title: Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements.
abstract: Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Yme U
authors.initials: YU
authors.lastname: van der Velden
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Wendy
authors.initials: W
authors.lastname: Kleibeuker
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Harwig
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Bep
authors.initials: B
authors.lastname: Klaver
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: Siteur-van Rijnstra
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Esmay
authors.initials: E
authors.lastname: Frankin
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address: a.t.das@amc.uva.nl.
authors.forename: Atze T
authors.initials: AT
authors.lastname: Das
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002478:Cells, Cultured; D004318:Doxycycline / Q000378:metabolism*; D029544:Foot-and-Mouth Disease Virus; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008822:Mice, Transgenic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015533:Transcriptional Activation; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D004318:Doxycycline
keywords: 2A peptide; HIS mouse; HIV; IRES; Nef; Protein production; Tet-On system; Ubiquitin