Molecular Modification Strategies of Nitrilase for Its Potential Application in Agriculture.

Some feed source plants will produce secondary metabolites such as cyanogenic glycosides during metabolism, which will produce some poisonous nitrile compounds after hydrolysis and remain in plant tissues. The consumption of feed-source plants without proper treatment affect the health of the animals' bodies. Nitrilases can convert nitriles and have been used in industry as green biocatalysts. However, due to their bottleneck problems, their application in agriculture is still facing challenges. Acid-resistant nitrilase preparations, high-temperature resistance, antiprotease activity, strong activity, and strict reaction specificity urgently need to be developed. In this paper, the application potential of nitrilase in agriculture, especially in feed processing industry was explored, the source properties and catalytic mechanism of nitrilase were reviewed, and modification strategies for nitrilase application in agriculture were proposed to provide references for future research and application of nitrilase in agricultural and especially in the biological feed scene.

BmNPV Orf 65 (Bm65) Is Identified as an Endonuclease Directly Facilitating UV-Induced DNA Damage Repair.

Baculoviruses have been used as biopesticides for the control of Lepidoptera larvae. However, solar UV radiation reduces the activity of baculovirus. In this study, an UV endonuclease, Bm65, was found encoded in the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV). Bm65 (the ortholog of AcMNPV orf79) was guided by a key nuclear localization signal to enter the nucleus and accumulated at UV-induced DNA damage sites. Subsequent results further showed that Bm65-mediated DNA damage repair was not the only UV damage repair pathway of BmNPV. BmNPV also used host DNA repair proteins to repair UV-induced DNA damage. In summary, these results revealed that Bm65 was very important in UV-induced DNA damage repair of BmNPV, and BmNPV repaired UV-damaged DNA through a variety of ways. IMPORTANCE Baculovirus biopesticides are environmentally friendly insecticides and specifically infect invertebrates. UV radiation from the sunlight greatly reduces the activity of baculovirus biopesticides. However, the molecular mechanisms of most baculoviruses to repair UV-induced DNA damage remain unclear. Nucleotide excision repair (NER) is a major DNA repair pathway that removes UV-induced DNA lesions. At present, there are few reports about the nucleotide excision repair pathway in viruses. Here, we showed for the first time that the baculovirus Bm65 endonuclease actually cleaved UV-damaged DNA. Meanwhile, we found that BmNPV used both viral-encoded enzymes and host DNA damage repair proteins to reverse UV-induced DNA damage. These results will provide a reference for the research of UV damage repair of other viruses.

Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System.

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)-insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 104 vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 1012 VG/larva and 4.6 × 1012 VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.

18 Additional Amino Acids of the Signal Peptide of the Bombyx mori Nucleopolyhedrovirus GP64 Activates Immunoglobulin Binding Protein (BiP) Expression by RNA-seq Analysis.

GP64 is the key membrane fusion protein of Group I baculovirus, and while the Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 contains a longer n-region (18 amino acid) of the signal peptide than does the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the function of the n-region has not been determined. In this study, we first showed that n-region is required for membrane protein localization in BmN cells, then the transcriptome sequencing was conducted on proteins guided by different signal peptide regions, and the results were analyzed and validated by quantitative PCR and luciferase assays. The results indicated that 1049 differentially expressed genes (DEGs) were identified among the different region of signal peptides and the control. With the n-region, the protein export pathway was upregulated significantly, the Wnt-1 signaling pathway was downregulated, and BiP was significantly activated by the GP64 full-length signal peptide. Furthermore, RNA interference on BiP efficiently increased luciferase secretion. These results indicate that the GP64 n-region plays a key role in protein expression and regulation.

Rapid detection of Bombyx mori bidensovirus by loop-mediated isothermal amplification based lateral flow dipstick assay for field applications.

