title: TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism.
abstract: HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1, is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4+ T cells, and knockout mice have normal immune systems. RNA interference (RNAi) has been useful for studying LEDGF/p75, but the potent cofactor activity of small protein residua can be confounding. Here, in human cells with utility for HIV research (293T and Jurkat), we used transcription activator-like effector nucleases (TALENs) to completely eradicate all LEDGF/p75 expression. We performed two kinds of PSIP1 knockouts: whole-gene deletion and deletion of the integrase binding domain (IBD)-encoding exons. HIV-1 integration was inhibited, and spreading viral replication was severely impaired in PSIP1-/- Jurkat cells infected at high multiplicity. Furthermore, frameshifting the gene in the first coding exon with a single TALEN pair yielded trace LEDGF/p75 levels that were virologically active, affirming the cofactor's potency and the value of definitive gene or IBD exon segment deletion. Some recent studies have suggested that LEDGF/p75 may participate in HIV-1 assembly. However, we determined that assembly of infectious viral particles is normal in PSIP1-/- cells. The potency of an allosteric integrase inhibitor, ALLINI-2, for rendering produced virions noninfectious was also unaffected by total eradication of cellular LEDGF/p75. We conclude that HIV-1 particle assembly and the main ALLINI mechanism are LEDGF/p75 independent. The block to HIV-1 propagation in PSIP1-/- human CD4+ T cells raises the possibility of gene targeting PSIP1 combinatorially with CCR5 for HIV-1 cure.
  
  
  OBJECTIVE
  LEDGF/p75 dependence is universally conserved in the retroviral genus Lentivirus. Once inside the nucleus, lentiviral preintegration complexes are thought to attach to the chromosome when integrase binds to LEDGF/p75. This tethering process is largely responsible for the 2-fold preference for integration into active genes, but the cofactor's full role in the lentiviral life cycle is not yet clear. Effective knockdowns are difficult because even trace residua of this tightly chromatin-bound protein can support integration cofactor function. Here, in experimentally useful human cell lines, we used TALENs to definitively eradicate LEDGF/p75 by deleting either all of PSIP1 or the exons that code for the integrase binding domain. HIV-1 replication was severely impaired in these PSIP1 knockout cells. Experiments in these cells also excluded a role for LEDGF/p75 in HIV-1 assembly and showed that the main ALLINI mechanism is LEDGF/p75 independent. Site-specific gene targeting of PSIP1 may have therapeutic potential for HIV-1 disease.
authors.affiliation: Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA Department of Medicine, Division of Infectious Diseases, Mayo Clinic College of Medicine, Rochester, Minnesota, USA fadel.hind@mayo.edu poeschla.eric@mayo.edu.
authors.forename: Hind J
authors.initials: HJ
authors.lastname: Fadel
authors.affiliation: Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: Morrison
authors.affiliation: Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
authors.forename: Dyana T
authors.initials: DT
authors.lastname: Saenz
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Fuchs
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Ekker
authors.affiliation: Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA Department of Medicine, Division of Infectious Diseases, Mayo Clinic College of Medicine, Rochester, Minnesota, USA fadel.hind@mayo.edu poeschla.eric@mayo.edu.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Poeschla
mesh_terms: D002460:Cell Line; D055786:Gene Knockout Techniques; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics*; D019065:Virus Assembly; D016662:Virus Integration; D014779:Virus Replication
chemical_list: C108381:GEMIN2 protein, human; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1