Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA.

Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions between neighboring SSB subunits on ssDNA. However, it is still challenging to perform structural studies on SSB-ssDNA filaments at high resolution using the most studied SSB models, largely due to the intrinsic flexibility of these nucleoprotein complexes. In this study, HaLEF-3, an SSB protein from Helicoverpa armigera nucleopolyhedrovirus, was used for in vitro assembly of SSB-ssDNA filaments, which were structurally studied at atomic resolution using cryo-electron microscopy. Combined with the crystal structure of ssDNA-free HaLEF-3 octamers, our results revealed that the three-dimensional rearrangement of HaLEF-3 induced by an internal hinge-bending movement is essential for the formation of helical SSB-ssDNA complexes, while the contacting interface between adjacent HaLEF-3 subunits remains basically intact. We proposed a local cooperative SSB-ssDNA binding model, in which, triggered by exposure to oligonucleotides, HaLEF-3 molecules undergo ring-to-helix transition to initiate continuous SSB-SSB interactions along ssDNA. Unique structural features revealed by the assembly of HaLEF-3 on ssDNA suggest that HaLEF-3 may represent a new class of SSB.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod.

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Viruses of the Fall Armyworm Spodoptera frugiperda: A Review with Prospects for Biological Control.

The fall armyworm (FAW), Spodoptera frugiperda, is a native pest species in the Western hemisphere. Since it was first reported in Africa in 2016, FAW has spread throughout the African continent and is now also present in several countries in Asia as well as Australia. The invasion of FAW in these areas has led to a high yield reduction in crops, leading to huge economic losses. FAW management options in the newly invaded areas are limited and mainly rely on the use of synthetic pesticides. Since there is a risk of resistance development against pesticides in addition to the negative environmental and human health impacts, other effective, sustainable, and cost-efficient control alternatives are desired. Insect pathogenic viruses fulfil these criteria as they are usually effective and highly host-specific with no significant harmful effect on beneficial insects and non-target organisms. In this review, we discuss all viruses known from FAW and their potential to be used for biological control. We specifically focus on baculoviruses and describe the recent advancements in the use of baculoviruses for biological control in the native geographic origin of FAW, and their potential use in the newly invaded areas. Finally, we identify current knowledge gaps and suggest new avenues for productive research on the use of viruses as a biopesticide against FAW.

Global Metabolic Profiling of Baculovirus Infection in Silkworm Hemolymph Shows the Importance of Amino-Acid Metabolism.

Viruses rely on host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Infection of the silkworm, Bombyx mori, by Bombyx mori nucleopolyhedrovirus (BmNPV), has been studied extensively in the past to unravel interactions between baculoviruses and their lepidopteran hosts. To understand the interaction between the host metabolic responses and BmNPV infection, we analyzed global metabolic changes associated with BmNPV infection in silkworm hemolymph. Our metabolic profiling data suggests that amino acid metabolism is strikingly altered during a time course of BmNPV infection. Amino acid consumption is increased during BmNPV infection at 24 h post infection (hpi), but their abundance recovered at 72 hpi. Central carbon metabolism, on the other hand, particularly glycolysis and glutaminolysis, did not show obvious changes during BmNPV infection. Pharmacologically inhibiting the glycolytic pathway and glutaminolysis also failed to reduce BmNPV replication, revealing that glycolysis and glutaminolysis are not essential during BmNPV infection. This study reveals a unique amino acid utilization process that is implemented during BmNPV infection. Our metabolomic analysis of BmNPV-infected silkworm provides insights as to how baculoviruses induce alterations in host metabolism during systemic infection.

Establishment and characterization of a new embryonic cell line from Helicoverpa armigera (Hübner).