Bombyx mori bidensovirus (BmBDV), is the only bipartite single-stranded DNA (ssDNA) insect virus reported to date. BmBDV causes fatal flacherie disease in silkworm, resulting in large economic losses to sericulture. We developed a loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD) method that can successfully and rapidly detect BmBDV DNA with a low limit of detection (5 fg, 400 copies of the BmBDV genome). The method was evaluated and improved for direct field diagnosis using silkworm faeces. In the field, the actual limit of detection was ∼50 fg (4000 copies of the BmBDV genome). The results demonstrated that, compared with traditional methods for BmBDV detection, our new LAMP-LFD method was much more rapid, sensitive and cost-effective, with less dependence on equipment, making it easier to use. The method has potential to be translated into a new diagnostic product for field applications in the sericulture industry.

The Motif of 76KRKCSK in Bm65 Is an Efficient Nuclear Localization Signal Involved in Production of Infectious Virions.

orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) codes for a putative 104-amino-acid protein containing three cysteine residues with a putative molecular mass of 12.2 kDa. Previous studies have showed that Bm65 accumulates mainly in nucleus and involved in the repair of UV-damaged DNA. However, the mechanism of nuclear import of Bm65 remains unclear. In this study, a SDS-stable Bm65 tetramer was found in BmNPV-infected BmN cells, and alanine substitutions for the three cysteine residues did not affect the formation of Bm65 tetramer. Additionally, a basic amino acid cluster of the Bm65 protein was identified as an efficient nuclear localization signal (NLS). Firstly, transient expression of GFP-fused truncated Bm65 variants revealed that the 76KRKCSK motif functions as the NLS. This was also confirmed by alanine substitution in the 76KRKCSK motif, which caused attenuated nuclear localization of Bm65. Next, the 76KRKCSK motif-mutated bacmid was generated and the 76KRKCSK motif was also found to be important for nuclear localization of Bm65 in BmNPV-infected conditions. Lastly, analyses of flag-tagged Bm65 expressing bacmids revealed that the mutations in 76KRKCSK motif did not affect the synthesis of Bm65 tetramer, but severely impaired production levels of infectious virions. In conclusion, Bm65 exists in mainly a tetrameric form in virus-infected cells, which may be involved with production levels of infectious virions.

Discovery of anti-viral molecules and their vital functions in Bombyx mori.

The silkworm Bombyx mori (B. mori), a lepidopteran model organism, has become an important model for molecular biology researches with its genome completely sequenced. Silkworms confront different types of virus diseases, mainly including those caused by Bombyx mori nucleopolyhedrovirus (BmNPV), Bombyx mori densovirus type 1 (BmDNV-1), Bombyx mori bidesovirus (BmBDV) which was termed as Bombyx mori densovirus type 2 (BmDNV-2) or Bombyx mori parvo-like virus (BmPLV) before in sericulture. B. mori offers excellent models to study the molecular mechanisms of insect innate immune responses to viruses. A variety of molecules and pathways have been identified to be involved in the immune responses in the silkworm to viruses, such as the antimicrobial peptides, prophenoloxidase-activating system, apoptosis, ROS, small RNA and related molecules. Here in this review, we summarize the current research advances in molecules involved in silkworm anti-virus pathways. Moreover, taking BmNPV as an example, we proposed a schematic model of molecules and pathways involved in silkworm immune responses against virus infection. We hope this review can facilitate further study of antiviral mechanisms in silkworm, and provide a reference for virus diseases in other organisms.

Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage.

It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage. The localization of Bm65 was studied using enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid. We also showed that after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light.

Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins.

