Circular DNAs derived from single-stranded RNA viruses play important roles in counteracting viral infection. However, whether double-stranded RNA viruses generate functional circular DNAs is still unknown. Using circDNA sequencing, divergent PCR, DNA in situ hybridization and rolling circular amplification, we presently confirmed that in silkworm, Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a double-stranded RNA virus belonging to cypovirus, is prone to produce a BmCPV-derived circular DNA termed as vcDNA-S7. We have also found that vcDNA-S7 formation is mediated by endogenous reverse transcriptase (RT), and the proliferation of BmCPV can be inhibited by vcDNA-S7 in vitro and in vivo . Moreover, we have discovered that the silkworm RNAi immune pathway is activated by vcDNA-S7, while viral small interfering RNAs (vsiRNAs) derived from transcribed RNA by vcDNA-S7 can be detected by small RNA deep sequencing. These results suggest that BmCPV-derived vcDNA-S7, mediated by RT, can serve as a template for the biogenesis of antiviral siRNAs, which may lead to the repression of BmCPV infection. To our knowledge, this is the first demonstration that a circular DNA, produced by double stranded RNA viruses, is capable of regulating virus infection.
Polymerases encoded by segmented negative-strand RNA viruses cleave 5’-m7G-capped host transcripts to prime viral mRNA synthesis (“cap-snatching”) to generate chimeric RNA, and trans-splicing occurs between viral and cellular transcripts. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an RNA virus belonging to Reoviridae, is a major pathogen of silkworm ( B . mori ). The genome of BmCPV consists of 10 segmented double-stranded RNAs (S1-S10) from which viral RNAs encoding a protein are transcribed. In this study, chimeric silkworm-BmCPV RNAs, in which the sequence derived from the silkworm transcript could fuse with both the 5’ end and the 3’ end of viral RNA, were identified in the midgut of BmCPV-infected silkworms by RNA_seq and further confirmed by RT-PCR and Sanger sequencing. A novel chimeric RNA, HDAC11-S4 RNA 4, derived from silkworm histone deacetylase 11 (HDAC11) and the BmCPV S4 transcript encoding viral structural protein 4 (VP4), was selected for validation by in situ hybridization and Northern blotting. Interestingly, our results indicated that HDAC11-S4 RNA 4 was generated in a BmCPV RNA-dependent RNA polymerase (RdRp)-independent manner and could be translated into a truncated BmCPV VP4 with a silkworm HDAC11-derived N-terminal extension. Moreover, it was confirmed that HDAC11-S4 RNA 4 inhibited BmCPV proliferation, decreased the level of H3K9me3 and increased the level of H3K9ac. These results indicated that during infection with BmCPV, a novel mechanism, different from that described in previous reports, allows the genesis of chimeric silkworm-BmCPV RNAs with biological functions.
The silk-gland cells of silkworm are special cells in which division occurs only at the embryonic stage. Throughout the larval stage, the silk-gland cells only replicate DNA in the nucleus without cell division. The extrachromosomal circular DNAs (eccDNAs) have not yet been reported in the silk-gland of the silkworms. Herein, we have explored the presence and characterization of eccDNAs in the posterior silk-gland of silkworms. A total of 35,346 eccDNAs were identified, representing all chromosomes, with sizes ranging from 0.05 kb to 13,569,549 kb, but with most of them being shorter than 1 kb. Motif analysis revealed that dual direct repeats are flanking the 5’ and 3’ breaking points of eccDNA. The sequences exceeding 1kb length in eccDNAs, present palindromic sequence characteristics flanking the 5’ and 3’ breaking points of the eccDNA. These motifs might support possible models for eccDNA generation. Genomic annotation of the eccDNA population revealed that most eccDNAs (58.6%) were derived from intergenic regions, whereas full or partial genes were carried by 41.4 % eccDNAs. Furthermore, most eccDNAs are derived entirely from introns (2.9%) and 38.2 % are derived from exons. Additionally, the genes related to silk protein synthesis carried by eccDNAs were analyzed. Moreover, it was found that silk protein genes fib-H, fib-L, and P25 could be carried by eccDNAs. Besides, the transcription factors SGF and sage, which play an important regulatory role in silk protein synthesis, were also carried by eccDNAs. Finally, GO and KEGG enrichment analyses showed that the genes carried by eccDNAs were mainly associated with the development and metabolism-related signaling pathways. Overall, the results of the present study not only provide a novel perspective on the mechanism of silk gland development and silk protein synthesis but also complement previously reported genome-scale eccDNA data supporting that eccDNAs are common in eukaryotes.
