BmCPV-Derived Circular DNA vcDNA-S7 Mediated by Bombyx mori Reverse Transcriptase (RT) Regulates BmCPV Infection

Figure 1. mori produces BmCPV-derived circular DNA vcDNA-S7. (A) vcDNA-S7 was verified by divergent PCR. left, Schematic of primer to validate the junction site of vcDNA-S7. Green arrow represents divergent primers. Right, electrophoresis of PCR product from flanking sequence of the junction site of vcDNA-S7. DNA from BmCPV infected silkworm midgut and healthy silkworm midgut were extracted. RNAs and linear DNAs were respectively removed with RNase A and Plasmid-Safe™ ATP-Dependent DNase. mtDNA representing mitochondrial DNA as a positive control; Potu representing genomic linear DNA as a negative control. (B) Sanger sequencing of the products of divergent PCR. The green arrow represents the divergent primer and the red arrow represents the junction site of vcDNA-S7. (C) RCA validated that vcDNA-S7 is a circular molecule. Upper, schematic of RCA of circular molecules. Under, electrophoretic detection of RCA products. ‘BmN+CPV’ represents the BmN cells infected with BmCPV. The plasmid pMD19T-S7 containing the sequence flanking the junction site of vcDNA-S7 used as positive control. The genomic DNA from BmN cells were used a negative control. (D) Validation of vcDNA-S7 by in situ…

Figure 2. Reverse transcriptase activity is involved in the formation of vcDNA-S7. (A) Endogenous reverse transcriptase activity was measured in BmN cells. BmN cells were pre-treated with 5mM AZT or 50nM GSK-LSD1 for 24 h and then the cells were lysed and the reverse transcriptase activity was measured. Relative reverse transcriptase activity was calculated to 1U of commercialized M-MLV reverse transcriptase. (B) Effect of AZT treatment on the formation of vcDNA-S7. 1×106 BmN cells were pre-treated with AZT at the indicated concentrations for 24 h and were infected with BmCPV for additional 48 h. The copies of vcDNA-S7 were determined by real-time PCR. (C, D) GSK-LSD1 treatment improved the level of histone H3K9me. 1×106 BmN cells were pre-treated with GSK-LSD1 at the indicated concentrations for 24 h. The level of histone H3K9me was analyzed by western blotting (C). The grayscale of Western blotting signal bands is analyzed by software Image J (D, E) Effect of GSK-LSD1 treatment on the formation of vcDNA-S7. 1×106 BmN cells were pre-treated with GSK-LSD1 at the indicated concentrations for 24 h and were infected with BmCPV for another 48 h. The copies of vcDNA-S7 were determined by…

Figure 3. The source of reverse transcriptase activity involved in vcDNA-S7 formation. (A) Cluster analysis of genes encoding reverse transcriptase in silkworm. reverse transcriptases genes in the silkworm were divided into 9 groups. (B) Silencing efficiency of siRNAs on 9 groups of genes encoding reverse transcriptase. siRNA. 1×106 BmN cells were transfected with siRNAs for 48 h, and were infected with BmCPV for another 48 h. Total RNA was extracted and the expression level of reverse transcriptase genes relative to housekeeper gene TIF-4A was detected by real-time PCR. (C) 1×106 BmN cells were transfected with siRNAs for 48 h, and were infected with BmCPV for another 48 h. The copies of vcDNA-S7 were determined by real-time PCR. (n=3, *, P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

Figure 4. The expression pattern of vcDNA-S7. (A) The express pattern of vcDNA-S7 in different tissues of BmCPV infected silkworm. (B) mRNA levels of BmCPV vp1 and vp7 genes in BmCPV infected BmN cells at different stages were determined by real-time PCR. (C) copies of vcDNA-S7 in BmCPV infected BmN cells at different stages were determined by real-time PCR. (D) mRNA levels of BmCPV vp1 and vp7 genes in BmCPV infected silkworm midgut at different stages were determined by real-time PCR. (E) copies of vcDNA-S7 in BmCPV infected silkworm midgut at different stages were determined by real-time PCR (n=3).

Figure 5. vcDNA-S7 inhibited BmCPV infection. (A–C) vcDNA-S7 down-regulated the expression level of BmCPV vp1 gene and polh protein in BmN cells. BmN cells (1×106) were transfected with 1 μg vcDNA-S7 for 24 h and were infected with BmCPV for another 48 h. The expression levels of vp1 gene relative to translation initiation factor (TIF-4A) were investigated by real-time PCR (A). The level of Polh protein was analyzed by western blotting (B). The grayscale of western blotting signal bands is analyzed by software Image J (C). (D–F) Injection of vcDNA-S7 into silkworm down-regulated the expression level of BmCPV vp1 gene and VP7 protein in BmN cells. The silkworm larvae of fourth instar were injected 1μg of vcDNA-S7 for 24 h and were infected with BmCPV for another 48 h. The expression levels of vp1 gene relative to translation initiation factor (TIF-4A) were investigated by real-time PCR (D). The protein level of VP7 was analyzed by western blotting (E). The grayscale of western blotting signal bands is analyzed by software Image J (F). (n=2, *P<0.05; **P<0.01).

Figure 6. Endogenous reverse transcriptase activity inhibited BmCPV infection. (A–C) Endogenous reverse transcriptase activity inhibited virus infection in BmN cells. 1×106 BmN cells were pre-treated with 5 mM AZT or 50 nM GSKLSD1 and infected with BmCPV for 24 h. The relative RNA level of BmCPV vp1gene was determined by real-time PCR (A). The protein level of VP7 was analyzed by western blotting (B). The grayscale of western blotting signal bands is analyzed by software Image J (C). (D–F) Effect of endogenous reverse transcriptase activity on the inhibitory effect of vcDNA-S7 on virus. 1×106 BmN cells were pre-treated with 5mM AZT or 50 nM GSKLSD1 and were transfected 1μg vcDNA-S7 for 24 h. Then, the cells were infected with BmCPV for another 48 h. The relative RNA level of BmCPV vp1gene was determined by real-time PCR (D). The level of VP7 protein was analyzed by western blotting (E). The grayscale of western blotting signal bands is analyzed by software Image J (F). (n=2, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

Figure 7. vcDNA-S7 can provide vsiRNAs for RNAi pathway. (A) Effects of vcDNA-S7 on the main immune pathways of mori. BmN cells (1×106) were transfected with 1μg vcDNA-S7 for 48 h. The relative RNA level of the key genes in the immune pathways (including RNAi, JAK-STAT, Imd, and Toll pathway) was determined by real-time PCR (n=2, ***P<0.001). (B) 1×107 BmN cells were transfected with 6 μg vcDNA-S7 for 48 h and small RNAs were deep sequenced. Size distribution of vsiRNAs derived from vcDNA-S7 was shown.

Figure 8. Mechanism of BmCPV-derived circular DNA vcDNA-S7 controlling BmCPV infection. Silkworm infected with BmCPV produce viral-derived vcDNA-S7 mediated by reverse transcriptase. vcDNA-S7 can be transcribed to RNA and produce vsiRNAs to control BmCPV infection by RNAi pathway.