Identification and characterization of a conserved baculoviral structural protein ODVP-6E/ODV-E56 from Choristoneura fumiferana granulovirus

A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicted molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with other baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N- and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the ChfuGV odvp-6e/odv-e56 gene. A slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV).     

Identification, characterization and phylogenic analysis of conserved genes within the odvp-6e/odv-e56 gene region of Choristoneura fumiferana granulovirus

The genes that are located within the odvp-6e/odv-e56 region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing the 11 kb BamHI restriction fragment on the ChfuGV genome. The global GC content that was calculated from the data obtained from this genomic region was 34.96%. The open-reading frames (ORFs), located within the odvp-6e/odv-e56 region, are presented and compared to the equivalent ORFs that are located at the same region in other GVs. This region is composed of 14 ORFs, including three ORFs that are unique to ChfuGV with no obvious homologues in other baculoviruses as well as eleven ORFs with homologues to granuloviral ORFs, such as granulin, CfORF2, pk-1, ie-1, odv-e18, p49, and odvp-6e/odv-e56. In this study, the conceptual products of seven major conserved ORFs (granulin, CfORF2, IE-1, ODV-E18, p49 and ODVP-6E/ODV-E56) were used in order to construct phylogenetic trees. Our results show that granuloviruses can be grouped in 2 distinct groups as follows: Group I; Choristoneura fumiferana granulovirus (ChfuGV), Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), and Adoxophyes orana granulovirus (AoGV). Group II; Xestia c-nigrum granulovirus (XcGV), Plutella xylostella granulovirus (PxGV), and Trichoplusia ni granulovirus (TnGV). The ChfuGV conserved proteins are most closely related to those of CpGV, PhopGV, and AoGV. Comparative studies, performed on gene arrangements within this region of genomes, demonstrated that three GVs from group I maintain similar gene arrangements.     

昆虫包涵体衍生病毒囊膜蛋白的分子生物学

了解杆状病毒的囊膜蛋白对揭示病毒入侵、 囊膜蛋白核定向转运机制以及研究控制昆虫新策略等方面具有重要意义。 目前研究表明,包涵体衍生病毒(occlusion-derived virus, ODV)的囊膜蛋白包括ODV-E25, ODV-E66, ODV-E56, ODV-E18, ODV-E28, P74, PIF1, PIF2, PIF3, GP41, ODV-EC27, ODV-E35, ODV-EC43,BV/ODV-E26,P91和ORF150。 本文结合国内外的研究成果系统的综述了囊膜蛋白的结构和功能,其在经口感染、调节细胞周期和囊膜蛋白的传送等方面起作用。 囊膜蛋白的核定向转运机制,ODV与昆虫中肠之间和包涵体基质之间相互作用以及ODV结构蛋白之间的相互作用等将是今后的研究重点。     

Proteomics analysis of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus identified two new occlusion-derived virus-associated proteins, HA44 and HA100

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.     

Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).     

Characterization of novel components of the baculovirus per os infectivity factor complex

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.     

Autographa californica multiple nucleopolyhedrovirus 38K is a novel nucleocapsid protein that interacts with VP1054, VP39, VP80, and itself

It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusion-derived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.     

Baculovirus Data Suggest a Common but Multifaceted Pathway for Sorting Proteins to the Inner Nuclear Membrane

Multiple unique protein markers sorted to the inner nuclear membrane (INM) from the Autographa californica nucleopolyhedrovirus occlusion-derived virus (ODV) envelope were used to decipher common elements of the sorting pathway of integral membrane proteins from their site of insertion into the membrane of the endoplasmic reticulum (ER) through their transit to the INM. The data show that during viral infection, the viral protein FP25K is a partner for all known ODV envelope proteins and that BV/ODV-E26 (designated E26) is a partner for some, but not all, such proteins. The association with the ER membrane of FP25K, E26, and the cellular INM-sorting protein importin-α-16 is not static; rather, these sorting proteins are actively recruited to the ER membrane based upon requirements of the proteins in transit to the INM. Colocalization analysis using an ODV envelope protein and importin-α-16 shows that during viral infection, importin-α-16 translocates across the pore membrane to the INM and then is incorporated into the virus-induced intranuclear membranes. Thus, the association of importin-α-16 and INM-directed proteins appears to remain at least through protein translocation across the pore membrane to the INM. Overall, the data suggest that multiple levels of regulation facilitate INM-directed protein trafficking, and that proteins participating in this sorting pathway have a dynamic relationship with each other and the membrane of the ER.     

