Transcriptional architecture of African swine fever virus

Figures

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  Fig 1. Comprehensive analysis of ASFV transcription and replication in infected PAMs. (A and B) Confirmation of ASFV infection in PAMs. (A) Immunofluorescence detection of the ASFV protein p72 in cells infected with ASFV. (B) Viral titers in the supernatants were measured via a hemadsorption assay and are expressed as log10 TCID50/mL. Data represent the mean value ± SD. (Scale bars, 200 μm.). (C) Read counts obtained from Oxford Nanopore RNA sequencing of total RNA from ASFV-infected PAMs. (D) Distribution of read lengths at 24 and 36 hpi. (E) Correlation between host mRNA length and read alignment coverage. (F) Correlation of transcript coverage in ASFV-infected PAMs at 24 hpi and 36 hpi. Log2-transformed coverage was calculated using non-overlapping 50-bp genomic bins (orange points). The red line represents the linear regression fit (Pearson’s r = 0.83). (G) Nanopore long-read sequencing combined with direct RNA sequencing enables the alignment of full-length reads to the viral genome, allowing accurate mapping of TSSs and TTSs.

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  Fig 2. Transcriptional landscape of ASFV. Mapped locations of TSSs and TTSs across the ASFV genome are shown. The novel transcripts are indicated by red lines, and the novel gene predictions are marked with red triangles. Data were obtained from long-read Nanopore RNA sequencing of infected PAMs and illustrate the complex architecture of ASFV gene expression.

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  Fig 3. Identification of transcriptional “read-through”, novel genes, and characterization of transcription regulatory motifs in ASFV. (A) Nanopore long-read sequencing reveals transcriptional “read-through” at the MGF_360-1L locus. The top panel displays the RNA-seq coverage track, while the bottom panel shows individual long-read alignments. Transcription initiates at a primary transcription start site (TSS 1946) but bypasses the canonical transcription termination site (TTS 794) to terminate at a distal site (TTS 407), generating multiple RNA transcripts of varying lengths. (B) RT-PCR validation of the long transcripts. Agarose gel electrophoresis confirms the continuous transcription across the indicated genomic coordinate spans. The left panel shows PCR products from untreated total RNA, while the right panel displays PCR products from total RNA subjected to oligo(dT) reverse transcription prior to amplification. (C) Schematic representation and RT-PCR validation of the transcriptional expression of predicted novel genes. Red arrows indicate the genomic locations and transcriptional orientations of the putative novel genes. (D) Sequence logo representing the conserved promoter…

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  Fig 4. Validation of potential novel genes and direct RNA sequencing uncovers translational activity, poly(A) dynamics, and RNA modifications of ASFV. (A) The left panel shows a schematic diagram of the IP workflow used to validate the expression of a novel candidate ASFV gene. The right panel shows the results of the Western blot analysis of proteins immunoprecipitated with virus-induced specific antibodies. Lanes indicate the original cell lysate (input), IP using positive infection serum (experimental group), and IP using negative control serum (control group). Target proteins were detected using an anti-ST tag monoclonal antibody. (B) Polysome profiling analysis of PAMs infected with ASFV at an MOI of 0.5 at 24 hours post-infection. Ribosome-bound RNAs were separated via 10–50% sucrose gradient ultracentrifugation. (C) DRS read statistics for ribosome-bound mRNAs. (D) Density distribution plot of poly(A) tail lengths for virally transcribed RNAs, categorized by their respective types. Arrows indicate the primary and secondary peaks located at ~42 nt and ~24 nt, respectively. (E and F) Representative raw nanopore squiggle plots displaying normalized ionic current signals across…

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  Fig 5. Construction of recombinant ASFV viruses ASFV-ΔMGF110-14L-promoter and ASFV-ΔI267L-promoter, and validation of RNA produced from transcription of multicistronic transcripts. (A-B) Generation of recombinant viruses with targeted promoter deletion and corresponding transcript structure schematics. (C) Fluorescence microscopy imaging of recombinant virus-infected PAMs. (Scale bars, 200 μm.). (D) PCR detection of purified and isolated recombinant virus genomes. The ASFV-ΔMGF110-14L-promoter (black) and the ASFV-ΔI267L-promoter (red) were tested to confirm the presence of the expected DNA fragments after deleting their promoters and inserting an eGFP-Pp72 cassette. The band sizes on the gel matched the expected fragment sizes of the recombinant virus genomes, confirming successful modification. The absence of wild-type ASFV bands verified that the recombinant viruses contained no wild-type contamination. (Left panel) PCR detection specific to the wild-type ASFV genome; (Right panel) PCR detection specific to the recombinant viral genomes. (E) RT‒PCR analysis of long transcripts spanning adjacent genes at the viral recombination sites. Blue indicates the detection of transcripts…

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  Fig 6. Validation of the expression of multicistronic downstream ORFs and potential novel viral proteins via eGFP-fusion recombinant viruses. (A) Construction of recombinant viruses harboring in-frame eGFP fusions targeted immediately upstream of the stop codons of the downstream multicistronic ORFs (MGF_110-11L and E66L). (B) Construction of recombinant viruses harboring in-frame eGFP fusions targeted immediately upstream of the stop codons of the potential novel viral genes (16848-F and 180627-R). (C) Representative fluorescence microscopy images showing the expression of eGFP-fusion proteins in PAMs infected with the indicated recombinant ASFV strains (ASFV-ΔMGF110-11L-IneGFP, ASFV-ΔE66L-IneGFP, ASFV-Δ16848-F-IneGFP, and ASFV-Δ180627-R-IneGFP). Scale bars: 200 μm. (D) RT-PCR validation of the target gene-eGFP junctions reveals that the transcripts of multicistronic downstream ORFs and potential novel viral genes in the recombinant viruses contain the eGFP sequence, generating transcripts containing the target gene–eGFP junction. Total RNA was extracted from PAMs infected with the respective recombinant viruses. Left: PCR amplification using non-reverse-transcribed RNA as template…