DNA replication curve analysis confirmed that the recombination did not affect the viral growth and DNA replication in host cells. There is no discernable differ- ence in occlusion-body morphogenesis between RjAalTf-HearNPV,, Egfp-HearNPV and HZ8-HearNPV, which was confirmed by transmission electron microscopy analysis. How- ever, the insecticidal activity of RjAal 7f-HearNPV is enhanced against the third instar H. armigera larvae according to the bioassay on virulence comparison. There is a dramatic reduction （56.9%） in median lethal dose （LD50） and also a reduction （13.4%） in median sur- vival time （ST50） for the recombinant RjAalTf-HearNPV compared to the HZ8-HearNPV, but only a 27.5% reduction in LD50 and 10.1% reduction in ST50 value when Egfp- HearNPV is compared with HZ8-HearNPV. The daily diet consumption analysis showed that the RjAal 7f-HearNPV was able to inhibit the infected larvae feeding compared with the egt minus HearNPV. These results demonstrated that this novel recombinant RjAa17f- HearNPV could improve the insecticidal effect against its host insects and RjAa17fcould be a considerable candidate for other recombinant baculovirus constructions.
基金National Nature Science Foundations ofChina (30325002, 30470075)National Basic ResearchPriorities Program of China (2003CB1140).
文摘Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.
基金National Nature Science Foundations of China (30325002,30670077)National Basic Research Priorities Program of China (2003CB1140).
文摘Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group II NPV. The HearNPV vfgf transcripts were detected from 18 to 96 h post-infection (hpi) of Hz-AM1 cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.
基金National Nature Science Foundations of China (31030027,30770085 and 30800044)
文摘Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Thr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.
基金supported by the National Science Foundation of China(31130058 to Z.H.).Monoclonal Antibodies against HearNPV P74
文摘P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.
基金supported by grants from the National Science Foundation of China (No.31200124 and 31321001)the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No.XDB11030400)
文摘Fibroblast growth factor(FGF) is found throughout multicellular organisms; however, fgf homologs(vfgf) have only been identified among viruses in lepidopteran baculoviruses. The function of v FGFs from Group I alphabaculoviruses, including Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) and Bombyx mori nucleopolyhedrovirus(Bm NPV), involves accelerated killing of infected larvae by both viruses. The v FGF of Group II alphabaculovirus is structurally different from that of Group I alphabaculovirus, with a larger C-terminal region and additional N-linked glycosylation sites. In this study, we characterized the Group II alphabaculovirus v FGF of Helicoverpa armigera single nucleopolyhedrovirus(Hear NPV). The transcription and expression of vfgf was detected at 3 h and 16 h post-infection in Hear NPV-infected cells. To further study v FGF function, we constructed vfgf-knockout and-repaired Hear NPV bacmids and investigated their affect in both cultured cells and insects. Deletion of vfgf had no effect on budded-virus production or viral DNA replication in cultured Hz AM1 cells. However, bioassays showed that Hear NPV vfgf deletion significantly increased the median lethal dose and delayed the median lethal time by ~12 h in the host insect when the virus was delivered orally. These results suggested that v FGF is an important virulent factor for HearN PV infection and propagation in vivo.
