BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Single-Nucleus Sequencing of Silkworm Larval Brain Reveals the Key Role of Lysozyme in the Antiviral Immune Response in Brain Hemocytes.

INTRODUCTION: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown. METHODS: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain. RESULTS: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects. CONCLUSION: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.

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.

Ras3 in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus.

Bombyx mori ras protein3 (BmRas3) is a small molecular protein in the GTPase superfamily, which has the activity of binding guanosine nucleotides and GTP enzymes. It acts as a molecular switch by coupling extracellular signal to different cellular response through the conversion between Ras-GTP conformation and Ras-GDP conformation, thus regulating signal pathways responsible for cell growth, migration, adhesion, survival and differentiation. However, few studies have been done on Ras3 in silkworm, and its function and mechanism are unclear. In this study, we found that the overexpression of BmRas3 inhibited the infection of BmNPV(B. mori nucleopolyhedrovirus), while knockdown of BmRas3 could promote the infection of BmNPV. In addition, after the BmRas3 in silkworm larvae was knockdown, the anti-BmNPV ability of silkworm decreased and the survival rate of silkworm was affected. Additionly in the cells with BmRas3 overexpression, the transcription level of BmMapkk6 、BmP38、BmJNK、BmERK1/2 and BmERK5 were significantly increased after BmNPV infection, and the transcript levels of BmMapkk6、BmP38、BmJNK、BmERK1/2 and BmERK5 were also inhibited to varying degrees This is the first report on the antiviral effect of BmRas3 in silkworm, which provides a new direction for further study on the anti-BmNPV mechanism of silkworm and screening and cultivation of anti-BmNPV silkworm strain.

Glucose-regulated protein 94 facilitates the proliferation of the Bombyx mori nucleopolyhedrovirus via inhibiting apoptosis.

Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.

The DEAD/H-box helicase DHX9 contributes to suppression of Bombyx mori nucleopolyhedrovirus propagation in B. mori cells.

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

Bombyx mori nucleopolyhedrovirus induces BmFABP1 downregulation to promote viral proliferation.

Fatty acid binding proteins (FABPs) play an important role as endogenous cytoprotectants. However, studies on FABPs in invertebrates are scarce. Previously, we discovered Bombyx mori fatty acid binding protein 1 (BmFABP1) through co-immunoprecipitation. Here, we cloned and identified BmFABP1 from BmN cells. The results of immunofluorescence indicated that BmFABP1 was localized in the cytoplasm. The tissue expression profile of silkworms showed that BmFABP1 was expressed in all tissues except hemocytes. The expression level of BmFABP1 gradually decreases in BmN cells and B. mori larvae after infection with B. mori nucleopolyhedrovirus (BmNPV). Upregulation of BmFABP1 expression through overexpression or WY14643 treatment significantly inhibited the replication of BmNPV, while downregulation of BmFABP1 expression by RNA interference promoted the replication of BmNPV. The same results were obtained in experiments on silkworm larvae. These results suggest that BmNPV induces BmFABP1 downregulation to promote its proliferation and that BmFABP1 has a potential anti-BmNPV role. This is the first report on the antiviral effect of BmFABP1 in silkworms and provides new insights into the study of the FABP protein family. Also, it is important to study BmNPV resistance in silkworms to breed transgenic silkworms with BmNPV resistance.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

BACKGROUND: Commensal microorganisms are widely distributed in insect gut tissues and play important roles in host nutrition, metabolism, reproductive regulation, and especially immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. RESULTS: We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that re-introduction of the HcM7 strain to germfree larvae preactivated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. CONCLUSION: These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest. © 2023 Society of Chemical Industry.

Uric acid metabolism promotes apoptosis against Bombyx mori nucleopolyhedrovirus in silkworm, Bombyx mori.

The white epidermis of silkworms is due to the accumulation of uric acid crystals. Abnormal silkworm uric acid metabolism decreases uric acid production, leading to a transparent or translucent phenotype. The oily silkworm op50 is a mutant strain with a highly transparent epidermis derived from the p50 strain. It shows more susceptibility to Bombyx mori nucleopolyhedrovirus (BmNPV) infection than the wild type; however, the underlying mechanism is unknown. This study analysed the changes in 34 metabolites in p50 and op50 at different times following BmNPV infection based on comparative metabolomics. The differential metabolites were mainly clustered in six metabolic pathways. Of these, the uric acid pathway was identified as critical for resistance in silkworms, as feeding with inosine significantly enhanced larval resistance compared to other metabolites and modulated other metabolic pathways. Additionally, the increased level of resistance to BmNPV in inosine-fed silkworms was associated with the regulation of apoptosis, which is mediated by the reactive oxygen species produced during uric acid synthesis. Furthermore, feeding the industrial strain Jingsong (JS) with inosine significantly increased the level of larval resistance to BmNPV, indicating its potential application in controlling the virus in sericulture. These results lay the foundation for clarifying the resistance mechanism of silkworms to BmNPV and provide new strategies and methods for the biological control of pests.

