Autographa Californica Multiple Nucleopolyhedrovirus Research Articles

A CRM1-dependent nuclear export signal in Autographa californica multiple nucleopolyhedrovirus Ac93 is important for the formation of intranuclear microvesicles.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93-null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93-null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCEAutographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway.

Enhanced baculoviral virulence by suppressing the degradation of an insect immune resolvin, epoxyoctadecamonoenoic acid, in three lepidopteran insects

Epoxyoctadecamonoenoic acids (EpOMEs) are produced from linoleic acid by a cytochrome P450 monooxygenase (CYP) and play a crucial role in terminating excessive and unnecessary immune responses during the late infection stage in insects. This suggests that an increase in the EpOME level may enhance the virulence of insect pathogens against pests. This study tested this hypothesis using a specific inhibitor against soluble epoxide hydrolase (sEH) to degrade EpOMEs, which leads to elevated endogenous EpOME levels. A baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was used to infect three different lepidopteran insects (Spodoptera exigua, Maruca vitrata, and Plutella xylostella) by oral feeding or hemocoelic injection treatments. Within one hour, the viral infection induced the expression of three different phospholipase A2 (PLA2) genes and, after 12 h, up-regulated the expressions of CYP and sEH genes in Spodopera exigua. As expected, AcMNPV virulence was suppressed by the addition of arachidonic acid (a catalytic product of PLA2) but was enhanced by the addition of either of the EpOME regioisomers. In addition, treatment with a specific sEH inhibitor (AUDA) increased AcMNPV virulence against three different lepidopteran insects, presumably by increasing endogenous EpOME levels. This enhanced effect of EpOMEs on virulence was further supported by specific RNA interference (RNAi), in which RNAi specific to CYP expression decreased AcMNPV virulence while a specific RNAi against sEH expression significantly enhanced virulence. In response to AcMNPV infection, TUNEL assay results showed that S. exigua larvae exhibited apoptosis in the midgut, fat body, and epidermis. Inhibition of apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, significantly increased virulence. Similarly, the addition of AUDA to the viral treatment suppressed the gene expression of five inducible caspases and cytochrome C to suppress apoptosis, which led to a significant increase in the tissue viral titers. These results indicate that EpOMEs play a role in terminating excessive and unnecessary immune responses against viral infection during the late stage by down-regulating antiviral apoptosis in lepidopteran insects.

Baculovirus entry into the central nervous system of Spodoptera exigua caterpillars is independent of the viral protein tyrosine phosphatase.

Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.

Multifaceted interactions between host ESCRT-III and budded virus-related proteins involved in entry and egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 Å helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.

Rapid production of chimeric silkworm/spider silk with improved mechanical properties by infection of nonpermissive Bombyx mori with recombinant AcMNPV harboring native-size of spidroin genes

Spider silks with excellent mechanical properties attract more attention from scientists worldwide, and the dragline silk that serves as the framework of the spider's web is considered one of the strongest fibers. However, it is unfeasible for large-scale production of spider silk due to its highly territorial, cannibalistic, predatory, and solitary behavior. Herein, to alleviate some of these problems and explore aneasy way to produce spider fibers, we constructed recombinant baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) simultaneously expressing Trichonephila clavipes native ampullate spidroin 2 (MaSp-G) and spidroin 1 (MaSp-C) driven by the promoters of silkworm fibroin genes, to infect the nonpermissive Bombyx mori larvae at the fifth instar. MaSp-G and MaSp-C were co-expressed in the posterior silk glands (PSGs) of infected silkworms and successfully secreted into the lumen of the silk gland for fibroin globule assembly. The integration of MaSp-G and MaSp-C into silkworm silk fibers significantly improved the mechanical properties of these chimeric silk fibers, especially the strength and extensibility, which may be caused by the increment of β-sheet in the chimeric silkworm/spider silk fiber. These results demonstrated that silkworms could be developed as the nonpermissive heterologous host for the mass production of chimeric silkworm/spider silk fibers via the recombinant baculovirus AcMNPV.

