A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication

J Biol Chem. 2009 Jul 17;284(29):19463-73. doi: 10.1074/jbc.M109.010033. Epub 2009 May 20.


Short interfering RNAs (siRNAs) have been used to inhibit HIV-1 replication. The durable inhibition of HIV-1 replication by RNA interference has been impeded, however, by a high mutation rate when viral sequences are targeted and by cytotoxicity when cellular genes are knocked down. To identify cellular proteins that contribute to HIV-1 replication that can be chronically silenced without significant cytotoxicity, we employed a shRNA library that targets 54,509 human transcripts. We used this library to select a comprehensive population of Jurkat T-cell clones, each expressing a single discrete shRNA. The Jurkat clones were then infected with HIV-1. Clones that survived viral infection represent moieties silenced for a human mRNA needed for virus replication, but whose chronic knockdown did not cause cytotoxicity. Overall, 252 individual Jurkat mRNAs were identified. Twenty-two of these mRNAs were secondarily verified for their contributions to HIV-1 replication. Five mRNAs, NRF1, STXBP2, NCOA3, PRDM2, and EXOSC5, were studied for their effect on steps of the HIV-1 life cycle. We discuss the similarities and differences between our shRNA findings for HIV-1 using a spreading infection assay in human Jurkat T-cells and results from other investigators who used siRNA-based screenings in HeLa or 293T cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Antigens, Neoplasm / genetics
  • Antigens, Surface / genetics
  • Cell Line
  • Cell Proliferation
  • DNA-Binding Proteins / genetics
  • Exoribonucleases / genetics
  • Exosome Multienzyme Ribonuclease Complex
  • Flow Cytometry
  • Gene Library
  • Genome, Human*
  • HIV Core Protein p24 / genetics
  • HIV Core Protein p24 / metabolism
  • HIV-1 / genetics*
  • HIV-1 / growth & development
  • HIV-1 / metabolism
  • HeLa Cells
  • Histone Acetyltransferases / genetics
  • Histone-Lysine N-Methyltransferase
  • Humans
  • Jurkat Cells
  • Munc18 Proteins / genetics
  • Nuclear Proteins / genetics
  • Nuclear Receptor Coactivator 3
  • Nuclear Respiratory Factor 1 / genetics
  • Oligonucleotide Array Sequence Analysis / methods
  • Proteins / genetics*
  • RNA, Messenger / genetics
  • RNA, Small Interfering / genetics*
  • RNA-Binding Proteins
  • Trans-Activators / genetics
  • Transcription Factors / genetics
  • Virus Replication / genetics*


  • Antigens, Neoplasm
  • Antigens, Surface
  • DNA-Binding Proteins
  • EXOSC5 protein, human
  • HIV Core Protein p24
  • Munc18 Proteins
  • NRF1 protein, human
  • Nuclear Proteins
  • Nuclear Respiratory Factor 1
  • Proteins
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • STXBP2 protein, human
  • Trans-Activators
  • Transcription Factors
  • Histone-Lysine N-Methyltransferase
  • PRDM2 protein, human
  • Histone Acetyltransferases
  • NCOA3 protein, human
  • Nuclear Receptor Coactivator 3
  • Exoribonucleases
  • Exosome Multienzyme Ribonuclease Complex