Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.

Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. The protocol steps include generation of viruses, virus quality control, and initial expression trials utilizing standard commercial baculovirus vector systems and are exemplified for G protein-coupled receptor targets. The viral quality, quantity, and recombinant protein expression are evaluated by microscopy, flow cytometry, fluorimetry, and SDS-PAGE, using either covalently fused fluorescent proteins or co-expressed fluorescence markers. Moreover, integral membrane protein expression levels, approximate molecular mass, and stability can be evaluated from small-scale expression and purification trials.

Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies.

BACKGROUND: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. RESULTS: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. CONCLUSIONS: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

Identification of specific B cell linear epitopes of mycoplasma hyorhinis P37 protein using monoclonal antibodies against baculovirus-expressed P37 protein.

BACKGROUND: Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development. However, there is little information concerning Mhr P37 protein epitopes. A precise analysis of the P37 protein epitopes should extend our understanding of the antigenic composition of the P37 protein and the humoral immune responses to Mhr infection. Investigating the epitopes of Mhr P37 will help to establish a detection method for Mhr in tissue and provide an effective tool for detecting Mhr infection. RESULTS: Western blot and indirect immunofluorescence assays (IFA) confirmed that the expressed P37 protein was recognized by Mhr-positive porcine and mouse sera. Furthermore, the P37 protein was purified using affinity chromatography and used to immunize mice for hybridoma cell fusion. Four monoclonal antibodies (mAbs) found to be positive for Mhr were detected in infected lung tissue. A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs. The core epitope was determined to be 206KIKKAWNDKDWNTFRNF222. CONCLUSIONS: In this study, we identified 17 critical amino acids that determine the epitope of the P37 protein of Mhr. This study identified mAbs that could provide useful tools for investigating the Mhr P37 antigenic core epitope (amino acids 206-222) and detecting Mhr-specific antigens in infected tissue.

Baculovirus entire ORF1629 is not essential for viral replication.

It is generally accepted that ORF1629 is essential for baculovirus replication, which has enabled isolation of recombinant viruses in a baculovirus expression system using linearized viral DNA. ORF1629-defective viruses cannot replicate in insect cells; only recombinant virus with complete ORF1629 restoration by recombination can propagate, allowing for pure isolation and the development of bacmids for easy selection of recombinant viruses. We inadvertently found proliferation in insect cells of a bacmid lacking a complete ORF1629. PCR indicated no other viruses but a lack of complete ORF1629 in the proliferated bacmid, suggesting that the baculovirus propagated without a complete ORF1629. Lack of ORF1629 decreased the virus growth rate and yield; it also increased the occlusion body (OB) size but decreased its yield. These results were confirmed for Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV). Thus, entire ORF1629 is not essential for viral replication, though it does affect the virus growth rate, yield, and size and OB production.

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing.

The clustered regularly interspaced short palindromic repeats (CRISPR) system-associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.

Preparing Antigens Using a Baculovirus Expression System.

Baculovirus-produced recombinant proteins circumvent many of the issues that limit bacterial protein production. Baculoviruses contain double-stranded, circular, supercoiled DNA in a rod-shaped capsid. The viral life cycle includes three major phases: (1) early (or virus synthesis) phase (0.5-6 h after infection), which includes viral attachment, uncoating early viral gene expression, and shutting off host gene expression; (2) late phase (6-12 h after infection), which includes viral replication and formation of budded virus containing host plasma membrane and glycoprotein (gp)64, which is required for virus entry; and (3) very late phase (24-96 h after infection), which includes formation of polyhedral inclusion bodies and cell lysis. Large proteins can thus be produced readily in abundant quantities. Furthermore, posttranslational modifications of the protein similar to those of mammals (such as phosphorylation, disulfide-bond formation, and even glycosylation) will occur; indeed, because typical baculovirus constructs produce secreted proteins, most products will be glycosylated. Baculovirus expression systems are safe, because baculoviruses are nonpathogenic to mammals and plants, and insect cells can be grown easily in suspension culture, which makes protein production easy to scale up. Counterbalancing this approach, however, is the need to perform a two-step process: first cloning the gene into the recombination donor vector and then generating the viral construct. At that point, the virus can be used as a mixture or plaque-purified. Insect cells are then infected with the virus, and the proteins are produced.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Expression- and genomic-level changes during passage of four baculoviruses derived from bacmids in permissive insect cell lines.

