Genome-Wide Characterization of Extrachromosomal Circular DNA in the Midgut of BmCPV-Infected Silkworms and Its Potential Role in Antiviral Responses

Abstract

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.

Keywords: BmCPV; circRNA; eccDNAs; mRNA; miRNA.

Figure 1

Characteristics of eccDNAs in midguts of B. mori. (A) Validation of BmCPV-infected silkworms. (0.0001< *** p < 0.001, n = 3). (B) A schematic overview of the Circle-Seq approach applied for genome-wide profiling of eccDNA from the silkworm midgut. (C) The quantity of eccDNAs across various chromosomes. (D) The number of formed eccDNAs per Mb on each chromosome. (E,F) Heatmap of the distribution of eccDNAs in silkworm midguts on the chromosome in BmCPV-uninfected (E) and BmCPV-infected silkworms (F). (G) The distribution of eccDNA among different genomic elements. (H) Distribution of sizes for eccDNA in BmCPV-infected and uninfected silkworm midguts. (I) Comparison of GC content in the eccDNA locus and its adjacent upstream and downstream regions to the genomic average.

Figure 2

Validation of eccDNAs. (A) For each predicted eccDNA, divergent PCR primers were designed to specifically amplify the circular molecules and their junction sites. Five eccDNAs were chosen for additional validation (including Chromosome 1: 9054503–9056429, Chromosome 12: 1637053–1639494, Chromosome 3: 4034829-4036205, Chromosome 10: 1758579–1761576 and Chromosome 1: 4197696–4199055). (B) PCR products validation by Sanger sequencing. Arrow indicated junction site. The red arrow indicates the junction site of eccDNAs.

Figure 3

Motif analysis of the sequences flanking the breaking points of eccDNAs. The picture on the left represents the motif of the 10 bp sequences located upstream and downstream of the 5′ breaking points of eccDNA. The picture on the right represents the motif of the 10 bp sequences located upstream and downstream of the 3′ breaking points of eccDNA.

Figure 4

Expression patterns of ecccDNA in BmCPV-infected and uninfected midgut and functional annotation of differentially expressed eccDNAs. (A) The quantity of eccDNA co-expressed in BmCPV-infected and uninfected silkworm midguts. CPV-JS represents BmCPV-infected group. Con-JS represents BmCPV-uninfected group. (B) The scatter plot of differentially expressed eccDNAs. (C) The volcano plots of differentially expressed eccDNAs. (D) Heat map and hierarchical clustering of eccDNAs. (E–J) The biological processes, cellular components, and molecular functions are associated with the up-regulated (E–G) and down-regulated (H–J) eccDNAs. (K,L) Analysis of KEGG pathways related to the up-regulated (K) and down-regulated (L) eccDNAs.

Figure 5

Association of DEGs with BmCPV infection. (A) The clustering plot of DEGs. (B) The scatter plot of DEGs. (C) The volcano plots of DEGs. (D–I) The biological processes, cellular components, and molecular function related to the up-regulated (D–F) and down-regulated (G–I) genes. (J,K) Analysis of KEGG pathways associated with the up-regulated (J) and down-regulated (K) genes. (L) qPCR validation of RNA-Seq results.

Figure 6

DEMs with BmCPV infection. (A) The clustering plot of DEMs. (B) The scatter plot of DEMs. (C) The volcano plots of DEMs. (D–I) The biological processes, cellular components, and molecular function related to the up-regulated (D–F) and down-regulated (G–I) miRNAs. (J,K) Analysis of the KEGG pathways associated with the up-regulated (J) and down-regulated (K) miRNAs. (L) qPCR validation of miRNA-Seq results.

Figure 7

Association analysis of eccDNA-mRNA. (A) Association analysis between eccDNA counts in each gene and gene expression profiling. The scatter plot shows the expression levels of individual genes compared to the total expression level of eccDNA linked to each gene. (B) eccDNA abundance and gene density distribution. The scatter plot shows the quantity of genes and eccDNAs per MB of length for each chromosome. (C,D) The sequences representing back-spliced junctions of some circRNAs were shared by junctions of eccDNAs in the BmCPV-infected (C) and -uninfected silkworms (D). (E) Visualization of the genomic locations of eccDNAs and circRNAs loci.