Here, a new cell line, Ha168, was established from Helicoverpa armigera eggs and has been stably subcultured for over 30 passages in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line consists of round and spindle-shaped cells and several giant cells. The round cells, with a cell diameter of 14.30 ± 2.804 µm, account for 77% of the cells. DNA amplification fingerprinting, random amplified polymorphic DNA analysis, and analysis of the mitochondrial cytochrome c oxidase subunit I gene confirmed that the Ha168 cells were derived from H. armigera. Karyotype analysis revealed the average chromosome number of Ha168 cells to be 71. Growth curves at passage 25 were determined and demonstrated that the cell population doubling time is 56.8 h. No mycoplasma contamination was detected in the cell line. Ha168 cells can be infected by recombinant baculovirus AcMNPV-EGFP, and exogenous protein expression level in this cell line is 70% of that in the Sf9 cell line.

Dynamic chromatin accessibility profiling reveals changes in host genome organization in response to baculovirus infection.

DNA viruses can hijack and manipulate the host chromatin state to facilitate their infection. Multiple lines of evidences reveal that DNA virus infection results in the host chromatin relocation, yet there is little known about the effects of viral infection on the architecture of host chromatin. Here, a combination of epigenomic, transcriptomic and biochemical assays were conducted to investigate the temporal dynamics of chromatin accessibility in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The high-quality ATAC-seq data indicated that progressive chromatin remodeling took place following BmNPV infection. Viral infection resulted in a more open chromatin architecture, along with the marginalization of host genome and nucleosome disassembly. Moreover, our results revealed that chromatin accessibility in uninfected cells was regulated by euchromatic modifications, whereas the viral-induced highly accessible chromatin regions were originally associated with facultative heterochromatic modification. Overall, our findings illustrate for the first time the organization and accessibility of host chromatin in BmNPV-infected cells, which lay the foundation for future studies on epigenomic regulation mediated by DNA viruses.

Functional analysis of the baculovirus per os infectivity factors 3 and 9 by imaging the interaction between fluorescently labelled virions and isolated midgut cells.

Baculovirus occlusion-derived viruses (ODVs) contain ten known per os infectivity factors (PIFs). These PIFs are crucial for midgut infection of insect larvae and form, with the exception of PIF5, an ODV entry complex. Previously, R18-dequenching assays have shown that PIF3 is dispensable for binding and fusion with midgut epithelial cells. Oral infection nevertheless fails in the absence of PIF3. PIF9 has not been analysed in much depth yet. Here, the biological role of these two PIFs in midgut infection was examined by monitoring the fate of fluorescently labelled ODVs when incubated with isolated midgut cells from Spodoptera exigua larvae. Confocal microscopy showed that in the absence of either PIF3 or PIF9, the ODVs bound to the brush borders, but the nucleocapsids failed to enter the cells. Finally, we discuss how the results obtained for PIF3 with dequenching assays and confocal microscopy can be explained by a two-phase fusion process.

Biopesticide synergy when combining plant flavonoids and entomopathogenic baculovirus.

Four crop plants known to be hosts for the lepidopteran Trichoplusia ni (soybean, green bean, cotton, and cabbage) were treated with the biopesticide AfMNPV baculovirus in a dosage response assay. Treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Leaf trichomes on soybeans were not found to be responsible for the observed increase of insecticidal activity. Three flavonoid compounds (daidzein, genistein, and kaempferol) were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The individual flavonoid compounds did not cause T ni. mortality in no-virus assays when incorporated into artificial insect diet. The combination of the three flavonoid compounds at leaf level concentrations significantly increased baculovirus activity in diet incorporation assays. When the daidzein, genistein, and kaempferol were added to artificial diet, at 3.5-6.5 × leaf level concentrations, virus activity increased 1.5, 2.3, and 4.2-fold for each respective flavonoid. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against T. ni.

Advances in Molecular Biology of Baculoviruses.

Baculoviridae constitutes a family of insect-specific, large DNA viruses with a unique life cycle characterized by the production of two morphologically distinct virions, the budded virus (BV) and the occlusion-derived virus (ODV). ODV and BV, with different tissue tropisms, have been widely applied in the areas of biological control and biotechnology, respectively. In nature, baculovirus infection of susceptible host larvae is initiated by ODV-mediated primary infection, followed by the production of BV for spreading infection within larval body. Across millions of years of co-evolution with their hosts, baculoviruses have developed dedicated mechanisms for efficient entry/egress, genome replication/transcription, and virion assembly by employing either their own proteins or host machineries. They have also adopted versatile strategies to precisely regulate the immunity, behaviors and physiology of hosts to facilitate their own replication and dispersal. In this chapter, research advances relating to key aspects of the baculovirus life cycle are reviewed, and the application of a newly-developed baculovirus synthetic biology technology is introduced. Finally, future avenues for baculovirus research are discussed.

Identification of four caspase genes from Spodoptera exigua (Lepidoptera: Noctuidae) and their regulations toward different apoptotic stimulations.

Apoptosis plays critical roles in multiple biological processes in multicellular organisms. Caspases are known as important participators and regulators of apoptosis. Here, four novel caspase genes of Spodoptera exigua were cloned and characterized, which were designated as SeCasp-1, SeCasp-6, SeCasp-7 and SeCasp-8. Analysis of the putative encoded protein sequences of these SeCasps indicated that SeCasp-1 and SeCasp-7 were possible homologs of executor caspases; SeCasp-8 was a possible homolog of initiator caspases; and SeCasp-6 was a unique caspase of S. exigua that shares low similarity with all the identified insect caspases. Based on baculovirus expression system analyses, SeCasp-1 exhibited similar caspase activity to human caspase-1, -3, -4, -6, -8 and -9; SeCasp-6 presented similar caspase activity to human caspase-2, -3, -4, -6, -8 and -9; SeCasp-7 exhibited similar caspase activity to human caspase-2, -3 and -6; and SeCasp-8 presented similar caspase activity only to human caspase-8. Induction with different chemicals revealed that SeCasp-1 showed extreme upregulation after 24 h in the treated fat body cell line (IOZCAS-Spex-II) of S. exigua. Developmental expression analysis revealed that SeCasp-1 was highly transcribed in the larval stages, while SeCasp-6, SeCasp-7, SeCasp-8 were down-regulated. The in vivo detection of the relative expression levels of SeCasps in S. eixgua larvae inoculated with different pathogens suggested that SeCasp-1 was sensitive to Bacillus thuringiensis infection and that SeCasp-6 was sensitive to baculovirus infection. SeCasp-7 and SeCasp-8 showed slight changes under either in vitro chemical apoptosis induction or in vivo pathogen infection.

Immersion immunization with recombinant baculoviruses displaying cyprinid herpesvirus 2 membrane proteins induced protective immunity in gibel carp.

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp.

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.

Nucleocapsid Assembly of Baculoviruses.

The baculovirus nucleocapsid is formed through a rod-like capsid encapsulating a genomic DNA molecule of 80~180 kbp. The viral capsid is a large oligomer composed of many copies of various protein subunits. The assembly of viral capsids is a complex oligomerization process. The timing of expression of nucleocapsid-related proteins, transport pathways, and their interactions can affect the assembly process of preformed capsids. In addition, the selection of viral DNA and the injection of the viral genome into empty capsids are the critical steps in nucleocapsid assembly. This paper reviews the replication and recombination of baculovirus DNA, expression and transport of capsid proteins, formation of preformed capsids, DNA encapsulation, and nucleocapsid formation. This review will provide a basis for further study of the nucleocapsid assembly mechanism of baculovirus.

Correlation in Expression between LTR Retrotransposons and Potential Host Cis-Targets during Infection of Antherea pernyi with ApNPV Baculovirus.

