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.     

家蚕生物反应器的研究进展及开发前景

目前,家蚕生物反应器的研究和开发主要是以BmNPV为载体,在家蚕体液中表达多种有用蛋白,其表达量比其它生化微生物高出许多倍;但是家蚕绢丝腺细胞表达外源蛋白比家蚕BmNPV表达系统有着更大的优越性。本文综述了家蚕BmNPV表达系统的研究现状及家蚕丝腺反应器表达外源基因的开发前景。     

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   

Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus

Abstract:  The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6× His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8  m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses.     

Enhanced production of secretory beta1,3-N-acetylglucosaminyltransferase 2 fusion protein into hemolymph of Bombyx mori larvae using recombinant BmNPV bacmid integrated signal sequence

The enhanced secretion of beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) fusion protein into the hemolymph of Bombyx mori larvae was studied using a recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid integrating seven signal sequences. When the BmNPV bacmid encoding the signal sequences from the silkworm B. mori bombyxin (bx) and B. mori prophenoloxidase-activating enzyme (ppae) was injected into silkworm larvae, 56.1 and 51.5mU/ml beta3GnT, respectively, were secreted into the hemolymph of silkworm larvae. For bx, 97.3% of the total beta3GnT activity was secreted into hemolymph, and only 1.1% remained in the intestines of silkworm larvae. For ppae, 90.8% of the total beta3GnT activity was secreted to the hemolymph, but 7.8% remained in the intestines of silkworm larvae. Using the BmNPV bacmid encoding bx, the amount of secreted beta3GnT was 91mug per larva, which was 2.5% of the total amount of protein in the hemolymph.     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene.B.mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo.The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E.coli DH10B.Recombinant bacmids were screened by kanamycin resistance,PCR and restriction enzyme(REN)digestion.One of the bacmid colonies,BmBacJS13,which had similar REN profiles to that of wild-type BmNPV,was selected for further research.To investigate the infectivity of BmBacJS13,the polyhedrin gene was introduced into the bacmid and the resultant recombinant(BmBacJS13-ph)was transfected to BmN cells.The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells.Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV.Bio-assays indicated that BmBacJS13-ph was also infectious to B.mori larvae.     

In vivo production of recombinant protein by a baculovirus vector inoculated perorally to the prefinal instar larvae of Bombyx mori L. (Lep., Bombycidae) aided by an optical brightener, Tinopal UNPA-GX

Abstract: Budded particles of nucleopolyhedrovirus (NPV) were found to infect perorally the 4th (prefinal) instar larvae of Bombyx mori L. that were treated by an optical brightener, Tinopal UNPA-GX (Tinopal). Host larvae were fed a diet containing 0.3% (w/w) Tinopal on day 1 in the 4th instar and then fed a diet contaminated by budded particles of NPV (1.0 × 10⁶ TCID₅₀ U/larva) that was pathogenic to B . mori (BmNPV) on day 2 (inoculation schedule 1). Another set of host larvae was fed a diet containing BmNPV budded particles (2.5 × 10⁶ TCID₅₀ U/larva) together with 0.3% (w/w) Tinopal on day 1 in the 4th instar (inoculation schedule 2). Host larvae treated by both schedules died of viral infection. The operation of schedule 2 is simpler than that of schedule 1, although the former required higher doses of the virus for satisfactory infection. We inoculated a baculovirus vector carrying human serum albumin (HSA) gene into 4th instar B . mori larvae by schedule 1. Recombinant HSA was detected in the homogenate of host larvae 4 days after inoculation. The peroral inoculation of BmNPV budded particles aided by Tinopal may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts.     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

Arginine kinase is highly expressed in a resistant strain of silkworm (Bombyx mori, Lepidoptera): Implication of its role in resistance to Bombyx mori nucleopolyhedrovirus

A gene encoding Bombyx mori arginine kinase (BmAK) has been indentified differentially expressed in the midguts of Bombyx mori strain NB which is resistant to nucleopolyhedrovirus (BmNPV), strain 306 which is susceptible to NPV and a near isogenic line BC(8) with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). In this study, we characterized the expression profiles of BmAK using RT-PCR and real-time quantitative PCR. The expression level of BmAK fluctuated in various developing stage and various tissue. Remarkably, the expression level of BmAK increased more than 10-fold 24 hours post inoculation (h p.i.) of NPV in strain NB and BC(8), while such increment was abraded in strain 306 although the basal expression level of BmAK in strain 306 was higher than that of strain NB and BC(8). Western blotting analysis using polyclonal antibody against BmAK verified such observation, and immunofluoresence analysis indicated for the first time that BmAK was mainly located to the cytoplasm or some structures in cytoplasm. These findings suggest that arginine kinase is involved in the antiviral process of Bombyx mori larvae against NPV infection.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3zeta and Bm14-3-3epsilon). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3zeta shared 90% identity with that in Drosophila, while Bm14-3-3epsilon shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3zeta expression levels are higher than Bm14-3-3epsilon in seven tissues and in four silkworm developmental stages examined. Bm14-3-3zeta was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3epsilon expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3zeta expression is universal and continuous, while Bm14-3-3epsilon expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