The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-β, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.
Extrachromosomal circular DNAs (eccDNAs) has been found to be widespread and functional in various organisms. However, comparative analyses of pre- and post-infection of virus are rarely known. Herein, we investigated the changes in expression patterns of eccDNA following infection with Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and explore the role of eccDNA in viral infection. Circle-seq was used to analyze eccDNAs in the midgut of BmCPV-infected and BmCPV-uninfected silkworms. A total of 5508 eccDNAs were identified, with sizes varying from 72 bp to 17 kb. Most of eccDNAs are between 100 to 1000 bp in size. EccDNA abundance in BmCPV-infected silkworms was significantly higher than in BmCPV-uninfected silkworms. GO and KEGG analysis of genes carried by eccDNAs reveals that most are involved in microtubule motor activity, phosphatidic acid binding, cAMP signaling pathway, and pancreatic secretion signaling pathways. Several eccDNAs contain sequences of the transcription factor SOX6, sem-2, sp8b, or Foxa2. Association analysis of eccDNA-mRNA/miRNA/circRNA revealed that some highly expressed genes are transcribed from relevant sequences of eccDNA and the transcription of protein coding genes influenced the frequency of eccDNA. BmCPV infection resulted in changes in the expression levels of six miRNAs, but no known miRNAs with altered expression levels due to changes in eccDNA abundance were identified. Moreover, it was found that 1287 and 924 sequences representing back-spliced junctions of circRNAs were shared by the junctions of eccDNAs in the BmCPV-infected and uninfected silkworms, respectively, and some eccDNAs loci were shared by circRNAs on Chromosomes 2, 7, 11, 14, and 24, suggesting some eccDNAs may exert its function by being transcribed into circRNAs. These findings suggest that BmCPV infection alter the expression pattern of eccDNAs, leading to changes in RNA transcription levels, which may play roles in regulating BmCPV replication. In the future, further experiments are needed to verify the association between eccDNA-mRNA/miRNA/circRNA and its function in BmCPV infection.
Abstract The silk gland cells of silkworm are special cells which only replicate DNA in the nucleus without cell division throughout the larval stage. The extrachromosomal circular DNAs (eccDNAs) have not yet been reported in the silk gland of silkworms. Herein, we have explored the characterization of eccDNAs in the posterior silk gland of silkworms. A total of 35 346 eccDNAs were identified with sizes ranging from 30 to 13 569 549 bp. Motif analysis revealed that dual direct repeats are flanking the 5′ and 3′ breaking points of eccDNA. The sequences exceeding 1 kb length in eccDNAs present palindromic sequence characteristics flanking the 5′ and 3′ breaking points of the eccDNA. These motifs might support possible models for eccDNA generation. Genomic annotation of the eccDNA population revealed that most eccDNAs (58.6%) were derived from intergenic regions, whereas full or partial genes were carried by 41.4% of eccDNAs. It was found that silk protein genes fib‐H , fib‐L , and P25 , as well as the transcription factors SGF and sage , which play an important regulatory role in silk protein synthesis, could be carried by eccDNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the genes carried by eccDNAs were mainly associated with the development and metabolism‐related signaling pathways. Moreover, it was found that eccDNA fib‐L could promote the transcription of fib‐L gene. Overall, the results of the present study not only provide a novel perspective on the mechanism of silk gland development and silk protein synthesis but also complement previously reported genome‐scale eccDNA data supporting that eccDNAs are common in eukaryotes.
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