Polyhedra can encapsulate other proteins and have potential applications as protein stabilizers. The extremely stable polyhedra matrix may provide a platform for future engineered micro-crystal devices. However, the protein composition of the polyhedra matrix remains largely unknown. In this study, the occlusion-derived virus (ODV)-removed BmNPV polyhedra matrix fraction was subjected to SDS-PAGE and then an LC-ESI-MS/MS analysis using a Thermo Scientific Q Exactive mass spectrometer. In total, 28 host and 91 viral proteins were identified. The host components were grouped into one of six categories, i.e., chaperones, ubiquitin and related proteins, host helicases, cytoskeleton-related proteins, RNA-binding proteins and others, according to their predicted Pfam domain(s). Most viral proteins may not be essential for polyhedra assembly, as evidenced by studies in the literature showing that polyhedra formation occurs in the nucleus upon the disruption of individual genes. The structural role of these proteins in baculovirus replication will be of significant interest in future studies. The immobilization of enhanced green fluorescent protein (eGFP) into the polyhedra by fusing with the C-terminus of BM134 that is encoded by open reading frame (ORF) 134 suggested that the polyhedra had a powerful capacity to trap foreign proteins, and BM134 was a potential carrier for incorporating proteins of interest into the polyhedra.

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx.

The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing.

Characterization of aggregate/aggresome structures formed by polyhedrin of Bombyx mori nucleopolyhedrovirus.

Virus infections often lead to formation of aggregates and aggresomes in host cells. In this study, production of aggregates and aggresomes by the highly expressed protein polyhedrin of Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h postinfection (p.i.) was detected with a fluorescent molecular dye, and verified by colocalization of polyhedrin with aggresomal markers, GFP-250 and γ-tubulin. Polyhedrin aggregates showed hallmark characteristics of aggresomes: formation was microtubule-dependent; they colocalized with heat shock cognates/proteins of the 70-kDa family (HSC/HSP70s), ubiquitinated proteins and recruited the mitochondria. Aggregated polyhedrin protein gradually gained its active conformation accompanying progress of BmNPV infection. At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production. These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17.

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.

Identification of a novel functional nuclear localization signal in the protein encoded by open reading frame 47 of Bombyx mori nucleopolyhedrovirus.

BM47 is encoded by open reading frame 47 of Bombyx mori nucleopolyhedrovirus (BmNPV). BM47 was localized in the nucleus of BmNPV-infected cells. In the present study, we investigated a novel nuclear localization signal (NLS) for BM47 transport and accumulation in the nucleus. By expressing various regions of BM47 fused to enhanced green fluorescent protein (EGFP), we demonstrated that residues 117-148 are necessary for mediating nuclear localization of BM47. Site-directed mutation analysis showed that the two basic residue clusters at positions 117-120 (¹¹7RKRR) and 144-148 (¹44RKR-K) constitute an authentic NLS for BM47 localization. Finally, we observed that two clusters of basic residues were conserved in BM47 homologues of group-I nucleopolyhedroviruses.

Open reading frame 60 of the Bombyx mori nucleopolyhedrovirus plays a role in budded virus production.

Open reading frame 60 (bm60) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a conserved gene among group I and some group II NPVs. bm60 encodes a late expressed protein that localizes to both the cytoplasm and nucleus of infected cells. This paper describes the characterization of a BmNPV mutant (vbm60-Null) lacking functional bm60. It was observed that the production of budded virus (BV) was reduced by nearly an order of magnitude relative to wt virus in vbm60-Null-infected BmN cells and B. mori larvae. Quantitative real-time PCR assay showed that the viral DNA replication was affected in infected cells due to disruption of bm60. Larval bioassays showed that the speed of kill of vbm60-Null virus was greatly reduced, as it took approximately 28-36 h longer to kill the fifth instar B. mori larvae. These results suggest that BmNPV bm60 is not essential for viral replication, but required for efficient BV production.

Cloning and partial characterization of a gene in Bombyx mori homologous to a human adiponectin receptor.

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.

Expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate).

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate), named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate). Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DH10Bac. The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3) isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BmN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.

[Expression of non-structural protein 1 of Bombyx mori densovirus-3].

