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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 29, pp. 19463–19473, July 17, 2009 Printed in the U.S.A.

A Genome-wide Short Hairpin RNA Screening of Jurkat T-cells for Human Proteins Contributing to Productive HIV-1 Replication*□
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Received for publication, February 9, 2009, and in revised form, April 17, 2009 Published, JBC Papers in Press, May 20, 2009, DOI 10.1074/jbc.M109.010033Man Lung Yeung, Laurent Houzet, Venkat S. R. K. Yedavalli, and Kuan-Teh Jeang1 From the Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460
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 highmutation 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, eachexpressing 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 siRNAbased screenings in HeLa or 293T cells.

The use of RNA interference (RNAi)2 to silencegenes holds potential applications for antiviral therapy (1, 2). RNAi, directed to viral and/or host RNA sequences, has been employed to inhibit HIV-1 replication (3– 6). These approaches are effective over the short term; however, over longer durations, they have elicited either a high rate of escape mutation when viral sequences are targeted or cytotoxicity when cellular genes are chronicallysuppressed (7–10). If cellular genes important for HIV-1 replication (11, 12), but dispensable for host cell viability, could be identified, then silencing the RNAs from these genes may remedy the proclivity

for HIV-1 mutational escapes when viral sequences are targeted. Toward this objective, Brass et al. (13), Konig et al. (14), and Zhou et al. (15) have recently used libraries of presynthesizedsiRNA to knock down transiently HeLa/293T cell RNAs. They reported 272, 278, and 304 annotated gene candidates that contribute to steps in the HIV-1 life cycle. Intriguingly, the identities of the gene candidates discovered by Brass et al. (13), Konig et al. (14), and Zhou et al. (15) are highly divergent with very little overlap when the three studies are compared with each other (16). HeLa and293T cells are highly efficient model cells for siRNA transfection. They are, however, poor representations of T-cells that are physiologically infected by HIV-1. In this respect, human T-cell lines, such as Jurkat, are better models, although the latter cells cannot be transfected efficiently with siRNAs. To perform a genome-wide RNAi screening in Jurkat T-cells, we decided to employ a shRNAlibrary cloned in a retroviral vector. Our approach was to transduce Jurkat cells with the shRNA library and then select for cell clones that are individually integrated for single constitutively expressed shRNA. These shRNA clones are then infected with HIV-1. Because Jurkat T-cells are killed by HIV-1 infection, a cell would survive only if it harbored a shRNA that has knocked down sufficiently a...