TALEN knockout of the PSIP1 gene in human cells: Analyses of HIV-1 replication and allosteric integrase inhibitor mechanism

Hind J. Fadel, James H. Morrison, Dyana T. Saenz, James R. Fuchs, Mamuka Kvaratskhelia, Stephen C. Ekker, Eric M. Poeschla

Research output: Contribution to journalArticle

51 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)9704-9717
Number of pages14
JournalJournal of virology
Volume88
Issue number17
DOIs
StatePublished - 2014

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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