We cloned the NS1 gene of BmDNV-3 into the prokaryotic expression vector pET-30a after we amplified the NS1 gene by PCR, and then we transformed the pET-30a-NS1 plasmid into BL21 star to express BmDNV-3 NS1. After we induced NS1 expression by IPTG, we used Western blot analysis to indentify the recombinant protein, the result indicated that the recombinant protein was BmDNV-3 NS1. After purification, we used NS1 to immunize New Zealand white rabbits following standard protocol to harvest anti-NS1 anti serum. On the other hand, we cloned the BmDNV-3 NS1 into pFastBacHTb-eGFP vector, and then transformed the pFastBacHTb-NS1-eGfp into BmDH10BAC to harvest recombinant bacmid genome. We obtained the recombinant virus from the cells, which was transfected by the recombinant bacmid genome using liposomes. We used the virus genome to infect Bombyx mori larvae. We observed the fluorescence in the cells and silkworm larvae at 2 days post infection, and then we used SDS-PAGE and fluorescence image analysis to identify the fusion protein. The result showed that the size of the fusion protein was not consistent with the expected size of NS1-eGFP, indicating the fusion protein was degraded randomly by the intrinsic digestive protease. To further confirm the fusion protein, we used Western blot with an anti-NS1 antibody.

Bombyx mori nucleopolyhedrovirus orf25 encodes a 30kDa late protein in the infection cycle.

Bombyx mori nucleopolyhedrovirus (BmNPV) orf25 gene was characterized for the first time. The coding sequence of Bm25 was amplified and subcloned into the prokaryotic expression vector pGEX-4T-2 to produce glutathione S-transferase-tagged fusion protein in the BL21 (DE3) cells. The GST-Bm25 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. Temporal expression analysis revealed a 30-kDa protein, which was detected beginning 24 hours post-infection using a polyclonal antibody against GST-Bm25 fusion protein. The transcript of Bm25 was detected by RT-PCR at 18-72 h p.i. In conclusion, the available data suggest that Bm25 encodes a 30kDa protein expressed in the late stage of infection cycle.

Characterization of a late gene, ORF67 from Bombyx mori nucleopolyhedrovirus.

Open reading frame 67 of Bombyx mori nucleopolyhedrovirus (BmORF67) is a homologue of Autographa californica multiple NPV ORF81. The gene is conserved among all baculoviruses and is thus considered a baculovirus core gene. The transcript of BmORF67 was detected at 18-72 h post-infection (p.i.). Polyclonal antiserum raised to a His-BmORF67 fusion protein recognized BmORF67 in infected cell lysates from 24 to 72 h p.i., suggesting that BmORF67 is a late gene. BmORF67 was not detected either in budded viruses or occlusion-derived virus. Immunofluoresence analysis showed that the protein located in the cytoplasm and interacted with host protein actin A3. In conclusion, BmORF67 is a late protein localized in the cytoplasm of infected cells that interacts with host protein.

[Rapid disruption of Bombyx mori nucleopolyhedrovirus orf60 by red recombination system].

BmNPV bacmid constructed recently and Red recombinant system were used to rapidly disrupted Bombyx monri nucleopolyhedrovirus (BmNPV) orf60 in Escherichia coli (E. coli) BW25113. BmNPV bacmid isolated from E. coli BmDH10Bac was electroporated into E. coli BW25113, which harbors plasmid pKD46 encoding lamda Red recombinase,to produce E. coli BW25113-Bac, which could be used for gene deletion of BmNPV. A linear fragment was amplified by PCR from plasmid pKD3 (containing a chloramphenicol acetyltransferase gene cat) using a pair of primers with length of 63bp,which had 45 bp homologous to the orf60 gene and 18bp homologous to cat sequences. The linear fragment was electroporated into E. coli BW25113-Bac and homologous recombination occurred between the linear fragment and orf60 with the help of lamda Red recombinase. Three specific primer pairs were used to confirm the replacement of orf60 by cat gene. Western blot analysis showed that orf60 was not expressed in BmN cells infected with knockout bacmid.