Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts
Fig 4
Novel fusion transcript between E4orf6 and DBP is expressed, translated, and conserved.
(A) Enlarged transcriptome map of Ad5 E4 and E2A transcriptional units. Promoter transcription start sites are indicated with left-facing arrows, and cleavage and polyadenylation sites (pA) labeled with downward facing arrows. Novel E4-derived transcripts that terminate in E2A are highlighted in red. Ad5 mutant viruses dl1004 and dl355 contain deletions (indicated by hashed boxes) that remove most of the E4 region and splice donors (ΔE4) or a 14-base deletion inside E4orf6 that only abrogates E4orf6 expression (ΔE4orf6). (B) Reverse-transcriptase PCR on cDNA derived from Ad5-infection of A549 cells reveals characteristic bands of both E4orf6/Unk and E4orf6/DBP. L denotes DNA ladder and triangle indicates increasing cDNA concentration. (C) A549 or W162 cells were uninfected (mock) or infected with WT Ad5, ΔE4orf6, or ΔE4 viruses for 40 hours and proteins detected by immunoblot analysis. When blotting with antisera raised against the N-terminus of E4orf6, a prominent band is detected at the predicted size of E4orf6/DBP. This band is absent during ΔE4 infection and not observed in W162 cells where only the E4 region is provided in trans. Kilodalton size markers are shown to the left of each blot. (D) Infections and immunoblot analysis were performed as described in panel C. Two independently derived anti-N-terminal E4orf6 antibodies (RSA3 and M45) detect E4orf6/DBP. (E) Proteins expressed over a time-course of Ad5 infection in A549 were detected by immunoblot analysis at indicated hpi. (F) A549 cells were infected with adenoviruses from four different serotypes in a time-course. All tested adenovirus serotypes express a protein corresponding to E4orf6/DBP. (G) Quantitative reverse-transcriptase PCR was performed to demonstrate mRNA accumulation of Ad5 E1A (a representative early transcript), Fiber (a representative late transcript), and three E4orf6 containing transcript isoforms. Transcripts were normalized to expression level at 48 hpi and internal HPRT1 housekeeping gene.