Reactive oxygen species-mediated phosphorylation of JNK is involved in the regulation of BmFerHCH on Bombyx mori nucleopolyhedrovirus proliferation.

c-Jun N-terminal kinase (JNK) phosphorylation is widely observed during virus infection, modulating various aspects of the virus-host interaction. In our previous research, we have proved that B. mori ferritin heavy-chain homolog (BmFerHCH), an inhibitor of reactive oxygen species (ROS), facilitates B. mori nucleopolyhedrovirus (BmNPV) proliferation. However, one question remains: Which downstream signaling pathways does BmFerHCH regulate by inhibiting ROS? Here, we first determined that silencing BmFerHCH inhibits BmNPV proliferation, and this inhibition depends on ROS. Then, we substantiated that BmNPV infection activates the JNK signaling pathway. Interestingly, the JNK phosphorylation during BmNPV infection is activated by ROS. Further, we found that the enhanced nuclear translocation of phospho-JNK induced by BmNPV infection was dramatically reduced by pretreatment with the antioxidant N-acetylcysteine (NAC), whereas there was more detectable phospho-JNK in the cytoplasm. Next, we investigated how changes in BmFerHCH expression affect JNK phosphorylation. BmFerHCH overexpression suppressed the phosphorylation of JNK and nuclear translocation of phospho-JNK during BmNPV infection, whereas BmFerHCH knockdown facilitated phosphorylation of JNK and nuclear translocation of phospho-JNK. By measuring the viral load, we found the inhibitory effect of BmFerHCH knockdown on BmNPV infection depends on phosphorylated JNK. In addition, the JNK signaling pathway was involved in BmNPV-triggered apoptosis. Hence, we hypothesize that ROS-mediated JNK phosphorylation is involved in the regulation of BmFerHCH on BmNPV proliferation. These results elucidate the molecular mechanisms and signaling pathways of BmFerHCH-mediated response to BmNPV infection.

BmCH25H, a vertebrate interferon-stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori.

Cholesterol-25-hydroxylase (CH25H) has been identified as an interferon-stimulated gene (ISG) in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol (25HC). However, invertebrates lack an antiviral system homologous to vertebrate interferons (IFNs) because the genomes of invertebrates do not encode IFN-like cytokines. Nevertheless, CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates. In this study, the Bombyx mori CH25H (BmCH25H) gene, of which the encoded protein has high homology with other lepidopteran species, was identified and located on chromosome 9. Interestingly, we found that the expression of BmCH25H was significantly upregulated in B. mori nucleopolyhedrovirus (BmNPV) -infected BmN cells and silkworm (B. mori) larvae at the early infection stage. The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC. More importantly, we demonstrated that during BmNPV infection, BmCH25H expression was increased through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, similar to the induction of ISGs following virus infection in vertebrates. This is the first report that CH25H has antiviral effects in insects; the study also elucidates the regulation of its expression and its mechanism of action.

LincRNA_XR209691.3 could promote Bombyx mori nucleopolyhedrovirus replication by interacting with BmHSP70.

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides and lacking protein coding potential, have been proven to play important roles in viral infection and host immunity. Bombyx mori nucleopolyhedrovirus (BmNPV) is an important pathogen, which causes the silkworm disease and leads to a huge challenge to the sericultural industry. At present, research on the roles of insect lncRNAs in host-virus interaction are relatively few. In this study, we explored the function of lincRNA_XR209691.3 that was significantly up-regulated in the silkworm fat body upon BmNPV infection. Firstly, the subcellular localization experiment confirmed that lincRNA_XR209691.3 was present in both the nucleus and cytoplasm. Enhancing the expression of lincRNA_XR209691.3 in BmN cells could promote the proliferation of BmNPV, while inhibition of lincRNA_XR209691.3 by RNA interference suppresses the proliferation of BmNPV. Combining RNA pull-down and mass spectrometry, we identified the host and BmNPV proteins that could interact with lincRNA_XR209691.3. Next, by using truncation experiment and RNA immunoprecipitation (RIP) assay, it was found that lincRNA_XR209691.3 could bind to the Actin domain of BmHSP70. Subsequently, overexpression of lncRNA_XR209691.3 in BmN cells promoted the expression of BmHSP70, while knockdown of BmHsp70 suppressed the replication of BmNPV. Based on the above results, it is speculated that lincRNA_XR209691.3 could promote the proliferation of BmNPV through interaction with BmHSP70, possibly by improving the stability of BmHSP70 and thereby enhancing the expression of BmHSP70. Our results shed light on the lncRNA function in insect-pathogen interactions and provide a new clue to elucidate the molecular mechanism of BmNPV infection.

