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Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture and causes huge economic loss annually. The roles of N6-methyladenosine (m6A) modification in silkworms following BmNPV infection are currently unclear. Here, methylated RNA immunoprecipitation with next-generation sequencing were applied to investigate the m6A profiles in silkworm midgut following BmNPV infection. A total of 9144 and 7384 m6A peaks were identified from the BmNPV-infected (TEST) and uninfected silkworm midguts (CON), respectively, which were distributed predominantly near stop codons. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common m6A peaks in nuclear genes revealed that these m6A-related transcripts were associated with crucial signaling pathways. Comparative transcriptome analysis showed that 1221 differential expressed m6A peaks were identified between TEST and CON, indicating that m6A modification is regulated following BmNPV infection. GO and KEGG pathway analysis of the differentially expressed m6A peaks showed their association with signal transduction, translation, and degradation. To understand further the effect of the m6A machinery on virus infection, expression levels of m6A-related genes were altered in silencing and overexpression experiments. Expression of viral structural protein VP39 was increased in BmN cells by siRNA-mediated depletion of methyltransferase-like (METTL) enzyme genes (BmMETTL3, BmMETTL14) and cytoplasmic YTH-domain family 3 (BmYTHDF3), while the reverse results were found after overexpression of the m6A-related enzymes in BmN cells. Overall, m6A modification might be a novel epigenetic mechanism that regulation BmNPV infection and interference with this mechanism may provide a novel antiviral strategy for preventing BmNPV disease. [ABSTRACT FROM AUTHOR]

Bombyx mori cypovirus (BmCPV) enters permissive cells via clathrin-mediated endocytosis pathway. However, the distinct entry mechanism for BmCPV is still ambiguous. The aim of this study is to investigate the role of gangliosides and cholesterol in BmCPV cell entry. The number of BmCPV virions attached to the cell surface and the expression level of BmCPV vp 1 gene was significantly decreased by digestion of terminal sialic acids in gangliosides with neuraminidase (NA). Preincubation of different concentration of ganglioside GM1, GM2 or GM3 with BmCPV prior to infection, the reduction of BmCPV infectivity was found by GM2-treated in a dose-depend manner. BmCPV virions were found to colocalize with GM2 in the cell surface. The infectivity of BmCPV was reduced by anti-GM2 antibody treatment cells. Moreover, BmCPV infection was impaired by depletion of membrane cholesterol with MβCD, but the inhibitory effect of MβCD was restored by supplementing with cholesterol. The number of viral particles attached on the BmN cells was significantly decreased by pretreated with MβCD, and BmCPV infection was inhibited by silencing the expression of 3-hydroxy-3-methylglutaryl-CoA reductase gene ( Hmg -r) in cholesterol biosynthesis pathway. These results indicate that ganglioside GM2 and cholesterol in membrane lipid rafts are essential for BmCPV attachment to cell surface for its cell entry. [ABSTRACT FROM AUTHOR]

• At least seven alternative splicing isoforms of BmYki were identified. • S97 phosphorylation contributes to the cellular location of BmYki1. • BmYki3 plays a major positive regulatory role in Hippo signaling pathway. • BmYki2 plays a role of microregulation and balance in Hippo signaling pathway. • BmYki1 plays a secondary positive regulatory role in the Hippo signaling pathway. Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm. [ABSTRACT FROM AUTHOR]

Background: In our previous study, we identified four isoforms of the Bmovo gene, Bmovo-1, Bmovo-2, Bmovo-3 and Bmovo-4 from the silkworm ovary and verified that ovarian development was regulated by the BmOVO proteins. Results: To understand the regulatory mechanisms of ovarian development, the regulation of four BmOVO isoforms on the B. mori ovarian tumor (Bmotu) promoter activity was investigated with luciferase reporter assays. The results showed the Bmotu promoter activity was positively regulated by BmOVO-1, BmOVO-2, BmOVO-3 and BmOVO-4 in a dose-dependent manner, of which BmOVO-2 had the highest transcriptional activation. However, the first (A1) and third acidic domains (A3) at the N-terminus of BmOVO-1 are transcriptional repression domains, while the fourth (A4) and fifth acidic domains (A5) are transcriptional activation domains. A recombinant BmOVO zinc-finger domain was found to bind to the GTACCGTTGTA sequence located at the Bmotu promoter. Furthermore, the Bmotu promoter activity was negatively regulated by 'Tal-like' peptide, which can trigger BmOVO-1 degradation at the N-terminus. Conclusions: These results will help us to further understand the regulatory mechanisms of BmOVO isoforms on Bmotu promoter activity and ovarian development in the silkworm. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β superfamily members inhibit Bombyx mori nucleohedrovirus (BmNPV) multiplication in silkworm are not determined. In this study, we first found that BmNPV RNA transcription and protein expression level were regulated by TGF-β members, Decapentaplegic (Bmdpp) and Dawdle (Bmdaw) in the domesticated silkworm, B. mori and silkworm ovary-derived cells. Furthermore, subcellular localization showed that Bmdpp and Bmdaw were mainly presented in cytomembrane of the cultured BmN cells. Tissues expression pattern analysis found that the highest expression levels of Bmdpp and Bmdaw genes were in the hemocyte of fifth instar larvae. During the immune response, the expression level of Bmdpp gene was elevated and Bmdaw gene was declined in BmNPV infected BmN cells and silkworm. The multiplication of BmNPV was inhibited by overexpression of Bmdpp and Bmdaw genes in BmN cells. RNA interference experiments found that the multiplication of BmNPV was raised with specific siRNAs of Bmdpp and Bmdaw genes in BmN cells. The antiviral immune pathways were not significantly regulated by the TGF-β superfamily members. Taken together, these findings provided a clue to understand the function of Bmdpp and Bmdaw gene in response to the BmNPV infection in silkworm. [ABSTRACT FROM AUTHOR]