The published genome sequence of Antheraeayamamai (Saturnnidae) was used to construct a library of long terminal repeat (LTR)-retrotransposons that is representative of the wild silkmoth (Antherea) genus, and that includes 22,666 solo LTRs and 541 full-length LTRs. The LTR retrotransposons of Antheraeayamamai (AyLTRs) could be classified into the three canonical groups of Gypsy, Copia and Belpao. Eleven AyLTRs contained the env gene element, but the relationship with the env element of baculovirus, particularly A. yamamai and pernyi nucleopolyhedrovirus (AyNPV and ApNPV), was distant. A total of 251 "independent" full-length AyLTRs were identified that were located within 100 kb distance (downstream or upstream) of 406 neighboring genes in A. yamamai. Regulation of these genes might occur in cis by the AyLTRs, and the neighboring genes were found to be enriched in GO terms such as "response to stimulus", and KEGG terms such as "mTOR signaling pathway" among others. Furthermore, the library of LTR-retrotransposons and the A. yamamai genome were used to identify and analyze the expression of LTR-retrotransposons and genes in ApNPV-infected and non-infected A. pernyi larval midguts, using raw data of a published transcriptome study. Our analysis demonstrates that 93 full-length LTR-retrotransposons are transcribed in the midgut of A. pernyi of which 12 significantly change their expression after ApNPV infection (differentially expressed LTR-retrotransposons or DELs). In addition, the expression of differentially expressed genes (DEGs) and neighboring DELs on the chromosome following ApNPV infection suggests the possibility of regulation of expression of DEGs by DELs through a cis mechanism, which will require experimental verification. When examined in more detail, it was found that genes involved in Notch signaling and stress granule (SG) formation were significantly up-regulated in ApNPV-infected A. pernyi larval midgut. Moreover, several DEGs in the Notch and SG pathways were found to be located in the neighborhood of particular DELs, indicating the possibility of DEG-DEL cross-regulation in cis for these two pathways.

A nuclear envelop-associated baculovirus protein promotes intranuclear lipid accumulation during infection.

Although it has been well-accepted that baculoviruses produce a virus envelop within the nucleus, the redistribution of membrane lipids in infected cells has not been demonstrated. Here, we characterize a baculovirus protein (Bm5/Ac13: renamed BION; baculovirus protein associated with both the inner- and outer nuclear membranes) that localizes to both the inner- and outer nuclear membranes and show that the nuclear membrane (NE) protein promotes formation of a virus-induced intranuclear structure, the peristromal region (PR). Consistent with its role in virus envelopment, the PR was found to contain viral membrane proteins and lipids, suggesting PR formation proceeds through intranuclear lipid accumulation. About 50% of the cells infected with a bion-deficient virus exhibited no polyhedra production due to lack of the PR. Association of BION with the NE rather than the PR may contribute to the formation of the PR and polyhedra via NE-to-PR lipid transport.

Cross-talking between baculoviruses and host insects towards a successful infection.

Baculoviridae is a family of large DNA viruses that infect insects. They have been extensively used as safe and efficient biological agents for the control of insect pests. As a result of coevolution with their hosts, baculoviruses developed unique life cycles characterized by the production of two distinctive virion phenotypes, occlusion-derived virus and budded virus, which are responsible for mediating primary infection in insect midgut epithelia and spreading systemic infection within infected insects, respectively. In this article, advances associated with virus-host interactions during the baculovirus life cycle are reviewed. We mainly focus on how baculoviruses exploit versatile strategies to overcome diverse host barriers and establish successful infections. For example, in the midgut, baculoviruses encode enzymes to degrade peritrophic membranes and use a series of per os infectivity factors to initiate primary infection. A viral fibroblast growth factor is expressed to attract tracheoblasts that spread the virus for systemic infection. Baculoviruses use different strategies to suppress host defence systems, including apoptosis, melanization and RNA interference. Additionally, baculoviruses can manipulate host physiology and induce 'tree-top disease' for optimal virus replication and dispersal. These advances in our understanding of baculoviruses will greatly inform the development of more effective baculoviral pesticides. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

MultiBac: Baculovirus-Mediated Multigene DNA Cargo Delivery in Insect and Mammalian Cells.

The baculovirus/insect cell system (BICS) is widely used in academia and industry to produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening and the provision of protein biologics and therapeutics. Multi-protein complexes have emerged as vital catalysts of cellular function. In order to unlock the structure and mechanism of these essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly tailored for heterologous multigene transfer and multi-protein complex production. Baculovirus is unique among common viral vectors in its capacity to accommodate very large quantities of heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality during heterologous protein expression, and developing customized MultiBac baculovirus variants in the process. By altering its tropism, recombinant baculovirions can be used for the highly efficient delivery of a customized DNA cargo in mammalian cells and tissues. Current advances in synthetic biology greatly facilitate the construction or recombinant baculoviral genomes for gene editing and genome engineering, mediated by a MultiBac baculovirus tailored to this purpose. Here, recent developments and exploits of the MultiBac system are presented and discussed.

