Bombyx mori Nucleopolyhedrovirus ORF94, A Novel Late Protein Is Identified to be a Component of ODV Structural Protein

Orf94 (Bm94) of Bombyx mori nucleopolyhedrovirus (BmNPV) potentially encodes 424-amino acids with a predicted molecular weight of 49.4 kDa, but its function remains unknown. Blast search results revealed that Bm94 homologues exist in 10 completely sequenced Lepidopteron NPVs with identities ranging from 95 to 37%. Results of our recent study showed that Bm94 was transcribed from 12 to 72 h and the corresponding protein was detected from 24 to 72 h post-infection. Furthermore, Western blot analysis revealed that Bm94 was present in occlusion-derived virus (ODV) and in total protein from BmNPV-infected BmN cells, but not in budded virus. Immunofluoresence analysis revealed that the protein located primarily in the cytoplasm and was also present in the nucleus in the later infection. In conclusion, these results together indicated that Bm94 was a late gene, which distributed both in the cytoplasm and in the nucleus, and was identified to be a component of BmNPV ODV.     

家蚕核型多角体病毒orf25基因在病毒感染过程中 编码一个30kDa的晚期蛋白

首次对家蚕核型多角体orf25基因进行了描述.扩增Bm25基因,亚克隆到原核表达载体pGEX-4T-2,在大肠杆菌BL21(DE3)中表达含有GST标签的融合蛋白.IPTG诱导后高效表达GST-Bm25融合蛋白.纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体.利用制备的抗GST-Bm25融合蛋白的多克隆抗体进行表达时相分析显示:24 h p.i.检测到30 kDa的蛋白条带.RT-PCR方法,在18-72 h p.i 检测到Bm25基因的转录本.结论:以上数据表明Bm25基因编码一晚期表达的30kDa蛋白.     

Identification and characterization of host factors interacting with Bombyx mori nucleopolyhedrovirus ORF8

The orf8 gene (Bm8) in Bombyx mori nucleopolyhedrovirus (BmNPV) is one of 17 genes unique to group I NPVs and is expressed as an early gene. We have reported that Bm8 may play an important role during viral infection and that Bm8 protein co-localized with IE1 to specific nuclear foci throughout infection. It was also demonstrated that both IE1 and BmNPV hr facilitate this localization of Bm8. To investigate further, host proteins interacting with Bm8 were screened using a yeast two-hybrid system. We identified 6 host clones as Bm8-interacting partners from three cDNA libraries derived from BmN cells or B. mori larvae. Further assays showed that the N-terminal region of Bm8 is important for the interaction with most host clones and that two of the clones can associate with IE1. Cloning and sequencing of full-length cDNAs revealed that most of the clones potentially encode either membrane-bound proteins or secreted proteins. Quantitative RT-PCR analysis revealed that some of these host genes were slightly induced during the early stage of infection in BmN cells, and that the expression of all genes was markedly reduced during the late stage of infection. Generation of mutant BmNPVs over-expressing these host genes also identified a gene that potentially functions as a negative factor during BmNPV infection. These features of Bm8-interacting host proteins strongly support that Bm8 is a multifunctional protein involved in multiple signaling pathways in host cells.     

Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain

Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90?% on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.     

Baculovirus-encoded protein BV/ODV-E26 determines tissue tropism and virulence in lepidopteran insects

Lepidopteran nucleopolyhedroviruses (NPVs) show distinct tissue tropism in host insect larvae. However, the molecular mechanism of this tropism is largely unknown. We quantitatively investigated NPV tissue tropism by measuring mRNA levels of viral genes in 16 tissues from Bombyx mori NPV (BmNPV)-infected B. mori larvae and found clear tissue tropism, i.e., BmNPV replicates poorly in the silk glands, midgut, and Malpighian tubule compared with other larval tissues. We next identified the viral genes determining tissue tropism in NPV infection by investigating the phenotypes of larvae infected with 44 BmNPV mutants in which one gene was functionally disrupted by a LacZ cassette insertion. We found that occlusion body (OB) production was markedly enhanced compared with that of the wild type in the middle silk glands (MSGs) of larvae infected with three mutants in which one of three tandemly arrayed genes (Bm7, Bm8, and Bm9) was disrupted. We generated additional mutants in which one or two genes of this gene cluster were partially deleted and showed that Bm8, also known as BV/ODV-E26, was solely required for the suppression of OB production in the MSGs of BmNPV-infected B. mori larvae. Western blotting showed that a LacZ cassette insertion in Bm7 or Bm9 resulted in aberrant expression of Bm8, presumably leading to abnormal OB production in the MSGs. Larval bioassays also revealed that disruption of Bm8 accelerated the death of B. mori larvae. These results suggest that the group I NPV-specific protein BV/ODV-E26 determines tissue tropism and virulence in host lepidopteran insects.     

BmNPV chitinase gene deletion enhances foreign gene expression in a BmN cell system

Bombyx mori nucleopolyhedrovirus (BmNPV) is extensively being studied as an expression vector for heterologous gene expression in silkworm-derived cells as well as in the host larvae or pupae. BmNPV chitinase is necessary for liquefaction of the virus-infected host insect. The influence of chitinase on the efficiency of foreign gene expression was studied to provide a scientific basis for improving the BmNPV expression system. The BmNPV chitinase gene ( chiA ) was deleted and the expression level of the polyhedrin promoter controlling the lac Z gene in BmN cells was determined. Sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) showed that β-galactosidase accounted for approximately 6.9 and 7.7% of the total protein in BmN cells infected with the chiA deficient Bm lac Z⁺ chiA ⁻ at 3 and 4 days post infection, while the total protein was 3.2 and 4.2% in cells infected with Bm lac Z⁺. The relative β-galactosidase activities in Bm lac Z⁺ chiA ⁻-infected cells improved 2.33- and 1.56-fold compared to those of Bm lac Z⁺-infected cells at 3 and 4 days post infection. The results of the present study suggest that chitinase deletion could improve the lacZ expression level in the BmNPV-BmN cell expression system.     

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.     

Colocalization of Baculovirus IE-1 and Two DNA-Binding Proteins, DBP and LEF-3, to Viral Replication Factories

We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107–3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.     

Helicoverpa armigera nucleopolyhedrovirus ORF80 encodes a late, nonstructural protein

The Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ORF80 (ha80) has 765 bp encoding a protein with approximately 254 amino acids and a predicted molecular weight of 30.8 kDa. Homologues of ha80 are found in most baculovirus sequences, including those from lepidopteran NPVs, lepidopteran granuloviruses (GVs), hymenopteran baculoviruses, and one dipteran baculovirus, yet their functions remain unclear. In this study we characterized ha80, and showed that it was transcribed late in infected host cells (HzAM1). The product of ha80 was a 31 kDa protein that was not a structural protein of budded virus (BV) or occlusion-derived virus (ODV) particles. Ha80 was first detected in the cytoplasm of infected HzAM1 cells at 12 h p.i., and was observed in the nucleus at later stages of infection, suggesting that it may be involved in transporting viral proteins into the host cell nucleus or play its roles in the nucleus.     

High-frequency homologous recombination between baculoviruses involves DNA replication

We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4% when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1.4 kb, the mean frequency of recombination was 2.7%. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was 相似文献   

Expression of <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> endoglucanase III in the larvae of silkworm, <Emphasis Type="Italic">Bombyx mori L.</Emphasis> and characteristic analysis of the recombinant protein

Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.     

Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene.