Identification and characterization of circular RNAs in the silkworm midgut following Bombyx mori cytoplasmic polyhedrosis virus infection.

The pathogenesis of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection is unclear, although accumulating evidence indicates that circular RNAs (circRNAs), which act as competing endogenous RNAs or positive regulators, play important roles in regulating gene expression in eukaryotes and, thus, may play a role in BmCPV infections. To explore the expression and biological functions of circRNAs in the silkworm midgut following BmCPV infection, silkworm circRNA expression profiles of normal midgut tissue (control) and BmCPV-infected midgut tissue (test) were determined using high-through sequencing. A total of 9,753 and 7,475circRNAs were detected from the control and test samples, respectively. The two samples shared 6,085 circRNAs, while 646 and 737 circRNAs were expressed specifically in the control and test samples, respectively. A total of 3,638 circRNAs were shown to be differentially expressed, and 400 circRNAs were substantially differentially expressed with a fold-change ≥ 2.0 (p< 0.05 and a false discover rate < 0.05), of which 294 were up-regulated and 106 were down-regulated following infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to determine the principal functions of the substantially differentially regulated genes. circRNA-miRNA interaction networks were constructed based on a correlation analysis between the differentially expressed circRNAs and the nature of their microRNA (miRNA) binding sites. The network inferred that 13 miRNAs interacting with 193 circRNAs were among the 300 most abundant relationships. bmo-miR-3389-5p, bmo-miR-745-3p, and bmo-miR-3262 were related to 30, 34, and 34 circRNAs, respectively. circRNA_8115, circRNA_9444, circRNA_4553, circRNA_0827, and circRNA_6649 contained six, five, four, four, and four miRNA binding sites, respectively. We further found that alternative circularization of circRNAs is a common feature in silkworms and that the junction sites of many silkworm circRNAs are flanked by canonical GT/AG splicing signals. Our study is the first to show the circRNA response to virus infection. Thus, it provides a novel perspective on circRNA-miRNA interactions during BmCPV pathogenesis, and it lays the foundation for future research of the potential roles of circRNAs in BmCPV pathogenesis.

Identification of tarsal-less peptides from the silkworm Bombyx mori.

The polycistronic and non-canonical gene tarsal-less (tal, known as pri) was reported to be required for embryonic and imaginal development in Drosophila; however, there are few reports of the tal gene in the silkworm Bombyx mori. Here, we cloned a tal-like (Bmtal) gene, and a sequence analysis showed that the Bmtal cDNA (1661 bp) contains five small open reading frames (smORFs) (A1, A2, A3, A4, and B) that encode short peptides of 11-12 (A1-A4) amino acid residues containing an LDPTG(E)L(Q)(V)Y motif that is conserved in Drosophila Tal, as well as a 32-amino-acid B peptide. Reverse transcription-quantitative polymerase chain reaction showed that the expression of the Bmtal gene was highest in the trachea, followed by the silk gland and Malpighian tubule, in day 3 fifth-instar larvae. Subcellular localization showed that BmTal localized in the nucleus. By regulating the expression of the full-length Bmtal gene and the functional smORFs of Bmtal, we showed that the expression levels of the Bmovo gene and genes related to the Notch, transforming growth factor-ß, and Hippo signaling pathways changed with changes in BmTal peptide expression. A co-immunoprecipitation assay showed that BmTal interacts with polyubiquitin, which triggered degradation and/or processing of the 14-3-3 protein zeta. A comparative transcriptome analysis showed that 2843 (2045) genes were up- (down)-regulated after Bmtal gene expression was up-regulated. The up- (down)-regulated differentially expressed genes were enriched in 326 (278) gene ontology terms (P ≤ 0.05) and 54 (59) Kyoto Encyclopedia of Genes and Genomes pathways (P ≤ 0.05), and the results indicated that the BmTal peptides could function as mediators of hormone levels or the activities of multiple pathways, including the peroxisome proliferator-activated receptor, Hedgehog, mitogen-activated protein kinase, adipocytokine, and gonadotropin-releasing hormone signaling pathways, as well as the innate immune response. These results increase our understanding of the function and mechanism of BmTal at the genome-wide level.

Circular RNA alterations in the Bombyx mori midgut following B. mori nucleopolyhedrovirus infection.

Thus far, no systematic studies have examined circRNA expression profiles in the silkworm following B.mori nucleopolyhedrovirus (BmNPV) infection. To explore the expression patterns of circRNAs in the silkworm midgut following BmNPV infection, circRNAs in normal midguts and BmNPV-infected midguts were analyzed by high-throughput sequencing. A total of 353 circRNAs were significantly differentially expressed, of which 241 were upregulated and 112 were downregulated following infection. GO annotation and KEGG pathways analyses of these circRNAs showed that many key immunity pathways and metabolism pathways were enriched in the BmNPV-infected midguts. The potential roles of the predicted targets of the miRNAs that interacted with the circRNAs showed that ubiquitin, apoptosis, and endocytosis signaling pathways were enriched significantly by BmNPV infection.

Both ganglioside GM2 and cholesterol in the cell membrane are essential for Bombyx mori cypovirus cell entry.

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 vp1 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 MßCD, but the inhibitory effect of MßCD was restored by supplementing with cholesterol. The number of viral particles attached on the BmN cells was significantly decreased by pretreated with MßCD, 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.