Study on physical properties of four pH responsive Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) microcapsules as controlled release carriers.

This study provides a promising controlled release form of nuclear polyhedrosis virus (NPV) for targeted control of lepidopteran pests. However, the application of NPV is limited due to its sensitivity to UV inactivation. This study investigated the anti-UV properties of microcapsules of SeMNPV occlusion bodies (OBs) encapsulated by calcium alginate (CA), and also the influence of the modification of CA by chitosan (CS), whey protein (WP), and polydopamine (PDA). These capsules were used to deliver, in a controlled release manner virions under alkaline pH conditions. Characterization of the structure, morphology, particle size, encapsulation efficiency, contact angle, insecticidal activity, UV resistance and in vitro release of the microcapsules was conducted. The modified microcapsules had better sphericity, and were devoid of SeMNPV OBs on the surface. The encapsulation rate was 84.76 ± 0.59%. PDA@CA-NPV had the highest wettability and the contact angle was 74.51 ± 0.53°. The 50% lethal concentration values (LC50) of CA-NPV, CS@CA-NPV, WP@CA-NPV and PDA@CA-NPV were 11.5, 10.7, 10.5 and 1.2 times that of SeMNPV OBs alone. The modified microcapsules all improved the anti-UV performance of the virus, and PDA@CA-NPV was the most UV-resistant. Using qPCR, it was observed that under alkaline conditions, a large number of virions were released from PDA@CA-NPV, CA-NPV and SeMNPV OBs. Microencapsulated virus under alkaline conditions did not change the release pattern of virions.

Ataxia Telangiectasia-Mutated Is Activated but Not Required for Productive Autographa californica Multiple Nucleopolyhedrovirus Infection.

Multiplication of the invertebrate DNA baculoviruses activates the host DNA damage response (DDR), which promotes virus DNA replication. DDR signaling is initiated by the host insect's phosphatidylinositol-3 kinase-related kinases (PIKKs), including ataxia telangiectasia-mutated kinase (ATM). Like other PIKKs, ATM phosphorylates an array of host DDR proteins at serine/threonine glutamine (S/TQ) motifs, the result of which leads to cell cycle arrest, DNA repair, or apoptosis. To define the role of host PIKKs in baculovirus replication, we compared replication levels of the baculovirus prototype species Autographa californica multiple nucleopolyhedrovirus in permissive Spodoptera frugiperda (SF21) cells with and without ATM function. Caffeine, which inhibits multiple DDR kinases, and the ATM-specific inhibitors KU-55933 and KU-60019 each prevented phosphorylation of Spodoptera histone H2AX (SfH2AX), a recognized indicator of ATM activity. However, only caffeine reduced autographa californica multiple nucleopolyhedrovirus (AcMNPV)-induced bulk phosphorylation of S/TQ protein motifs. Furthermore, only caffeine, not KU-55933 or KU-60019, reduced AcMNPV yields, suggesting a limited role for ATM. To investigate further, we identified and edited the Spodoptera ATM gene (sfatm). Consistent with ATM's known functions, CRISPR/Cas9-mediated knockout of sfatm eliminated DNA damage-induced phosphorylation of DDR marker SfH2AX in SF21 cells. However, loss of sfatm failed to affect the levels of AcMNPV multiplication. These findings suggested that in the absence of the kinase SfATM, another caffeine-sensitive host DDR kinase promotes S/TQ phosphorylation and baculovirus multiplication. Thus, baculoviruses activate and utilize the host insect DDR in an ATM-independent manner. IMPORTANCE The DDR, while necessary for the maintenance and fidelity of the host genome, represents an important cellular response to viral infection. The prolific DNA baculoviruses activate and manipulate the invertebrate DDR by using mechanisms that positively impact virus multiplication, including virus DNA replication. As the key DDR initiator kinase, ATM was suspected to play a critical role in this host response. However, we show here that baculovirus AcMNPV activates an ATM-independent DDR. By identifying the insect host ATM ortholog (Spodoptera frugiperda SfATM) and evaluating genetic knockouts, we show that SfATM is dispensable for AcMNPV activation of the DDR and for virus replication. Thus, another PIKK, possibly the closely related kinase ATR (ATM- and Rad3-related kinase), is responsible for efficient baculovirus multiplication. These findings better define the host pathways used by invertebrates to engage viral pathogens, including DNA viruses.