Baculoviral infection reduces the expression of four allergen proteins of silkworm pupa.

Silkworm (Bombyx mori) larvae are widely used to express exogenous proteins. Moreover, some silkworm pupal proteins can be used as drug-loading materials for selfexpressed oral tolerance drugs. However, several proteins expressed in silkworm pupae cause severe allergic reactions in humans and animals. Interestingly, some baculovirus vectors have been shown to alter the host gene and its expression in insect cells, but this has not been confirmed in silkworm. Here, we analyzed the effects of infection with an empty B. mori baculovirus (BmNPV) vector on silkworm pupal protein expression. Using a proteomics approach, the allergens thiol peroxiredoxin (Jafrac1), 27-kDa glycoprotein (p27k), arginine kinase, and paramyosin as well as 32 additional differentially expressed proteins were identified. Downregulation of the messenger RNA expression of the four known allergens was observed after BmNPV infection; subsequent changes in protein expression were confirmed by the western blot analysis using polyclonal antibodies prepared with recombinant proteins of the four allergens. Collectively, these data indicate that the four known allergens of silkworm pupae can be reduced by infection ith an empty BmNPV vector to increase the safety of silkworm pupa-based exogenous protein expression and drug delivery of oral pharmaceuticals. In addition, the four recombinant allergen proteins may contribute to the diagnosis of allergic diseases of silkworm pupa.

Resistant silkworm strain block viral infection independent of melanization.

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, the role of melanization on viral infection has not been wildly revealed in Bombyx mori (B. mori), silkworm. Here, we investigated the extent of melanization of susceptible (871) and resistant (near-isogenic line 871C) B. mori strains. The result showed that the extent of melanization was significantly higher in the susceptible strain than in the resistant strain. A majority of Serpins were up-regulated in the resistant strain through iTRAQ-based quantitative proteomics, comparing with susceptible strain. Our data further identified that Serpin-5, Serpin-9 and Serpin-19 reduced PO activity, indicating that the menlanization pathway was inhibited in the resistant strain. Moreover, our results indicated that the hemolymph of 871 reduced viral infection in the presence of PTU, indicating that melanization of 871 strain hemolymph blocked viral infection. However, viral infection was significantly suppressed in the hemolymph of 871C strain regardless of the presence of PTU or not, which implied that the resistant strain inhibited viral infection independent of the melanization pathway. Taken together, our findings indicated that the melanization pathway was inhibited in resistant strain. These results expend the analysis of melanization pathway in insects and provide insights into understanding the antiviral mechanism.

Expressing Double-Stranded RNAs of Insect Hormone-Related Genes Enhances Baculovirus Insecticidal Activity.

Baculoviruses have already been used for insect pest control, but the slow killing speed limits their further promotion and application. Here we provide a strategy for improving baculovirus insecticidal activity using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) to express double-stranded RNAs (dsRNAs) targeting cotton bollworm (Helicoverpa armigera) juvenile hormone (JH)-related genes. Droplet-feeding bioassays show that the 50% lethal concentration (LC50) values of recombinant baculoviruses expressing the dsRNA of JH acid methyl transferase gene (HaJHAMT) and the JH acid binding protein gene (HaJHBP) were 1.24 × 104 polyhedral inclusion bodies (PIB)/mL and 2.26 × 104 PIB/mL, respectively. Both were much lower than the control value (8.12 × 104 PIB/mL). Meanwhile, the LT50 of recombinant baculovirus expressing dsRNA of HaJHBP was only 54.2% of the control value, which means that larval death was accelerated. Furthermore, the mRNA level of target genes was reduced in recombinant baculovirus-treated cotton bollworm larvae. Transcription of several key genes involved in hormone signaling pathways-for example, ecdysone receptor gene (HaEcR)-was also altered. This study establishes a new strategy for pest management by interfering with insect hormone-related gene expression via baculoviruses, and the engineered baculoviruses have great potential application in cotton production.