Fig 1.
Chimeric RNAs generated in the silkworm midgut infected with BmCPV.
A, Circos diagram of chimeric RNAs in the midgut at 48 h (BmCPV_48), 96 h (BmCPV_96) and 144 h (BmCPV_144) post-infection with BmCPV; B, Fusion types of chimeric RNAs detected in the midgut infected with BmCPV. “BMSK_X” indicates the individual silkworm chromosomes. Each line in the Circos plots represents a fusion event; the red line represents the fusion event between two RNA molecules originating from the same chromosome, while the blue line represents the fusion event between RNA molecules originating from different chromosomes/BmCPV genomic dsRNA segment. The circle at one end of the wavy line represents the 5’-end of RNA molecule.
Fig 2.
Identification of chimeric RNAs by RT-PCR and Sanger sequencing.
A and B, fragments from the antisense RNAs of BmCPV S3 (A) and S4 (B) dsRNAs fused with a fragment of the larger subunit of ribosomal RNA (LSrRNA) from silkworm, the sequence indicated with a blue background is a common sequence shared by the S3/S4 and LSrRNA antisense sequences. C, Fragment of antisense RNA of BmCPV S7 dsRNA fused with a fragment of the silkworm mRNA clone: fcaL43P13. D, Fragment of silkworm U6 ncRNA fused with a fragment of sense RNA BmCPV S7 dsRNA. E and F, a fragment of silkworm U6 ncRNA/Bm_160 RNA fused with a fragment of the sense RNA of BmCPV S9 dsRNA. G, Fragment of antisense RNA of BmCPV S7 dsRNAs fused with a fragment of the antisense RNA of BmCPV S5 dsRNAs. H, A fragment of the sense RNA of BmCPV S9 dsRNAs fused with a fragment of the sense RNA of BmCPV S7 dsRNAs. The sequences indicated with a blue background are sequences shared by two parental RNAs.
Fig 3.
Isoforms of chimeric silkworm HDAC11-S4 RNA.
A, B, C, D, E and F represent the junction sites of isoforms of chimeric silkworm HDAC11-S4 RNA.
Fig 4.
HDAC11-S4 RNA 4 was validated by Northern blotting and in situ hybridization.
A, Validation of HDAC11-S4 RNA 4 by Northern blotting. The total RNA extracted from the BmCPV-infected midgut was separated on 1% agarose-formaldehyde gels (left) and transferred to Hybond-N+ nylon membranes. Northern blotting was conducted with biotin-labeled DNA targeting the junction site of chimeric HDAC11-S4 RNA 4 (Right). Total RNAs extracted from the non-BmCPV-infected midgut was used as a control. Lane M, DNA marker; Lane Con, total RNAs extracted from the non-BmCPV-infected midgut; Lane BmCPV, total RNAs extracted from the BmCPV-infected midgut. B, Validation of HDAC11-S4 RNA 4 by in situ hybridization. BmN cells with/without BmCPV infection at 48 h post-infection were digested with proteinase K and hybridized with biotin-labeled DNA targeting the junction site of chimeric HDAC11-S4 RNA 4. Hybridization signals were detected using CY3-labeled streptavidin. Cell nuclei were counterstained with DAPI.
Fig 5.
Formation and expression pattern of chimeric HDAC11-S4 RNA 4.
A, Formation of chimeric HDAC11-S4 RNA 4. pIZT-CS4 and a mixture of pIZT-CS4 and pIZT-CS2 were transfected into BmN cells, and total RNAs were extracted at 48 h posttransfection to determine the formation of chimeric HDAC11-S4 RNA 4 with RT-PCR. B, Expression pattern of chimeric HDAC11-S4 RNA 4. The total RNAs extracted from the midgut infected with BmCPV at 0–144 h postinfection were used as templates, and the expression level of chimeric HDAC11-S4 RNA 4 was determined by qRT–PCR. The TIF-4A gene was used as an internal reference. Each biological experiment was repeated three times. ***, p<0.001.
Fig 6.
Effect of chimeric HDAC11-S4 RNA 4 on BmCPV viral gene expression and H3K9me3/H3K9ac of histone.
A, Schematic diagram of various RNA molecules in the chimeric HDAC11-S4 RNA 4, HDAC11-S4 RNA 4, a chimeric RNA derived from HDAC11 RNA and the BmCPV S4 RNA; partial HDAC11 RNA, the upstream sequence of the junction site of chimeric HDAC11 S4 RNA 4; CPV S4 RNA, the RNA derived from BmCPV segment S4 RNA. B, Effect of chimeric HDAC11-S4 RNA 4 on BmCPV VP7 and H3K9me3/H3K9ac of histone 3 levels. BmN cells (106) transfected with 1, 2 or 3 μg of chimeric HDAC11-S4 RNA 4 were inoculated with BmCPV at 24 h post-transfection, and the VP7 protein level and H3K9me3/H3K9ac of histone 3 were determined at 48 h post-infection by Western blotting with anti-VP7, anti-H3K9me3 and anti-H3K9ac antibodies. The transfection with the corresponding doses of GFP RNA, partial HDAC11 RNA and CPV S4 RNA (produced by in vitro transcription) were used as controls, respectively. C, D, E, The grayscale intensity of the Western blot signaling bands in Fig 6B were analyzed by Image J software. F, G, H, Effect of chimeric HDAC11-S4 RNA 4 on BmCPV vp1 gene expression levels. BmN cells (106) transfected with 1, 2 or 3 μg of chimeric HDAC11-S4 RNA 4 were inoculated with BmCPV at 24 h posttransfection, and the relative expression level of the BmCPV vp1 gene was determined at 48 h posttransfection by qRT–PCR. GFP RNA, partial HDAC11 RNA and CPV S4 RNA-transfected cells infected with BmCPV were used as controls. Each biological experiment was repeated three times. **, p<0.01; *, p<0.05.
Fig 7.
Chimeric HDAC11-S4 RNA 4 encodes a truncated viral VP4 with N-terminal extensions derived from host HDAC11 RNA.
A, Chimeric HDAC11-S4 RNA 4 sequence. Lowercase letters represent the HDAC11 gene mRNA fragment with HDAC11-S4 RNA 4. Uppercase letters represent the BmCPV S4 RNA fragment with HDAC11-S4 RNA 4. Blue letters represent the speculated open reading frame, and the box represents the hypothesized IRES-like sequence. B, Deduced sequence of the NesVP4 protein encoded by chimeric HDAC11-S4 RNA 4. Red letters represent the amino acid sequence derived from HDAC11 mRNA, and blue letters represent the amino acid sequence derived from sense RNA of the BmCPV S4 segment. C, Identification of NesVP4 encoded by chimeric HDAC11-S4 RNA in the BmCPV-infected midgut by Western blotting. Proteins from the midguts of silkworms infected with BmCPV at 24–144 h postinfection were separated by SDS–PAGE. After the proteins on the gel were transferred to a nitrocellulose membrane, Western blotting was performed. An anti-VP4 antibody was used as a primary antibody, and HRP-conjugated goat anti-mouse IgG was used as a secondary antibody. α-tubulin was used as an internal reference. Lane M, molecular marker; lane con, midgut uninfected with BmCPV; lane 24-lan144, midgut infected with BmCPV at 24–144 h postinfection with BmCPV. D, Identification of NesVP4 in BmN cells transfected with the chimeric HDAC11-S4 RNA 4 by Western blotting. Lane M, molecular marker; lane Mock, cells without transfection; lane HDAC11-S4 RNA 4, BmN cells transfected with HDAC11-S4 RNA 4 at 48 h.