Nuclear Factor Kappa B Promotes Ferritin Heavy Chain Expression in Bombyx mori in Response to B. mori Nucleopolyhedrovirus Infection.

Ferritin heavy chain (FerHCH) is a major component of ferritin and plays an important role in maintaining iron homeostasis and redox equilibrium. Our previous studies have demonstrated that the Bombyx mori ferritin heavy chain homolog (BmFerHCH) could respond to B. mori nucleopolyhedrovirus (BmNPV) infection. However, the mechanism by which BmNPV regulates the expression of BmFerHCH remains unclear. In this study, BmFerHCH increased after BmNPV infection and BmNPV infection enhanced nuclear factor kappa B (NF-κB) activity in BmN cells. An NF-κB inhibitor (PDTC) reduced the expression of the virus-induced BmFerHCH in BmN cells, and overexpression of BmRelish (NF-κB) increased the expression of virus-induced BmFerHCH in BmN cells. Furthermore, BmNPV infection enhanced BmFerHCH promoter activity. The potential NF-κB cis-regulatory elements (CREs) in the BmFerHCH promoter were screened by using the JASPAR CORE database, and two effective NF-κB CREs were identified using a dual luciferase reporting system and electrophoretic mobility shift assay (EMSA). BmRelish (NF-κB) bound to NF-κB CREs and promoted the transcription of BmFerHCH. Taken together, BmNPV promotes activation of BmRelish (NF-κB), and activated BmRelish (NF-κB) binds to NF-κB CREs of BmFerHCH promoter to enhance BmFerHCH expression. Our study provides a foundation for future research on the function of BmFerHCH in BmNPV infection.

Bombyx mori transcription factor, E74A, beneficially affects BmNPV infection through direct interaction.

BACKGROUND: Nucleopolyhedrovirus (NPV), one of the baculoviruses, is a promising biopesticide for pest control. Lepidopteran account for 70% of pests, therefore investigation on highly conserved genes associated with viral infections in the lepidopteran model, the silkworm, will serve as a valuable reference for improving the effectiveness of pest management. BmE74A is a member of the erythroblast transformation-specific (ETS) family of transcription factors in Bombyx mori, which we previously found to be highly conserved and closely associated with BmNPV. This study aimed to elucidate the role of BmE74A in viral infection. RESULTS: A significantly high expression of BmE74A in eggs indicated its important role in embryonic development, as did relatively high expressions in the hemolymph and midgut. Significant differences in BmE74A expression in different resistant strains after BmNPV infection suggested its involvement as a response to viral infection. Moreover, RNA interference (RNAi) and overexpression experiments confirmed the important role of BmE74A in promoting viral infection. BmNPV infection was significantly suppressed and enhanced by BmE74A knockdown and overexpression, respectively. Besides, BmE74A was found to regulate the expression of BmMdm2 and Bmp53. Furthermore, the binding of ETS, the functional domain of BmE74A, to occlusion-derived virus proteins was confirmed by far-western blotting, and four viral proteins that may interact with ETS proteins were identified by mass spectrometry. Similarly, a homolog of BmE74A in Spodoptera litura was also found to be involved in larval susceptibility to BmNPV. CONCLUSION: BmE74A promotes BmNPV proliferation by directly interacting with the virus, which may be related to the suppression of the p53 pathway. © 2022 Society of Chemical Industry.

microRNA-34-5p encoded by Spodoptera frugiperda regulates the replication and infection of Autographa californica multiple nucleopolyhedrovirus by targeting odv-e66, ac78 and ie2.

