Tight junction protein claudin-2 promotes cell entry of Bombyx mori cypovirus.

Claudin-2 is a major component of tight junctions (TJs), which play an important role in reovirus entry into host cells. The Bombyx mori cytoplasmic polyhedosis virus (BmCPV) relates to the cypovirus strain of the reovirus family. So far, the role of claudin-2 in the process of BmCPV infection is not known. In the present study, it was observed that increasing expression of the claudin-2 gene (CLDN2) may concomitantly elevate BmCPV infection. Contrarily, knockdown of CLDN2 expression by siRNAs can reduce BmCPV infection. Similarly, antibody-based blockage of claudin-2 could also decrease BmCPV cell entry. These results suggest that claudin-2 can promote BmCPV infection in vitro. Moreover, immunofluorescence (IF) assays showed that claudin-2 can interact with BmCPV during viral infection. Specifically, co-immunoprecipitation experiments indicated that claudin-2 binds the BmCPV VP7 (instead of VP3 proteins). The interaction between VP7 and claudin-2 was further confirmed by bimolecular fluorescence complementation (BIFC). Altogether, our results suggest that BmCPV cell entry can be promoted upon interaction of VP7 with claudin-2. These findings provide new mechanistic insights related to BmCPV infection. KEY POINTS: •Claudin-2 could promote BmCPV infection of cells. •Claudin-2 interacted with BmCPV during BmCPV infection. •Claudin-2 could interact with BmCPV VP7 protein, but not with VP3 proteins.

Identification and characterization of novel type of RNAs, circRNAs in crucian carp Carassius auratus gibelio.

Circular RNAs (circRNAs) with regulatory potency activity was identified from varieties of species. Crucian carp (Carassius auratus gibelio) is one of the most freshwater aquaculture species in China. Every year, huge economic damage to the farming was caused by the virus and bacterial infection. Until now, there is any information about circRNA reported from the Crucian carp. In this study, the expression pattern of circRNA in Crucian carp was investigated with transcriptomic analysis. The results showed that only 37 circRNAs were identified from the Crucian carp, and these circRNAs biogenesis was formed with canonical GU-AG splicing mechanism with unevenly distributed on the chromosomes. Wherein, most of the circRNAs were derived from the sense overlapping strategy. Reverse transcript PCR and Sanger sequencing data indicated that these circRNAs were existed authenticity in Crucian carp. The bioinformatics analysis indicated that circRNAs identified from the Crucian carp with potential miRNA sponge regulate the expression level of mRNAs. GO annotation and KEGG pathway analysis of these circRNAs showed that more than 20% circRNAs were related with catalytic activity and binding in the category of molecular function, and these circRNAs were enriched in 9 signaling pathways, such as, Wnt signaling pathway, MAPK signaling pathway, Ubiquitin mediated proteolysis et al. 220 mRNAs would be regulated by the circRNAs via miRNAs mediation. These target mRNAs were further analyzed with functional annotation and KEGG analysis. GO annotation analysis showed that several genes were related with function of nucleotide binding, transcription regulatory activity. KEGG pathway analysis showed that 5 genes were enriched in the pathway of Endocytosis. The circRNA-miRNA-mRNA regulation network indicated that one miRNA can link one or more circRNA and one or more mRNA. Overall, these results will not only help us to further understand the novel RNA transcripts in Crucian carp, but also provide the novel clue to investigate the interaction between host and pathogens from this novel circRNA molecule.

N 6-Methyladenosine Level in Silkworm Midgut/Ovary Cell Line Is Associated With Bombyx mori Nucleopolyhedrovirus Infection.

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.