The baculovirus-based bacmid expression vector system has been widely used for protein production in basic research and biotechnological laboratories. Since the first construction of the Autographa californica multiple nucleopolyhedrovirus bacmid (AcBacmid), three more bacmids have been created from Bombyx mori nucleopolyhedrovirus (BmBacmid), Spodoptera exigua nucleopolyhedrovirus (SeBacmid) and Helicoverpa armigera nucleopolyhedrovirus (HaBacmid). Each of these bacmid-derived viruses replicates efficiently in a range of specific and permissive cell types. Here, we investigated the relative stability of each virus derived from the bacmid during passage in permissive cell lines through assessment of their expression level and genome structure changes. Using two different reporters, the expression levels of the viruses from the AcBacmid-Sf9, AcBacmid-Tn5, BmBacmid-BmN and SeBacmid-SeE1 bacmid-cell systems were significantly reduced after five passages of the viruses, whereas the reductions were not detected in the AcBacmid-Sf21 and HaBacmid-HzAM1 systems. Pulse field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis of viral DNA isolated from passaged viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 systems showed no major genomic changes. In contrast, the genomes from passaged viruses in the AcBacmid-Tn5 and AcBacmid-Sf9 systems displayed reduced genome size and various mutations at individual loci, including genotypes missing one at least or more viral RNA polymerase subunits and fp25k. These genotypic changes were correlated with reduced protein expression. RFLP analysis of viral DNA from passaged viruses in the BmBacmid-BmN and SeBacmid-SeE1 systems exhibited changes in genome size, including excision of particular EcoRI fragments containing the mini-F replicon. Collectively, our data suggest that the viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 bacmid-cell systems are better for large-scale protein expression in continuous culture. Further study is needed to investigate the mechanism(s) behind the protein expression reduction in these bacmid-derived virus/cell systems.

Production and Purification of Baculovirus for Gene Therapy Application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

Quantitative proteomics of Sf21 cells during Baculovirus infection reveals progressive host proteome changes and its regulation by viral miRNA.

System level knowledge of alterations in host is crucial to elucidate the molecular events of viral pathogenesis and to develop strategies to block viral establishment and amplification. Here, we applied quantitative proteomics approach to study global proteome changes in the host; Spodoptera frugiperda upon infection by a baculovirus, Spodoptera litura NPV at two stages i.e. 12 h and 72 h post infection. At 12 hpi, >95% of host proteins remained stable, however at 72 hpi, 52% host proteins exhibited downregulation of 2-fold or more. Functional analysis revealed significant upregulation of transposition and proteasomal machinery while translation, transcription, protein export and oxidative phosphorylation pathways were adversely affected. An assessment of perturbed proteome after viral infection and viral miRNA expression led to the identification of 117 genes that are potential targets of 10 viral miRNAs. Using miRNA mimics, we confirmed the down regulation of 9 host genes. The results comprehensively show dynamics of host responses after viral infection.

Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus.

BACKGROUND: Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. METHOD: Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. RESULTS: Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. CONCLUSION: The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

Deltabaculoviruses encode a functional type I budded virus envelope fusion protein.

Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cell culture. An f homologue gene is absent in gammabaculoviruses. Here we characterized the putative F-homologue (Cuni-F), encoded by (ORF) 104 of Culex nigripalpus nucleopolyhedrovirus (CuniNPV), the only deltabaculovirus member. When expressed alone, this protein seems to locate on the cell surface and is able to induce cell-cell fusion. When expressed by an alphabaculovirus (Autographa california nucleopolyhedrovirus), it was found to be incorporated into BVs. Western blot analyses detected the uncleaved Cuni-F0 and the furin-cleaved F1 forms. Treatment of infected cells with tunicamycin showed that Cuni-F contains N-glycans. Mutagenesis analysis identified the canonical furin cleavage site 126RARR129 as being responsible for the cleavage of Cuni-F in insect cells. The collective evidence suggests that CuniNPV encodes a functional F protein.

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV immediate early-1 (ie-1) and me53 genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy.IMPORTANCE Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species.

A Conserved Glycine Residue Is Required for Proper Functioning of a Baculovirus VP39 Protein.

The baculovirus VP39 protein is a major nucleocapsid protein essential for viral propagation. However, the critical domains or residues of the VP39 protein have not yet been identified. Here, we performed mutagenesis experiments with Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2'-deoxyuridine and isolated a BmNPV mutant that produced fewer occlusion bodies than the wild-type virus. This mutant also produced fewer infectious budded viruses (BVs) than the wild-type virus in both cultured cells and B. mori larvae. Marker rescue experiments using genomic libraries identified a single nucleotide mutation in the vp39 gene. This mutation resulted in an amino acid substitution at glycine 276 (Gly-276) to serine, which was required for all the defective phenotypes observed in the mutant. Sequence comparison revealed that this residue is completely conserved among the VP39 proteins of the sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. Although early viral gene expression was not significantly affected, the level of expression of a late gene, vcath, was reduced. In addition, two of the very late genes were markedly downregulated in cells infected with this mutant. Western blot and quantitative PCR analyses revealed that the BVs produced from cells infected with this mutant contained smaller amounts of the VP39 protein and viral genomic DNA than those produced from wild-type virus-infected cells. Combined with the results of transmission electron microscopy, VP39 Gly-276 can be concluded to be essential for correct nucleocapsid assembly, viral DNA packaging, and viral gene expression, especially of very late genes.IMPORTANCE The major nucleocapsid protein gene vp39 is one of the most well-known baculovirus genes. Although several viral and host proteins that interact with the VP39 protein have been identified, the functionally important domains or residues of this protein remain unknown. The present study revealed that the glycine residue at residue 276, which is completely conserved among sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses, is important for the VP39 function, i.e., structural assembly of nucleocapsids and viral DNA packaging. Moreover, our results provide evidence for the link between nucleocapsid formation and the transcription of viral very late genes.