Vegetable waste extracts as enhancers of baculovirus infections

Vegetable waste extracts (VWE) contain a great variety of antioxidants such as polyphenols, which have shown to potentiate baculovirus infections, making them ingredients for pest control ingredients. In the present study, the mortality enhancement of different vegetable extracts obtained from food residues when combined with baculoviruses was evaluated. Extracts from spent coffee (E2), rosehip (E17), asparagus (E28), artichoke (E29), beet stalks (E32) and banana peel (E37) were selected as they increased mortality of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) in second instar S. littoralis larvae, when comparing with the virus inoculation alone. Extracts were assayed at 1 % w/v. In S. littoralis-SpliNPV system, the selected extracts reduced the median lethal concentration (LC50) of SpliNPV against second instar larvae. The E37 extract presented the highest potentiation, as it reduced the LC50 13.61 times, while the rest of the extracts presented LC50 reductions from 3.71 to 7.72-fold. In Spodoptera exigua-SeMNPV (Spodoptera exigua multiple nucleopolyhedrovirus) system, none of the extracts decreased the LC50 of SeMNPV. In contrast, in Spodoptera frugiperda-SfMNPV (Spodoptera frugiperda multiple nucleopolyhedrovirus) system, E2 showed the greatest potentiating effect. In the heterologous systems, none of the extracts tested increased the effective host range of SfMNPV, AcMNPV (Autographa californica multiple nucleopolyhedrovirus), and MbMNPV (Mamestra brassicae multiple nucleopolyhedrovirus) in second instar S. littoralis larvae. Thus, the viral enhancing effect of VWE was host-pathogen and instar dependent. However, the potentiation effect of the extracts could not be directly related with the antioxidants content of the extracts.

Architecture of the baculovirus nucleocapsid revealed by cryo-EM

Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used as a bioinsecticide and a protein expression vector. Despite their importance, very little is known about the structure of most baculovirus proteins. Here, we show a 3.2 Å resolution structure of helical cylindrical body of the AcMNPV nucleocapsid, composed of VP39, as well as 4.3 Å resolution structures of both the head and the base of the nucleocapsid composed of over 100 protein subunits. AcMNPV VP39 demonstrates some features of the HK97-like fold and utilizes disulfide-bonds and a set of interactions at its C-termini to mediate nucleocapsid assembly and stability. At both ends of the nucleocapsid, the VP39 cylinder is constricted by an outer shell ring composed of proteins AC104, AC142 and AC109. AC101(BV/ODV-C42) and AC144(ODV-EC27) form a C14 symmetric inner layer at both capsid head and base. In the base, these proteins interact with a 7-fold symmetric capsid plug, while a portal-like structure is seen in the central portion of head. Additionally, we propose an application of AlphaFold2 for model building in intermediate resolution density.

A Baculovirus Expression Vector Derived Entirely from Non-Templated, Chemically Synthesized DNA Parts.

Baculovirus expression system1s are a widely used tool in recombinant protein and biologics production. To enable the possibility of genome modifications unconstrained through low-throughput and bespoke classical genome manipulation techniques, we set out to construct a baculovirus vector (>130 kb dsDNA) built from modular, chemically synthesized DNA parts. We constructed a synthetic version of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) through two steps of hierarchical Golden Gate assembly. Over 140 restriction endonuclease sites were removed to enable the discrimination of the synthetic genome from native baculovirus genomes. A head-to-head comparison of our modular, synthetic AcMNPV genome with native baculovirus vectors showed no significant difference in baculovirus growth kinetics or recombinant adeno-associated virus production-suggesting that neither baculovirus replication nor very-late gene expression were compromised by our design or assembly method. With unprecedented control over the AcMNPV genome at the single-nucleotide level, we hope to ambitiously explore novel AcMNPV vectors streamlined for biologics production and development.

A DNA Plasmid-Based Approach for Efficient Synthesis of Sacbrood Virus Infectious Clones within Host Cells.

