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

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.

FIGURE 1.

A screening strategy using a shRNA library in a genome-wide identification of candidates important for HIV-1 replication in Jurkat cells. A, a VSV-G packaged shRNA virus (black) library was used to transduce Jurkat cells. Puromycin selection was employed to cull cells that did not integrate stably a shRNA. Clones that integrated a shRNA that targets a gene whose loss elicits cytotoxicity would similarly be eliminated. After selection and propagation, the shRNA Jurkat clones were divided into two pools, one infected with high titer HIV-1 NL4-3 (red) and the other carried as control. In the infected pool, only cells that have acquired a shRNA that knocked down a cellular mRNA important for HIV-1 replication would be protected from virus-induced lytic cell death. Small RNAs were isolated from survivor cells, and RT-PCR was performed using a specially designed biotin-labeled reverse primer. λ-Exonuclease was then applied to remove the non-biotin-labeled strand from the duplex. The remaining biotin-labeled strand was then hybridized to Affymetrix microarrays and then labeled with Cy3. By comparing the data extracted from microarrays of HIV-1-infected and uninfected samples, shRNAs enriched in the HIV-1-infected Jurkat cells identified virus-important candidate genes. B, confirmation of the representative complexity of shRNA virus library transduced Jurkat cells. To determine the shRNA complexity represented in the transduced cells (Transduction 1 and 2), RNAs were extracted and hybridized to microarrays as described in A. Results from the microarray hybridizations are represented as linear heat maps. The microarray results compared virus library-transduced cells (Transduction 1 and 2) with the starting vector library (Library 1 and 2). As a negative control (Background neg Control), Jurkat mock transduced cells were used in the same microarray analysis. Example raw microarrays images from each of the categories (Transduction, Library, and Background neg Control) are shown on the right. Spot density varied with the intensity threshold of detection.

FIGURE 2.

Verification of the effect from selected candidate genes on HIV-1 replication. A, Jurkat cells were stably transduced with the indicated individual shRNA, and the resulting cell clones were tested for their restriction of HIV-1 replication. Successful restriction of HIV-1 replication in the shRNA clones was shown by a reduction in RT activity in the cell culture supernatants compared with infection of control Jurkat cells (set as 1). The dotted line indicates a 50% reduction in RT activity achieved in the clonal cells. 63 selected clones (as discussed in the text) are shown. B, RT-PCR confirmation of the specific knockdown of the targeted mRNAs. The indicated mRNAs in selected clones were quantified by RT-PCR using gene-specific primers; the data were normalized to cellular 18 S RNA. The RT-PCR amount for each of the indicated mRNA in the parental Jurkat cell was set as 1.

FIGURE 3.

Flow cytometric analysis of cell surface CD4 in shRNA Jurkat clones. Expression of cell surface CD4 in selected shRNA Jurkat clones (PRMT3, GALC, STXBP2, SNX10, EPAS1, PRDM2, NRF1, NCOA3, and EXOSC5) was compared with that of normal Jurkat cells. An IgG_κ1 isotype control phycoerythrin staining of Jurkat cells was used as a negative control. Dotted lines are drawn to indicate the peak background intensity of the isotype control. MFI, mean fluorescent intensity.

FIGURE 4.

CCK-8 measurement of the proliferation of Jurkat and shRNA Jurkat clones. The CCK-8 assay measures the dehydrogenase metabolic activity of proliferating cells. The 450 nm absorbance increases proportionally with the number of dividing cells. The indicated shRNA Jurkat clones (EXOSC5, CAPN3, HTR4, BTF3, PRMT3, GALC, PVALB, FGF7, STXBP2, CLDN3, ACT6LB, SNX10, PRDM2, CTBP1, NRF1, CREBP3, and NR2F1) showed CCK-8 profiles similar to that from control Jurkat cells (boxed).

FIGURE 5.

Analyses of the function contributed by candidate genes to the replicative life cycle of HIV-1. A, knockdown of NRF1 reduced the cell surface expression of HIV-1 co-receptor CXCR4. CXCR4 expression from the average values of three independent NRF1-shRNA clones was compared with parental Jurkat cells (set as 100) and with the average values from three of the other indicated shRNA clones. B, two different STXBP2-shRNA clones (A35 and A36) illustrate the reduced amount of reverse transcribed proviral DNA after HIV-1 infection. The proviral DNA level of infected control Jurkat cells was set as 100. Other shRNA clones, as indicated, did not show significant reduction. C, reduced HIV-1 transcription was seen in NCOA3 and PRDM2 shRNA clones compared with control Jurkat and the other indicated clones. Values are the averages from three independent clones. D, EXOSC5-shRNA cell clone showed a changed profile of transfected HIV-1 GFP-Gag. A “speckled” pattern for GFP-Gag was seen in the parental Jurkat cell (upper panel), whereas in an EXOSC5-shRNA clone a diffuse amorphous EGF-Gag pattern was visualized (lower panel). Images shown in the upper and lower panels represent “slices” from a single cell.

FIGURE 6.

Overexpression reconstitution of the cognate mRNAs targeted by shRNA knockdown rescued HIV-1 replication in shRNA Jurkat clones. The overexpression of the cognate full-length cDNA, but not a control vector plasmid, increased HIV-1 replication as measured by supernatant RT production (top) and p24 quantification (bottom) in the indicated shRNA Jurkat clones.