title: Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation.
abstract: BACKGROUND
  Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation.
  
  
  RESULTS
  To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells.
  
  
  CONCLUSIONS
  We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. alessia.landi@ugent.be.
authors.forename: Alessia
authors.initials: A
authors.lastname: Landi
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. jolien.vermeire@ugent.be.
authors.forename: Jolien
authors.initials: J
authors.lastname: Vermeire
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. veronica.iannucci@mssm.edu.|Present address: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. veronica.iannucci@mssm.edu.
authors.forename: Veronica
authors.initials: V
authors.lastname: Iannucci
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. hanne.vanderstraeten@ugent.be.
authors.forename: Hanne
authors.initials: H
authors.lastname: Vanderstraeten
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. evelien.naessens@ugent.be.
authors.forename: Evelien
authors.initials: E
authors.lastname: Naessens
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. mostafa.bentahir@uclouvain.be.|Present address: Centre de Technologies Moléculaires Appliquées, Ecole de Santé Publique, Brussels, Belgium. mostafa.bentahir@uclouvain.be.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Bentahir
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. bruno.verhasselt@ugent.be.
authors.forename: Bruno
authors.initials: B
authors.lastname: Verhasselt
mesh_terms: D015704:CD4 Antigens / Q000096:biosynthesis*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology*; D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis; D005786:Gene Expression Regulation; D005820:Genetic Testing; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D014779:Virus Replication
chemical_list: D015704:CD4 Antigens; D034741:RNA, Small Interfering