Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

Molecular characterization and expression analysis of BmNOX in two strains of Bombyx mori with contrasting viral resistance phenotype

We recently documented the identification of a 26.5 kDa protein named BmNox in the gut fluid of Nistari strain of Bombyx mori, which possessed antiviral activity against BmNPV in vitro. In this report, we report the characterization of the full‐length gene encoding BmNOX and the levels of expression of this gene in select tissues of silkworm larvae from a BmNPV‐susceptible and a BmNPV‐resistant strain to the defense capability in Bombyx mori larvae challenged with BmNPV. We also evaluated the BmNox expression in various stages of larval life of a resistant and a susceptible strain of Bombyx mori selected from among a panel of strains of silkworm. Nistari, a multivoltine strain of silkworm, expressed BmNOX during all five larval stages, and were highly resistant to BmNPV infection. In sharp contrast, CSR₂, a bivoltine strain, showed weaker expression of BmNOX in the anterior midgut in larval life and was highly susceptible to BmNPV infection. BmNOX is a secretory protein with dual expression in gut fluid and mid gut tissue. BmNOX is expressed heavily in the posterior mid gut, with weaker expression in the fore‐ and mid‐gut regions. © 2010 Wiley Periodicals, Inc.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.     

Low METTL3 level in midgut of the Bombyx mori inhibit the proliferation of nucleopolyhedrovirus

N6-methyladeosine (m⁶A) plays an important role in virus infection and replication. Bombyx mori nuclear polyhedrosis is caused by Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Expression levels of m⁶A-modification-related genes after the infection of BmNPV were detected at first. Then, expression levels of BmNPV nucleocapsid protein gene VP39 and envelope fusion protein gene GP64 after knockdown of METTL3in vitro were quantified to identify the effect of m⁶A modification on BmNPV. BmNPV firstly infects the larval midgut in case of oral infection. Subsequently, to clarify the relationship between m⁶A modification and resistance of the silkworm to BmNPV, we detected the expression levels of m⁶A-modification-related genes invivo before and after infection of BmNPV. The results indicated that low METTL3 level hindered the proliferation of BmNPV to some extent, and silkworm strain with low METTL3 level showed stronger resistance against BmNPV. This study will accumulate new experimental data for elucidating the resistance mechanism of silkworm against BmNPV.     

Cloning and Characterization of Bombyx mori PP-BP a Gene Induced by Viral Infection

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12₇₈₂) was obtained from the hemolymph RNA pools. Using 5′-RACE with a specially designed primer based on the FDD study, a 1 401 bp cDNA clone was obtained containing a 1 311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.     

Effects of overexpression and inhibited expression of thymosin,an actin‐interacting protein from Bombyx mori,on BmNPV proliferation and replication

Previous study showed that exogenously applied recombinant thymosin from Bombyx mori (BmTHY) reduces B. mori nucleopolyhedrovirus (BmNPV) proliferation in silkworm. Which stands to reason that BmTHY in B. mori is crucial for the defense against BmNPV. However, little is known about the effect of endogenously overexpressed or repressed BmTHY on B. mori resistance to virus infection. To study this issue, we constructed an overexpression and inhibited expression systems of BmTHY in BmN cells. The viral titer and the analysis from the quantitative real‐time polymerase chain reaction (PCR) revealed that overexpression of BmTHY decreased the copies of BmNPV gene gp41, which goes over to inhibit the proliferation of BmNPV in BmN cells, while the inhibited expression of BmTHY significantly enhanced viral proliferation in infected BmN cells. These results indicated that endogenous BmTHY can inhibit BmNPV proliferation and replication in infected BmN cells. Furthermore, Co‐IP showed that BmTHY could bind to actin in BmN cells. Also, the overexpression or inhibited expression of BmTHY shifted the ratio of F/G‐actin in infected BmN cells. Lastly, the BmTHY, an actin‐interacting protein, might be one of the key host factors against BmNPV, which inhibits viral proliferation and replication in BmN cells.     

Genome-Wide Analysis of Differentially Expressed microRNA in Bombyx mori Infected with Nucleopolyhedrosis Virus

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV.     

Evolutionary and functional analyses of the interaction between the Bombyx mori inhibitor of apoptosis (IAP) and nucleopolyhedrovirus IAPs

As an important insect immune response, apoptosis plays a critical role in the interaction between baculoviruses and insect hosts. Previous reports have identified inhibitor of apoptosis (IAP) proteins in both insects and baculoviruses, but the relationship between these proteins is still not clearly understood. Here, we found that insect IAP proteins were clustered with baculovirus IAP3, suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera. We demonstrated that Bombyx mori inhibitor of apoptosis (Bmiap) gene had an inhibitory effect on apoptosis in silkworm cells. Further analysis of the effects of Bmiap genes on the proliferation of B. mori nucleopolyhedrovirus (BmNPV) showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells. Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells, implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection. Taken together, our results provide important insights into the functional relationships of iap genes, and improve our knowledge of apoptosis in baculoviruses and insect hosts.     

Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.     

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.     

杆状病毒凋亡抑制基因IAP1促进家蚕CyclinB的核内积累

[目的]家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是生产上危害最严重的病原之一。BmNPV感染BmN-SWU1细胞将细胞周期阻滞于G2/M期。CyclinB是调控细胞周期G2期向M期转换的重要细胞周期蛋白。因此,研究BmNPV感染后CyclinB变化对解析病毒调控细胞周期的机制具有重要意义,同时探究这个过程中与CyclinB互作的病毒蛋白,可为构建家蚕转基因品系提供分子靶标。[方法]qRT-PCR检测BmNPV感染后BmCyclinB的表达变化;免疫荧光观察病毒感染前后BmCyclinB的定位变化,通过细胞质细胞核蛋白分离实验验证。免疫共沉淀钓取与BmCyclinB互作的病毒蛋白。BmNPV感染期间敲除BmNPV IAP1观察BmCyclinB的入核比例。[结果]BmNPV感染后BmCyclinB转录水平下调。BmNPV感染前BmCyclinB主要定位于细胞质,而感染后主要定位于细胞核。BmNPV感染BmN-SWU1细胞后促进BmCyclinB在核内积累。共钓取了7个与BmCyclinB互作的病毒蛋白,免疫共沉淀和细胞共定位证明BmNPV IAP1与BmCyclinB之间存在相互作用。敲除BmNPV IAP1后BmCyclinB进入细胞核的数量显著减少。[结论]BmNPV IAP1可通过与BmCyclinB互作,促进BmCyclinB在核内积累。     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.