title: Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing.
abstract: Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1(89.6) produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Ocwieja
authors.forename: Scott
authors.initials: S
authors.lastname: Sherrill-Mix
authors.forename: Rithun
authors.initials: R
authors.lastname: Mukherjee
authors.forename: Rebecca
authors.initials: R
authors.lastname: Custers-Allen
authors.forename: Patricia
authors.initials: P
authors.lastname: David
authors.forename: Michael
authors.initials: M
authors.lastname: Brown
authors.forename: Susana
authors.initials: S
authors.lastname: Wang
authors.forename: Darren R
authors.initials: DR
authors.lastname: Link
authors.forename: Jeff
authors.initials: J
authors.lastname: Olson
authors.forename: Kevin
authors.initials: K
authors.lastname: Travers
authors.forename: Eric
authors.initials: E
authors.lastname: Schadt
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
mesh_terms: D017398:Alternative Splicing; D002460:Cell Line; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D013601:T-Lymphocytes / Q000821:virology
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D012367:RNA, Viral