Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells

Abstract

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

Figure 1. shRNA screen in Jurkat cells.

A. Schematic representation of the shRNA screen. A pool of shRNA-encoding-lentiviral particles was used to transduce Jurkat cells and after 48 h they were challenged with HIV-HSA. After 7 days of infection, the shRNA transduced cells were negatively selected with magnetic beads conjugated with biotinylated anti-HSA. After 3 rounds of infection/selection, the HIV-1 resistant clones were recovered and isolated. Seven-hundred shRNA Jurkat clones were obtained, expanded and allowed to growth for 2 months to identify cellular proteins essential for HIV-1 replication in Jurkat cells but not essential for the cell viability. We obtained 180 viable shRNA clones. B. Resistance of shRNA clones to HIV-1 replication, measured by p24^CA expression in the cell culture supernatant after 7 days of infection with HIV-1_NL4-3 (MOI of 1). Percentage values are relative to Jurkat cells infected with HIV-1_NL4-3. Values indicated in graph represent the number of clones isolated in each subgroup.

Figure 2. shRNA clones are resistant to HIV-1 replication.

A. Two different shRNA clones for each target gene were infected with HIV-1_NL4-3 (MOI of 1) and after 7 days of infection, HIV-1 replication was measured by p24^CA levels in the cell culture supernatant. Values are relative to Jurkat cells infected with HIV-1_NL4-3 and represent the mean ± SEM (n = 6). *** corresponds to P<0,0001. B. Viability of shRNA clones after 7 days of infection with HIV-1_NL4-3. Values are relative to Jurkat cells infected with HIV-1_NL4-3 and correspond to mean ± SEM (n = 4). C. Immunoblotting of intracellular Gag protein in different shRNA clones after 7 days of HIV-1_NL4-3 infection (MOI of 1). This figure is representative of three independent experiments.

Figure 3. Neutralization of HIV-1 replication by shRNAs is cumulative over time.

A. Kinetics of HIV-1_NL4-3 replication in shRNA Jurkat clones during 7 days of infection. shRNA clones were infected with HIV-1_NL4-3 (MOI of 1) and p24^CA antigen was measured at day 2, 4 and 7. Values are relative to control shSCRAM cells infected with HIV-1 (▪) and represent mean ± SEM (n = 3). B. Immunoblotting of Vif protein in the different shRNA clones after 48 h of HIV-1_NL4-3 infection (MOI of 1). This figure is representative of three independent experiments. C. Flow cytometry analysis of HSA surface expression in shRNA clones infected with HIV-HSA (MOI of 1) during a time course assay of 7 days of infection. Cells were membrane stained with anti-HSA antibody for detection of HIV-1 infection. Percentage of infected cells was analysed by flow cytometry. Values correspond to mean ± SEM (n = 2).

Figure 4. Knockdown of host-proteins do not affect integration but affect an early step in HIV-1 replication.

Jurkat shRNA clones were transduced with EGFP-expressing lentiviral particles (FugW-EGFP) pseudptyped with VSV-G and HIV-1 gp120. After 48 h, EGFP expression was measured by flow cytometry. Values are relative to the percentage of shSCRAM EGFP positive cell and represent mean ± SEM (n = 3). Black bars indicate values for shRNA clones transduced with a VSV-G-lentivirus and white bars indicate values to shRNA clones transduced with a GP120-lentivirus.

Figure 5. HIV-1 LTR-driven transcription is affected by host-proteins.

Transient assays were performed in HeLa-P4 cells co-transfected with pHIV-1_NL4-3 and the different shRNA plasmids. After 48 h, cells were harvested and LTR transcription was measured by quantification of the β-Galactosidase activity in cell lysates. Cell supernatant was also collected to measure viral production by p24^CA ELISA. Black bars indicate values for measurements of β-Galactosidase activity in cell lysates and white bars indicate values for measurements of p24^CA in the supernatant. Values are relative to the control shSCRAM and represent mean ± SEM (n = 3).