In vitro production of baculoviruses: identifying host and virus genes associated with high productivity.

Baculoviruses are recognized as viral workhorses of biotechnology, being used for production of vaccines, complex recombinant proteins, gene delivery vectors' and safe biological pesticides. Improving production yields and understanding the interactions of the virus and its host cell are important aspects of ensuring baculovirus-based processes are commercially competitive. This study aims at potential optimization of host cells used in in vitro virus production by systemically investigating changes in host gene expression in response to virus replication and transcription inside host cells. The study focuses on in vitro interactions of the Helicoverpa armigera virus with Helicoverpa zea insect cells. We used 22 genome-wide microarrays to simultaneously measure both virus and host genes in infected cells in multiple batch suspension cultures, representing high- and low-producing infection conditions. Among 661 differentially expressed genes, we identified a core set of 59 host genes consistently overexpressed post infection, with strong overrepresentation of genes involved in retrotransposition, protein processing in the endoplasmic reticulum, and ubiquitin-mediated proteolysis. Applying a whole genome correlation network analysis to link gene expression to productivity, we revealed 18 key genes significantly associated to virus yield. In addition, this study is among the first to perform a genome-wide expression study for a major baculovirus group II strain, the H. armigera virus, extending current understanding of baculovirus-insect interactions, which mainly focuses on group I viruses.

[Research Advances in Baculovirus Occlusion-derived Virions].

Baculoviruses are a family of arthropod-specific viruses that produce two morphologically distinct types of virions (budded and occlusion-derived) in their lifecycle. Baculoviruses establish infection in the midgut of their host via the oral route: occlusion-derived virions have pivotal roles in these processes. This review summarizes the basic characteristics of baculoviruses, and discusses the composition and classification of baculovirus occlusion-derived virions. The latter focuses mainly on the evolution and role of multiple occlusion-derived virions in the lifecycle of baculoviruses. These achievements should aid understanding the evolution and infection mechanisms of baculoviruses.

Nanostructural characterization of Sf9 cells during virus-like particles generation.

Sf9 cells (a clonal isolate of Spodoptera frugiperda Sf21 cells) are commonly used to generate recombinant virus-like particles (VLPs). For VLPs generation, Sf9 cells are infected with recombinant baculoviruses (rBV) expressing desired proteins. During rBV infections, Sf9 cells have changes in cell diameters and surface structures. In this study, for the first time, we investigated nanostructural changes of Sf9 cells using atomic force microscopy (AFM) during VLPs generation containing Toxoplasma gondii rhoptry protein 18 (ROP 18). As results, Sf9 cells were changed to be larger at 2 days after rBV infections. They maintained their sizes and morphologies on day 3 and 4. Based on morphological (perimeter and diameter) and surface roughness (roughness average and root mean square) changes of Sf9 cells observed by AFM, we inferred that these nanostructural changes in Sf9 cell membranes might be due to the production and extrusion of VLPs after rBV infection. Our results suggest that shape and roughness parameters of Sf9 cell morphology and membrane surface by AFM could be very effective for quantitative analysis of VLP production. This study provides important information about structural and mechanochemical properties of Sf9 cells which are closely related with biological function. SCANNING 38:735-742, 2016. © 2016 Wiley Periodicals, Inc.

Recombinant Baculovirus Isolation.

Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

Virus and calcium: an unexpected tandem to optimize insecticide efficacy.

The effective control of insect pests is based on the rational use of the most efficient and safe insecticide treatments. To increase the effects of classical insecticides and to avoid the ability of certain pest insects to develop resistance, it is essential to propose novel strategies. Previous studies have shown that calcium-dependent phosphorylation/dephosphorylation is now considered as a new cellular mechanism for increasing the target sensitivity to insecticides. Because it is known that virus entry is correlated with intracellular calcium concentration rise, this report attempts to present the most important data relevant to the feasibility of combining an insect virus such as baculovirus or densovirus with an insecticide. In this case, the insect virus is not used as a bioinsecticide but acts as a synergistic agent able to trigger calcium rise and to activate calcium-dependent intracellular signalling pathways involved in the increase of the membrane receptors and/or ion channels sensitivity to insecticides. This virus-insecticide mixture represents a promising alternative to optimize the efficacy of insecticides against insect pests while reducing the doses.

Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin.

Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi. Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a "head-to-tail" shape. Cit1a fused to polyhedrin gene (polh) (polh-cit1a) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT50) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.