RNA viruses are often cited as a significant factor affecting the populations of both domestic honey bees and wild pollinators. To expedite the development of effective countermeasures against these viruses, a more comprehensive understanding of virus biology necessitates extensive collaboration among scientists from diverse research fields. While the infectious virus clone is a robust tool for studying virus diseases, the current methods for synthesizing infectious clones of bee-infecting RNA viruses entail the in vitro transcription of the viral genome RNA in 8-10 kb, presenting challenges in reproducibility and distribution. This article reports on the synthesis of an infectious clone of the Chinese variant sacbrood virus (SBV) using a DNA plasmid containing an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein (IE1) promoter to trigger transcription of the downstream viral genome within hosts. The results demonstrate that the IE1-SBV plasmid can synthesize SBV clones in a widely used lepidopteran immortal cell line (Sf9) and honey bee pupae. Furthermore, the negative strand of the clone was detected in both Sf9 cells and honey bee pupae, indicating active infection and replication. However, the transfection of Sf9 cells was observed in only a limited proportion (less than 10%) of the cells, and the infection did not appear to spread to adjacent cells or form infective virions. The injection of honey bee pupae with 2500 ng of the IE1-SBV plasmid resulted in high infection rates in Apis cerana pupae but low rates in A. mellifera pupae, although the dosage was comparatively high compared with other studies using in vitro transcribed viral RNA. Our findings suggest that the synthesis of bee-infecting RNA viruses using DNA plasmids is feasible, albeit requiring additional optimization. However, this method holds substantial potential for facilitating the production of clones with various sequence modifications, enabling the exploration of viral gene functions and biology. The ease of distributing infectious clones in DNA plasmid form may foster collaboration among scientists in applying the clone to bee biology, ecology, and behavior, ultimately offering a comprehensive approach to managing virus diseases in the future.

The Expression of P35 Plays a Key Role in the Difference in Apoptosis Induced by AcMNPV Infection in Different Spodoptera exigua Cell Lines.

Baculovirus infection induces apoptosis in host cells, and apoptosis significantly affects virus production. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can regulate apoptosis, but the regulatory mechanism is unclear. Here, we found that AcMNPV infection induced different apoptosis responses in different Spodoptera exigua cell lines. In the early stages of viral infection (1-6 h), Se-1 cells underwent severe apoptosis, while Se-3 cells underwent very slight apoptosis. In the late stages of viral infection (12-72 h), Se-1 cells continued to undergo apoptosis and formed a large number of apoptotic bodies, while the apoptosis of Se-3 cells was inhibited and no apoptotic bodies were formed. To determine the reasons for the apoptosis differences in the two cell lines, we measured the expression of the six S. exigua cysteine-dependent aspartate specific protease genes (SeCaspase-1 to -6) and the three AcMNPV antiapoptotic protein genes (iap1, iap2 and p35) during viral infection. We found that SeCaspase-1 to -6 were all activated in Se-1 cells and inhibited in Se-3 cells, whereas iap1, iap2 and p35 were all inhibited in Se-1 cells and normally expressed in Se-3 cells. And p35 was expressed earlier than iap1 and iap2 in Se-3 cells. Otherwise, Se-1 and Se-3 cells would all be apoptotic when infected with the recombinant p35 knockout AcMNPV, whereas only Se-1 cells were apoptotic, but Se-3 cells were not apoptotic when infected with the recombinant p35 repair AcMNPV. Combined with the fact that the expression of P35 protein is inhibited in Se-1 cells but normally expressed in Se-3 cells during the infection of recombinant p35 repair AcMNPV, we proposed that the different expression of P35 is an important reason for the apoptosis differences between the two cell lines. We also found that some genes associated with apoptosis can probably regulate the expression of P35. However, the major upstream regulators of P35 and their mechanisms are still unclear and will be studied in the future.

Cysteines 128 and 250 are essential for the functions of the baculovirus core gene ac109

The open reading frame 109 (ac109) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is one of the 38 core baculovirus genes. Ac109 was shown to be essential for the production of infectious budded virions (BV), envelopment of the nucleocapsid, and embedding of occlusion-derived virions (ODVs) into occlusion bodies (OBs). Herein, the roles of five cysteines with high conservation (C3, C116, C128, C250, and C325) in Ac109 function were investigated. AcMNPV bacmids lacking ac109 or containing single-mutated ac109 were generated. Transfection/infection assays showed that C128 and C250 in Ac109 were important for infectious BV production. Electron microscopy analysis further confirmed that these two cysteines played critical roles in nucleocapsid assembly, ODV envelopment, and embedding of ODVs into OBs. Altogether, these results demonstrate that the conserved residues Ac109 C128 and C250 are critical for baculovirus infection.

A sensitive assay for scrutiny of Autographa californica multiple nucleopolyhedrovirus genes using CRISPR-Cas9.

Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.

Microscopic Observation of Membrane Fusion between Giant Liposomes and Baculovirus Budded Viruses Activated by the Release of a Caged Proton.

Baculovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) is an envelope virus possessing a fusogenic protein, GP64, which can be activated under weak acidic conditions close to those in endosomes. When the budded viruses (BVs) are bathed at pH 4.0 to 5.5, they can bind to liposome membranes with acidic phospholipids, and this results in membrane fusion. In the present study, using the caged-proton reagent 1-(2-nitrophenyl)ethyl sulfate, sodium salt (NPE-caged-proton), which can be uncaged by irradiation with ultraviolet light, we triggered the activation of GP64 by lowering the pH and observed membrane fusion on giant liposomes (giant unilamellar vesicles, GUVs) by visualizing the lateral diffusion of fluorescence emitted from a lipophilic fluorochrome (octadecyl rhodamine B chloride, R18) that stained viral envelopes of BVs. In this fusion, entrapped calcein did not leak from the target GUVs. The behavior of BVs prior to the triggering of membrane fusion by the uncaging reaction was closely monitored. BVs appeared to accumulate around a GUV with DOPS, implying that BVs preferred phosphatidylserine. The monitoring of viral fusion triggered by the uncaging reaction could be a valuable tool for revealing the delicate behavior of viruses affected by various chemical and biochemical environments.

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome.

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

Baculovirus Actin Rearrangement-Inducing Factor 1 Can Remodel the Mammalian Actin Cytoskeleton.

The actin rearrangement-inducing factor 1 (Arif-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an early viral protein that manipulates the actin cytoskeleton of host insect cells. Arif-1 is conserved among alphabaculoviruses and is responsible for the accumulation of F-actin at the plasma membrane during the early phase of infection. However, the molecular mechanism underlying Arif-1-induced cortical actin accumulation is still open. Recent studies have demonstrated the formation of invadosome-like structures induced by Arif-1, suggesting a function in systemic virus spread. Here, we addressed whether Arif-1 is able to manipulate the actin cytoskeleton of mammalian cells comparably to insect cells. Strikingly, transient overexpression of Arif-1 in B16-F1 mouse melanoma cells revealed pronounced F-actin remodeling. Actin assembly was increased, and intense membrane ruffling occurred at the expense of substrate-associated lamellipodia. Deletion mutagenesis studies of Arif-1 confirmed that the C-terminal cytoplasmic region was not sufficient to induce F-actin remodeling, supporting that the transmembrane region for Arif-1 function is also required in mammalian cells. The similarities between Arif-1-induced actin remodeling in insect and mammalian cells indicate that Arif-1 function relies on conserved cellular interaction partners and signal transduction pathways, thus providing an experimental tool to elucidate the underlying mechanism. IMPORTANCE Virus-induced changes of the host cell cytoskeleton play a pivotal role in the pathogenesis of viral infections. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is known for intervening with the regulation of the host actin cytoskeleton in a wide manner throughout the infection cycle. The actin rearrangement-inducing factor 1 (Arif-1) is a viral protein that causes actin rearrangement during the early phase of AcMNPV infection. Here, we performed overexpression studies of Arif-1 in mammalian cells to establish an experimental tool that allows elucidation of the mechanism underlying the Arif-1-induced remodeling of actin dynamics in a well-characterized and genetically accessible system.

Transcriptional Reprogramming of Autographa Californica Multiple Nucleopolyhedrovirus Chitinase and Cathepsin Genes Enhances Virulence.

The baculoviral chitinase (CHIA) and cathepsin (V-CATH) enzymes promote terminal insect host liquefaction, which aids viral progeny dissemination. Recombinant Autographa californica nucleopolyhedrovirus (AcMNPV)-derived viruses were previously generated with reprogrammed chiA transcription by replacing the native promoter with the AcMNPV polyhedrin (polh) or core protein (p6.9) promoter sequences, but of both these chiA-reprogrammed viruses lacked v-cath transcription and V-CATH enzymatic activity. Here, we report that dual p6.9/polh promoter reprogramming of the adjacent chiA/v-cath genes resulted in modulated temporal transcription of both genes without impacting infectious budded virus production. These promoter changes increased CHIA and V-CATH enzyme activities in infected Spodoptera frugiperda-derived cultured cells and Trichoplusia ni larvae. In addition, larvae infected with the dual reprogrammed virus had earlier mortalities and liquefaction. This recombinant baculovirus, lacking exogenous genomic elements and increased chiA/v-cath expression levels, may be desirable for and amenable to producing enhanced baculovirus-based biopesticides.

