A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication.
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.
Yeung
Man Lung
ML
Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460, USA.
Houzet
Laurent
L
Yedavalli
Venkat S R K
VS
Jeang
Kuan-Teh
KT
D000951:Antigens, Neoplasm / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics; D063326:Exosome Multienzyme Ribonuclease Complex; D005434:Flow Cytometry; D015723:Gene Library; D015894:Genome, Human; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D019169:Jurkat Cells; D051938:Munc18 Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D056921:Nuclear Receptor Coactivator 3; D051266:Nuclear Respiratory Factor 1 / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D004268:DNA-Binding Proteins; C467564:EXOSC5 protein, human; D016655:HIV Core Protein p24; D051938:Munc18 Proteins; C495633:NRF1 protein, human; D009687:Nuclear Proteins; D051266:Nuclear Respiratory Factor 1; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C497940:STXBP2 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D011495:Histone-Lysine N-Methyltransferase; C093563:PRDM2 protein, human; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex