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

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

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  • 1Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460, USA.

Abstract

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.

PMID:
19460752
[PubMed - indexed for MEDLINE]
PMCID:
PMC2740572
Free PMC Article

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