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Abstract: Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV. • At present, there have been studies on small RNA regulation by using sensitive silkworm varieties, but no studies have conducted whole transcriptome analysis by comparing resistant and sensitive silkworm strans inoculated with virus. • The mRNA-miRNA-lncRNA ceRNA regulatory network was constructed, from which the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened, and the whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. • From the perspective of lncRNA to explain the significantly different genes, our research results may provide new clues to the molecular mechanism that reveals the interaction between silkworm and virus. [ABSTRACT FROM AUTHOR]

Uridine diphosphate (UDP)‐glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT‐interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms. [ABSTRACT FROM AUTHOR]

Interacting with cell surface attachment factors or receptors is the first step for virus infection. Glycans cover a thick layer on eukaryotic cells and are potential targets of various viruses. Bombyx mori nuclear polyhedrosis viruses (BmNPV) is a baculovirus that causes huge economic loss to the sericulture industry but the mechanism of infection is unclear. Looking for potential host receptors for the virus is an important task. In this study, we investigated the role of glycosaminoglycan (GAG) modifications, including heparan sulfate (HS) and chondroitin sulfate (CS), during BmNPV infection. Enzymatic removal of cell surface HS and CS effectively inhibited BmNPV infection and replication. Exogenous HS and CS can directly bind to BmNPV virion in solution and act as neutralizers for viral infection. Furthermore, the expression of enzymes involved in GAG biosynthesis was upregulated in the BmNPV susceptible silkworm after virus administration, but down-regulated in the resistant strain after virus treatment, suggesting that BmNPV was able to utilize host cell machinery to promote the biosynthesis of GAGs. This study demonstrated HS and CS as important attachment factors that facilitate the viral entry process, and targeting HS and CS can be an effective means of inhibiting BmNPV infection. • BmNPV infection was suppressed after cell surface heparin sulfate and chondroitin sulfate were reduced. • Heparan sulfate and chondroitin sulfate can directly bind to BmNPV in solution. • GAG biosynthesis was promoted in the susceptible silkworm but suppressed in the resistant strain upon BmNPV treatment. [ABSTRACT FROM AUTHOR]

Interacting with cell surface attachment factors or receptors is the first step for virus infection. Glycans cover a thick layer on eukaryotic cells and are potential targets of various viruses. Bombyx mori nuclear polyhedrosis viruses (BmNPV) is a baculovirus that causes huge economic loss to the sericulture industry but the mechanism of infection is unclear. Looking for potential host receptors for the virus is an important task. In this study, we investigated the role of glycosaminoglycan (GAG) modifications, including heparan sulfate (HS) and chondroitin sulfate (CS), during BmNPV infection. Enzymatic removal of cell surface HS and CS effectively inhibited BmNPV infection and replication. Exogenous HS and CS can directly bind to BmNPV virion in solution and act as neutralizers for viral infection. Furthermore, the expression of enzymes involved in GAG biosynthesis was upregulated in the BmNPV susceptible silkworm after virus administration, but down-regulated in the resistant strain after virus treatment, suggesting that BmNPV was able to utilize host cell machinery to promote the biosynthesis of GAGs. This study demonstrated HS and CS as important attachment factors that facilitate the viral entry process, and targeting HS and CS can be an effective means of inhibiting BmNPV infection. • BmNPV infection was suppressed after cell surface heparin sulfate and chondroitin sulfate were reduced. • Heparan sulfate and chondroitin sulfate can directly bind to BmNPV in solution. • GAG biosynthesis was promoted in the susceptible silkworm but suppressed in the resistant strain upon BmNPV treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • Trends of metabolite levels in silkworm hemolymph upon BmNPV treatment were revealed. • BmNPV reprogrammed metabolic pathways involving trehalose, glutamine, and tryptophan. • Tryptophan inhibited BmNPV replication while glutamine promoted it. • Trehalose and riboflavin affected BmNPV replication in a complex manner. 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. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we constructed a near-isogenic line (BC8) with BmNPV resistance using highly resistant (NB) and highly susceptible parental strains (306). We investigated variations in the gene expression in the midguts of BmNPV-infected BC8 and 306 at 12h pi using the microarray. 92 differentially expressed genes were identified. Real-time qPCR analysis confirmed that 10 genes were significantly up-regulated or down-regulated in the midguts of BC8 and NB compared to 306. To our knowledge, we first defined the role of the amino acid transporter and 26S proteasome in insect antiviral. However, serine protease was not completely consistent with data of reported previously in insect antiviral. The role of the 5 genes (Bm123, Bm122, COP β′, aquaporin, glycoside hydrolases) was also demonstrated in insect antiviral. Our results provided new insights into the molecular mechanism of the Bombyx mori immune response against BmNPV infection. [Copyright &y& Elsevier]

Abstract: The silkworm Bombyx mori is of great economic value. The B. mori nuclear polyhedrosis virus (BmNPV) is one of the most common and severe pathogens for silkworm. Although certain immune mechanisms exist in silkworms, most silkworms are still susceptible to BmNPV infection. Interestingly, BmNPV infection resistance in some silkworm strains is varied and naturally existing. We have previously established a silkworm strain NB by genetic cross, which is highly resistant to BmNPV invasion. To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we employed proteomic approach and genetic cross to globally identify proteins differentially expressed in parental silkworms NB and 306, a BmNPV-susceptible strain, and their F1 hybrids. In all, 53 different proteins were found in direct cross group (NB♀, 306♂, F1 hybrid) and 21 in reciprocal cross group (306♀, NB♂, F1 hybrid). Gene ontology and KEGG pathway analyses showed that most of these different proteins are located in cytoplasm and are involved in many important metabolisms. Caspase-1 and serine protease expressed only in BmNPV-resistant silkworms, but not in BmNPV-susceptible silkworms, which was further confirmed by Western blot. Taken together, our data suggests that both caspase-1 and serine protease play a critical role in silkworm resistance against BmNPV infection. [Copyright &y& Elsevier]

The biological functions of protein glycosylation have been increasingly recognized but not yet been very well understood, especially in lower organisms. Silkworm as a model lepidopteran insect and important economic insect, has been widely studied in life science, however, the current knowledge on the glycosylation status of its proteome is not satisfactory, and little is known about how pathogenic infections could affect the glycosylation status. This study performed large scale glycosite mapping for the silkworm Bombyx mori P50 strain, and quantitatively compared with that infected with the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). Some 400 glycoproteins were mapped in the silkworm, including N- and O-glycoproteins. Upon virus infection, the glycosylation levels of 41 N-glycopeptides were significantly changed, some of them belonging to transmembrane glycoproteins. The O-glycosylation profiles were also affected. In addition, 4 BmCPV-encoded viral proteins were found to be glycosylated for the first time, including polyhedrin, P101, VP3, and the NS protein. This study drafted a silkworm protein glycosylation map and underlined the potential impact of virus infection on glycosylation. This study reveals the characteristics of the glycoproteome in the silkworm strain P50, and quantitatively compared to that infected by the virus BmCPV, which underlines the impact of virus infection on the alteration of protein glycosylation in invertebrate species. Our findings add to the knowledge of the post translational modifications of this model organism, and also uncovered for the first time the glycosylation status of the viral proteins expressed by BmCPV. Unlabelled Image • Total N- and O-glycoproteins in silkworm strain P50 were revealed. • Alterations in glycosylation profiles were noted after BmCPV virus infection. • The glycosylation status of the BmCPV-encoded proteins was uncovered. [ABSTRACT FROM AUTHOR]

• Large scale and precision mapping of silkworm N-glycoproteome. • Silkworm O-glycoproteins are mapped with glycosylation sites and glycan compositions. • Some glycoproteins identified are unique to the virus susceptible or resistant strains. Protein glycosylation plays important roles in protein structure, function, and immune recognition, among many other activities. One of the major roles of glycans and glycoconjugates on the cell surface is acting as the receptor for outside pathogens such as viruses. During the initial stage of viral replication, viruses interact with cell membrane receptors, which are in many cases glycoproteins. Identifying such glycoproteins is essential to understanding the mechanisms of viral infection, as well as developing antiviral strategies. Silkworm is an important economic insect as well as a model organism for molecular biology, yet current knowledge on its glycoproteome is far from complete due to both analytical challenges and perceived lack of importance. In this study, we performed a large-scale glycoproteomic survey for two silkworm Bombyx mori strains 306 and NB, which are susceptible and resistant to the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), respectively. More than 400 silkworm N- and O- glycoproteins were identified with high confidence, demonstrating that this organism employs extensive glycosylation. We mapped some glycoproteins only to the BmNPV susceptible or resistant strain, underlining the potential relationship between glycosylation and viral susceptibility. We predicted O-glycoproteins and O-glycan compositions for the first time for this organism. The variations in glycan site occupancy, as well as glycan diversity between the two silkworm strains, provide an insight into role of glycosylation in viral recognition and infection processes. [ABSTRACT FROM AUTHOR]