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.2014 Dec 13:11:118.
doi: 10.1186/s12977-014-0118-4.

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

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Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

Alessia Landi et al. Retrovirology..

Abstract

Background: Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation.

Results: To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells.

Conclusions: We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

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Figures

Figure 1
Figure 1
Schematic representation of the genome-wide shRNA screening workflow. HeLa CD4++ cells were transduced with a pooled shRNA lentiviral library. Productively transduced cells were selected with puromycin and transduced with a retroviral vector for the expression of HIV-1 Nef and eGFP as a reporter gene. Cells that maintained high CD4 surface levels despite the expression of Nef (Nef(eGFP)+CD4hi, red box) and the Nef(eGFP)+CD4low population (green box) were sorted by FACS. The shRNA hairpins present in the cDNA obtained from the two sorted populations were amplified by nested PCR with biotinylated primers. The samples obtained were hybridized on different Affymetrix U133 v2-plus microarrays and the results compared. shRNA sequences enriched in the Nef(eGFP)+CD4hi cells were selected and further analyzed with pathway analysis tools to obtain a final list of 55 genes to be further validated together with 20 genes selected from literature.
Figure 2
Figure 2
Results of validation 1: CD4 residual expression in HeLa CD4++ expressing Nef and the shRNAs targeting the selected 20 genes. Bar graph shows the residual CD4 expression in HeLa CD4++ expressing the shRNAs for the knock-down of the 20 selected hits and the Nef retroviral construct. The values are representative of two independent confirmation experiments and for the shRNA clone with most activity among the ones tested. The gray bar indicates the value obtained in cells transduced with the non-targeting scrambled shRNA control.
Figure 3
Figure 3
Effect of the knock-down of the selected genes on CD4 expression in HIV-1-infected SupT1 cells. A. The bar graph shows the residual CD4 surface expression levels in HIV-1-infected (eGFP+) SupT1 cells (calculated as described in Methods) normalized to the non-targeting shRNA scrambled control. Error bars represent standard deviation between 7 to 17 independent experiments. The values are representative of the shRNA clone with most activity among the ones tested. P values: ***p ≤ 0.0004; **p ≤ 0.0099; *p ≤ 0.038.B. Flow cytometry dot plots showing the CD4 surface levels in HIV-1-infected cells in a representative experiment. eGFP is the reporter for HIV-1 infection, while CD4 levels are measured by surface staining with an anti-CD4 monoclonal antibody conjugated to APC. The numbers on the FACS plots indicate the mean fluorescence intensity of CD4 gated on the upper right and lower right quadrants.C. The bar graph shows the residual CD4 surface expression levels in shRNA expressing SupT1 cells normalized to the non-targeting shRNA scrambled controls (calculated as described in Methods), infected with the wild type (black bars, indicated as HIV-1 WT) and the dVpu HIV-1 NL4-3 IRES-EGFP (grey bars, indicated as HIV-1 dVpu). Error bars represent standard deviation between 3 independent experiments.D. The bar graph shows the residual CD4 surface expression levels (calculated as described in Methods) in SupT1 cells transduced by a retroviral vector to express Nef, normalized to the non-targeting shRNA scrambled control. Error bars represent standard deviation between 7 to 16 independent experiments. P values: ***p ≤ 0.0006 ; **p ≤ 0.0011.
Figure 4
Figure 4
Knock-down of some genes involved in CD4 down-regulation decreases replication of HIV-1 in SupT1 cells. A. Knock-down of four genes involved in CD4 down-regulation (EPS15, DNM3, ATP6AP1, IDH3G) decreases HIV-1 replication: graphs show levels of HIV-1 infection at different time points (2, 4 and 7 days post-infection), as measured by the percentage of eGFP expressing cells in the population. The grey lines represent HIV-1 replication in the negative control (cells expressing the non-targeting shRNA), the black line represents cells expressing the target shRNAs. Bar graphs represent the standard deviations for 5 to 6 independent experiments (p values: *p ≤ 0.03, **p ≤0.008).B. Knock-down of HRBL, SNX22, HSP90B1 does not affect HIV-1 replication: graph represent levels of HIV-1 infection as described for Figure 4A.
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References

    1. Pitcher C, Honing S, Fingerhut A, Bowers K, Marsh M. Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol Biol Cell. 1999;10:677–691. doi: 10.1091/mbc.10.3.677. - DOI - PMC - PubMed
    1. Landi A, Iannucci V, Van Nuffel A, Meuwissen P, Verhasselt B. One protein to rule them all: modulation of cell surface receptors and molecules by HIV Nef. Curr HIV Res. 2011;9:496–504. doi: 10.2174/157016211798842116. - DOI - PMC - PubMed
    1. Watkins RL, Zou W, Denton PW, Krisko JF, Foster JL, Garcia JV. In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis. Retrovirology. 2013;10:125. doi: 10.1186/1742-4690-10-125. - DOI - PMC - PubMed
    1. Casartelli N, Di Matteo G, Potesta M, Rossi P, Doria M. CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 nef protein in pediatric AIDS progression. J Virol. 2003;77:11536–11545. doi: 10.1128/JVI.77.21.11536-11545.2003. - DOI - PMC - PubMed
    1. Chen BK, Gandhi RT, Baltimore D. CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef. J Virol. 1996;70:6044–6053. - PMC - PubMed

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