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
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
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
Helicoverpa armigera single nucleocapsid nucleopolyhedrovirns open reading frame 101 （ha101） is 762 nts in length and encodes a 254 amino acid peptide with predicted 29 kDa molecular weight. The homologues of ha101 ...Helicoverpa armigera single nucleocapsid nucleopolyhedrovirns open reading frame 101 （ha101） is 762 nts in length and encodes a 254 amino acid peptide with predicted 29 kDa molecular weight. The homologues of ha101 were explored using BLASTP searching tool in the updated GenBank/EMBL and SWlSS-PROT databases. The results showed that the homologues of ha101 were present in all the completely sequenced lepidopteran nucleopolyhedrovirnses and granulovirnses, suggesting that ha101 might be a functional gene associated with their lepidopteran hosts. Sequence alignment of ha101 and its homologues revealed that 10 amino acids were completely conserved. RT-PCR analysis of ha101 manifested that the transcript of ha101 was first detected at 24 hpi and remained detectable at up to 122 hpi, suggesting that ha101 was transcribed during late stages of infection. Ha101 was expressed using Bac to Bac system in Tn5B-1-4 cells. The product of ha101 expressed in Tn5B-1-4 cells was approximately 29kDa, consistent with the predicted molecular weight, and the results were confirmed by western blot analysis. The subcellular localization indicated that ha101 was aggregated along nuclear envelope during the early stages of infection and spread out to the entire nucleus including virogenic stroma in late stages of infection, suggesting that ha101 may play a specific role in virion assembly process or virogenic stroma arrangement.展开更多
Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80-180 kb （Lu et al., 2012）. Two distinct types of viri- ons have been identified during the infectious cycle...Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80-180 kb （Lu et al., 2012）. Two distinct types of viri- ons have been identified during the infectious cycle of baculoviruses, namely budded virions （BVs） and occlu- sion-derived virions （ODVs）. BVs mediate infection from cell to cell, while ODVs initiate oral infection in the insect midgut （Braunagel and Summers, 2007）.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
对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆...对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。展开更多
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 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.
文摘Helicoverpa armigera single nucleocapsid nucleopolyhedrovirns open reading frame 101 （ha101） is 762 nts in length and encodes a 254 amino acid peptide with predicted 29 kDa molecular weight. The homologues of ha101 were explored using BLASTP searching tool in the updated GenBank/EMBL and SWlSS-PROT databases. The results showed that the homologues of ha101 were present in all the completely sequenced lepidopteran nucleopolyhedrovirnses and granulovirnses, suggesting that ha101 might be a functional gene associated with their lepidopteran hosts. Sequence alignment of ha101 and its homologues revealed that 10 amino acids were completely conserved. RT-PCR analysis of ha101 manifested that the transcript of ha101 was first detected at 24 hpi and remained detectable at up to 122 hpi, suggesting that ha101 was transcribed during late stages of infection. Ha101 was expressed using Bac to Bac system in Tn5B-1-4 cells. The product of ha101 expressed in Tn5B-1-4 cells was approximately 29kDa, consistent with the predicted molecular weight, and the results were confirmed by western blot analysis. The subcellular localization indicated that ha101 was aggregated along nuclear envelope during the early stages of infection and spread out to the entire nucleus including virogenic stroma in late stages of infection, suggesting that ha101 may play a specific role in virion assembly process or virogenic stroma arrangement.
基金supported by the grants from the National Science Foundation of China(No.31570153,31130058,31321001 and 31400142)the Strategic Priority Research Program of the Chinese Academy of Sciences(Grant No.XDB11030400)the Core Facility and Technical Support of Wuhan Institute of Virology for technical assistance
文摘Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80-180 kb （Lu et al., 2012）. Two distinct types of viri- ons have been identified during the infectious cycle of baculoviruses, namely budded virions （BVs） and occlu- sion-derived virions （ODVs）. BVs mediate infection from cell to cell, while ODVs initiate oral infection in the insect midgut （Braunagel and Summers, 2007）.
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.
文摘对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。