A novel way to purify recombinant baculoviruses by using bacmid

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.     

Conservation of a pair of serpin 2 genes and their expression in Amphiesmenoptera

Silk secreted by the larvae of Hydropsyche angustipennis (Trichoptera) contains serpins HaSerp2A and HaSerp2B that are homologous to serpin 2 known from several lepidopterans and some other insects. The gene HaSerp2A is 2684 bp downstream from the HaSerp2B gene. The genes possess identical exon/intron segmentation (9 exons) and their sequences are nearly identical: only 8 out of 1203 nt differ in the coding region, 4 out of 567 nt in the introns and 2 out of 52 nt in 3' UTR. Both genes are highly expressed in the silk glands whereas expression in larval carcass devoid of the silk glands is hard to detect. Translation products of the genes consist of 401 amino acids, are 98.8% identical, and are secreted as 45 kDa proteins into silk. Homologous genes in similar tandem arrangement occur on chromosome 15 of Bombyx mori (Lepidoptera). The upstream gene BmSerp2B is modified in several exons and does not seem to produce functional mRNA. The gene BmSerp2A contains two copies of exon 9, of which only the second one is used. One kind of mRNA does and the other does not include exon 1, which encodes a signal peptide. The mRNA yielding secreted BmSerp2A is expressed in the posterior, and that encoding the cytoplasmic BmSerp2A in the middle silk gland region; both kinds are strongly expressed in the anterior region. The data indicate that (1) A duplication of serpin 2 gene occurred either before Trichoptera and Lepidoptera diverged as separate orders or independently in early phylogeny of either order; (2) In the caddisfly H. angustipennis, both genes are expressed specifically in the silk glands and generate proteins deposited in the silk; (3) Only one gene seems to be functional in B. mori and is expressed in a cytoplasmic and in a secreted forms in diverse organs, including the silk glands.     

30K蛋白对家蚕细胞及幼虫存活的作用

利用杆状病毒表达系统在家蚕BombyxmoriL.细胞BmN中表达家蚕30K蛋白,以亲本病毒BmPAK6为对照,将重组病毒Bm/r-30K分别感染BmN细胞及家蚕幼虫,观察其感染后不同时间的凋亡及存活率。与对照相比,重组病毒感染后的BmN细胞的存活率明显高于对照组,并且感染的家蚕幼虫存活时间也较长,表明体内过量表达家蚕30K蛋白有助于延长其细胞及幼虫的存活。     

Disintegration of the peritrophic membrane of silkworm larvae due to spindles of an entomopoxvirus

Mode of action by which entomopoxvirus (EPV) spindles enhance nucleopolyhedrovirus (NPV) infection remains unclear. Spindles of Anomala cuprea entomopoxvirus (AcEPV), a coleoptran EPV, are known to enhance Bombyx mori NPV (BmNPV) infection in silkworm larvae. AcEPV spindles were orally administered to silkworm larvae with or without BmNPV polyhedra, and the peritrophic membranes (PMs) were observed using a binocular microscope. Soon after the larvae's access to spindles with or without the polyhedra had been terminated, some PMs disappeared wholly and some were observed in partial form. Some of the partial PMs observed were very fragile. The disintegration of the PM due to spindles also was observed by the histological sectioning of the midgut. However, a day after the larvae had terminated their access to the spindles, the PM regenerated partially or wholly. In contrast, the administration of AcEPV spheroids caused neither the disintegration of PMs nor the enhancement of BmNPV infection in silkworm larvae. These findings strongly suggest that the enhancement of NPV infection occurs due to that a greater number of NPV virions reaching the microvilli of midgut susceptible to NPV, since spindles lead to the disintegration of the PM as a barrier against NPV virions.     

DNA polymerase-delta from the silk glands of Bombyx mori.

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.     

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C‐terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth‐instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

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.