BACKGROUND: Spodoptera frugiperda is one of the significant migratory pests in the Global Alert issued by the Food and Agriculture Organization of the United Nations. As an insect-specific microbial insecticide, baculovirus has been used to control various pests. MicroRNA-34-5p (miR-34-5p) is involved in regulating growth, reproduction and innate immunity to pathogens in insects, playing an essential role in host-virus interactions. In this study, we explored the critical function of miR-34-5p encoded by S. frugiperda in the anti-Autographa californica multiple nucleopolyhedrovirus (AcMNPV), providing a reference for the design of a miR-34-5p target biopesticide against S. frugiperda and a theoretical basis for the wide application of microRNAs (miRNAs) in green pest control technology. RESULTS: We focused on miR-34-5p identified as downregulated in Sf9 cells and S. frugiperda larvae infected by AcMNPV. The regulatory function of miR-34-5p in AcMNPV-S. frugiperda interactions was studied by transfecting synthetic mimics and inhibitors, and constructing recombinant bacmids with miR-34-5p overexpression. miR-34-5p inhibited the production of infectious budded virions at the cellular and insect levels, inhibited the replication of the viral DNA and glucose metabolism, and increased the transcription of the antimicrobial peptide gloverin. Furthermore, the virus genes odv-e66, ac78 and ie2 were shown to be direct targets. CONCLUSION: We systematically revealed the mechanism by which miR-34-5p is involved in the insect antiviral process. miR-34-5p inhibited the replication and infection of AcMNPV by directly targeting AcMNPV genes, especially ac78 and ie2. Our study provides a new direction and thinking for the prevention and green control of lepidopteran pests. © 2022 Society of Chemical Industry.

An Autographa californica nucleopolyhedrovirus-encoded microRNA, AcMNPV-miR-4, downregulates the expression of host gene alg-2.

Autographa california multiple nucleopolyhedrovirus (AcMNPV)-encoded microRNAs (miRNAs) that regulate viral genes to achieve infection have been reported previously. Here, we report another AcMNPV encoded miRNA, AcMNPV-miR-4 (Ac-miR-4), which downregulated the host gene, apoptosis-linked gene (alg-2). This regulation was verified by dual-luciferase reporter assays. The effects of Ac-miR-4 on virus infection were assessed. The results showed that the production of infectious budded virions (BV) was decreased and the occlusion-derived virion (ODV) embedding into polyhedra was delayed when Sf9 cells were administered an overdose of Ac-miR-4. All these findings suggest that Ac-miR-4 prolongs cell lifespan and reduces virus virulence at a relatively early stage but increases ODV at a very late stage. This finding may be attributed to the downregulation effects of alg-2, which lead to weakened ALG-2 related functions, such as cell apoptosis, vesicle budding and protein transport.

Involvement of the neddylation modification system in Bombyx mori nucleopolyhedrovirus replication.

Neddylation is a posttranslational modification that is similar to ubiquitination, and involved in some critical biological processes, such as DNA repair, transcription regulation, and ubiquitin-proteasome pathway. Recently, it was found that neddylation inhibitor MLN4924 has potent antiviral activity against human viruses including herpes simplex virus (HSV)-1, HSV-2, and influenza viruses. Here, we report that MLN4924 could dramatically and dose-dependently inhibits the propagation, formation of budding virus (BV) and occlusion body (OB) of a lepidopteran virus-Bombyx mori nucleopolyhedrovirus (BmNPV), impaired OB assembly. In addition, the neddylation modification protein NEDD8 is colocalized with aggresome and autophagosome. Our findings suggest that inhibiting neddylation could be an antibaculovirus strategy and MLN4924 may be used as candidate drug for that purpose.

Identification of key metabolic pathways reprogrammed by BmNPV in silkworm Bombyx mori.

Elucidating the mechanism of infection of Bombyx mori nuclear polyhedrosis virus (BmNPV) and host antiviral response remains a major scientific task in sericulture. Virus invasion causes a series of antiviral immune responses in the host, and successful infection leads to massive changes in the host's physiological and biochemical state. Current research mainly focuses on silkworm genes and proteins associated with viral infection and resistance, but little is known regarding the host metabolic pathways that the virus utilizes for optimal replication. In this work, key metabolites involved in viral infection were identified, including trehalose, riboflavin, tryptophan, tyrosine, and phenylalanine. The genes associated with metabolite biosynthesis and catabolism were analyzed, and their expression levels were found to be largely consistent with their respective metabolite levels before and after viral treatment in both strains. The screened metabolites were further investigated for their roles in viral replication using exogenous metabolite addition into the culture medium. The results showed that tryptophan effectively inhibited BmNPV replication, while glutamine promoted viral replication in a dose-dependent manner. Trehalose and riboflavin exhibited a complex effect on BmNPV replication. This study outlines the critical metabolites and metabolic pathways required for BmNPV to proliferate and infect the host, indicting the potential of metabolite-based treatment for viral inhibition.