Baculovirus displaying SARS-CoV-2 spike RBD promotes neutralizing antibody production in a mouse model

BackgroundThere is always a need for a safe and efficient vaccine platform, especially when facing a pandemic such as COVID-19. Most of the SARS-CoV-2-based vaccines are based on the full spike protein, which is presented as a trimerized protein, and many viral vector vaccines express the spike protein into the host cells and do not display it on virus surfaces. However, the spike receptor-binding domain (RBD)-based vaccines are efficient and are currently under investigation and clinical trials. MethodologyIn this study, we are testing the efficacy of the RBD displayed on a baculovirus as a mean to formulate a safe and stable carrier to induce the immune system against SARS-CoV-2. Therefore, two pseudotyped baculoviruses were constructed to display the RBD, AcRBD-sfGFP-64, and AcRBD-sfGFP-V, using two different displaying strategies based on gp64 and VSV-G envelope glycoproteins, from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and vesicular stomatitis virus (VSV), respectively. BALB/C mice were immunized with the pseudotyped baculoviruses in a dose-optimized manner. Dot blot and Western blot were used to screen and validate the polyclonal antibodies’ specificity to the SARS-CoV-2 RBD. A plaque reduction neutralization test (PRNT) was used to measure the sera neutralization capacity against a SARS-CoV-2 wild-type isolate from Egypt. ELISA was used to quantify certain cytokines for the assessment of the immune response. ResultThe outcome of our investigation showed that the monomeric RBD proteins were properly displayed on baculovirus and efficiently triggered the mouse immune system. The produced sera efficiently neutralized about 50% of SARS-CoV-2 in more than 100-fold serum dilution. The immunized mice showed a significant increase (p<0.01) in the levels of IL-2 and IFN-γ and a significant decrease (p<0.01) and (p<0.001) in the levels of IL-4 and IL-10, respectively, which suggest that AcRBD-sfGFP-64 and AcRBD-sfGFP-V induce Th1 cellular immune response. ConclusionThe produced recombinant viruses can induce the immune response without adjuvant, which needs dose optimization and further stability tests. Neutralizing antibodies were induced without affecting the health of immunized mice. Th1 response can be attainable through the system, which is of great benefit in SARS CoV-2 infection and the system can be tested for future applications including vaccine development and polyclonal antibody production.

Genome-wide nonessential gene identification of Autographa californica multiple nucleopolyhedrovirus

The Baculovirus Expression Vector System (BEVS) is an insect cell-based heterologous protein expression system that possesses powerful potential in the development of protein drugs and vaccines. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most widely-used vector in BEVS with 151 open reading frames (ORFs) containing essential and nonessential genes. Deletion of nonessential genes has many advantages including increased foreign gene insertion. In this study, the λ red recombination system was used to knock out genes in a modified AcMNPV that carried an enhanced yellow fluorescent protein (eYFP) at the Ac126-Ac127 locus. Eighty genes were almost completely deleted respectively and 69 gene knockout AcMNPVs (KOVs) were obtained to evaluate their infection efficiency. After infecting Spodoptera frugiperda 9 (Sf9) cells, 51 KOVs including 62 genes showed similar infectivity as wide type (WT) and hence were defined as nonessential genes. However, 18 KOVs produced fewer infectious virions, indicating that these genes were influential in the production of progeny viruses. Combining our research with previous studies, a desired minimal AcMNPV genome containing 86 ORFs and all of the homologous regions (hrs) was brought up, facilitating genetic modification of baculovirus vectors and improvement of recombinant protein expression in the future.

Evaluation of Virus-Free Manufacture of Recombinant Proteins Using CRISPR-Mediated Gene Disruption in Baculovirus-Infected Insect Cells.

The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp64 open reading frame, which encodes the major envelope protein of AcMNPV. After comfirming the site-specific targeting of gp64 leading to reduced budded virus (BV) release, the gag gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release.