Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses
Fig 5
Detection of novel, conserved splicing events that expand PyV coding capacity.
A-D. Schematics illustrating identified ORFs. Each row is a reading frame (except for ST and the LT 1st exon, which are in the same frame), and unannotated amino acids are represented by grey boxes. The measured intron is indicated by the red arrow. Colored ORFs are annotated, while grey ORFs are unannotated. Percentages on the right side of the Fig are the percentage of spliced viral transcripts on the same strand as determined from short-read short-RNAseq (total) data. Numbers after each virus name indicate the transcript class within each short-RNAseq (total) dataset. The measured intron is indicated by the red arrow. A) ST2: This ORF is generating from a splicing event that uses the LT first exon donor and an acceptor within the ST ORF. In HPyV7 and BKPyV Dunlop, the splice lands in frame and results in an internal deletion within ST. In MPyV and MCPyV the splice lands out of frame, resulting in an ORF that contains the N-terminal region of ST and novel amino acids at the C terminus. B) MT: MPyV encodes a MT following splicing connecting the end of the ST ORF with an ORF in an alternate frame of the LT second exon. In BKPyV, a similar splice occurs connecting ST with an MT-like ORF in an alternative frame of the LT second exon. C) VP1X: JCPyV encodes two VP1X ORFs generated by splicing within VP1 and landing in an alternative frame of VP1, or earlier in the late region due to wraparound transcription. While predominant in JCPyV, VP1X is likewise present in many other PyV. D) superT: The superT-specific splice utilizes the splice donor canonically associated with truncated T antigens such as 17kT in SV40 and truncT in BKPyV. Due to wraparound transcription, a LT second exon acceptor is available to the 3’ of this donor and acts as the acceptor. For the superT ORF to form, an initial LT splice is required. Ultimately, superT contains a duplication in part of the LT second exon that includes the RB-binding LxCxE motif. E. Schematics detailing BKPyV Dik isolates used for querying the existence of superT. BKPyV WT is wild type virus. M1 contains a LT intron that has been replaced with an intron from the plasmid pCI. Both WT and M1 are expected to generate LT and superT of expected sizes. M2 has a completely removed LT intron, and the pCI intron is located directly 5’ of the LT ORF. M2 is expected to encode LT of expected size, but a larger superT variant due to incorporation of a second copy of the LT first exon. F. Western blot of cells infected with BKPyV Dik WT, M1, or M2 and probed with an antibody reactive against LT. The lower molecular weight band is LT, and the higher molecular weight bands are consistent with superT.