title: HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome.
abstract: To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein- but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Baeyens
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Naessens
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Anouk
authors.initials: A
authors.lastname: Van Nuffel
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Karin E
authors.initials: KE
authors.lastname: Weening
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Reilly
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Eva
authors.initials: E
authors.lastname: Claeys
authors.affiliation: HIV Translational Research Unit, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University and Ghent University Hospital, Ghent, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Trypsteen
authors.affiliation: HIV Translational Research Unit, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University and Ghent University Hospital, Ghent, Belgium.
authors.forename: Linos
authors.initials: L
authors.lastname: Vandekerckhove
authors.affiliation: VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Sven
authors.initials: S
authors.lastname: Eyckerman
authors.affiliation: VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Bruno
authors.initials: B
authors.lastname: Verhasselt
mesh_terms: D017398:Alternative Splicing / Q000502:physiology*; D002460:Cell Line; D016679:Genome, Viral / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059370:RNA Folding / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1