The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of approximately 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.     

P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens Larvae

P74, an envelope protein of the occlusion-derived virus (ODV) of Autographa californica M nucleopolyhedrovirus (AcMNPV), is critical for oral infection of Trichoplusia ni larvae. The role of P74 during primary infection, however, is unknown. Here we provide evidence that P74 facilitates binding of AcMNPV ODV to a specific receptor within the larval midgut epithelia of another host species, Heliothis virescens. We adapted a fluorescence dequenching assay to compare binding, fusion, and competition of wild-type AcMNPV ODV in vivo with itself and with the ODV of a p74-deficient AcMNPV mutant. We found that relative to wild-type ODV, binding and fusion of ODV deficient in P74 were both qualitatively and quantitatively different. Unlike wild-type ODV, an excess of P74-deficient ODV failed to compete effectively with wild-type ODV binding, and the overall binding level of the mutant ODV was one-third that of the wild type. These results implicated P74 as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut.     

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

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-1, 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.     

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

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.     

Novel characteristics of the function and induction of murine p56 family proteins

The interferon-stimulated gene 56 (ISG56) family is induced strongly in response to virus infection, interferons (IFNs) and double-stranded RNA (dsRNA). In the mouse, this family comprises three members, ISG56, ISG54, and ISG49, which are clustered on chromosome 19 and encode the corresponding proteins p56, p54, and p49. Here, we report differential properties of these proteins and their distinct induction patterns in different cell types. All three murine proteins bound to the c-subunit of the translation initiation factor eIF3, but unlike the other members, p49 did not inhibit protein synthesis. Using a newly raised antibody, we demonstrated that both in vitro and in vivo, p49 expression was strongly induced by IFN, dsRNA, and Sendai virus. However, in kidney mesangial cells, as opposed to podocytes, encephalomyocarditis virus, vesicular stomatitis virus, or extracellular dsRNA did not induce any of the p56 family proteins, although they were robustly expressed after Sendai virus infection or dsRNA transfection. Furthermore, protein-specific differences in the regulation of p56 family members became evident in various leukocyte types: all three proteins were induced by IFN in T cells, but in B cells p56 and ISG56 mRNA could not be detected. Similarly, p56 was selectively uninducible in plasmacytoid dendritic cells, whereas in myeloid dendritic cells, all three family members were expressed. These results revealed novel cell type-, inducer-, and gene-specific regulation of the ISG56 family of genes.     

Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.     

杆状病毒的结构蛋白及其功能

综述了昆虫杆状病毒的主要结构蛋白及其功能。髓核是由大约120 kb的双链DNA分子和与其密切相关的碱性蛋白所组成,碱性蛋白同DNA紧密结合以维持其复杂有序的超螺旋结构,并且能够增强杆状病毒DNA的感染性;衣壳蛋白是杆状病毒粒子的主要结构蛋白,主要有VP 39、VP 78/83、VP 87、VP 80、VP 24;囊膜蛋白有PDV-E 25、PDV-6 e、PDV-E 66、PDV-E 56、PDV-E 18、PDV-EC 27、PDV-E 35、PDV-EC 27、BV/PDV-E 26、PDV-43、gp 64-67、VP 40/41;包涵体的基质蛋白;多角体（或颗粒体）膜主要是由糖类构成,VP 32-36是与之相关的蛋白。