Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Alexander M. Price; Katharina E. Hayer; Daniel P. Depledge; Angus C. Wilson; Matthew D. Weitzman

AdV are capable of infecting non-dividing cells and reprogramming cellular processes for 48 productive viral infection. This rewiring involves a highly regulated cascade of viral gene 49 expression over various kinetic classes [5] . The first viral gene to be expressed after infection is 50 E1A, a multi-functional transcription factor that activates downstream viral transcription, liberates 51 E2F from RB proteins, as well as alters host transcriptional responses to the virus [11] [12] [13] [14] . While 52 all E1A molecules have identical 5' and 3' nucleotide sequences, splicing of differently sized 53 internal introns allows for the production of discrete proteins that lack specific functional domains 54 conserved across serotypes [15] . Early after infection, E1A is expressed mainly as large and small 55 isoforms, but later in infection alternative splicing leads to the production of a 9 Svedberg E1A 56 isoform (E1A-9s) as well as low abundance doubly-spliced E1A-11s and E1A-10s. The second 57 viral gene to be activated is E1B, consisting of predominantly two spliced isoforms producing 19-58 kilodalton and 55-kilodalton proteins, with two less abundant isoforms generating putative ORFs 59 of 156 and 93 residues [16] . While E1B-19K acts to block cellular apoptosis [17] , E1B-55K is 60 polyadenylation sites leading to E3A and E3B transcription units, and these gene products are 69 primarily involved in modulating the host innate immune system [28] [29] [30] . Like E1A, the E4 region 70 on the reverse strand has identical 5' and 3' regions, and encodes up to six ORFs by removal of 71 a first intron of varying length. E4 region transcripts encode for multifunctional proteins that are 72 involved in regulation of transcription, splicing, and translation of viral RNAs, as well as 73