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

Landi A, Vermeire J, Iannucci V, Vanderstraeten H, Naessens E, Bentahir M, Verhasselt B - Retrovirology (2014)

Bottom Line: 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.The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. alessia.landi@ugent.be.

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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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.
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Fig3: 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.

Mentions: To confirm the biological relevance of the selected gene candidates, we used an independent validation strategy using different shRNA sequences (Mission RNAi consortium) to target the genes identified during the screening. For each of the selected genes, we individually evaluated 2 to 5 lentiviral clones, each expressing different shRNA sequences to target the gene transcript (depending on the validation status and availability by the supplier). The validation set-up involved two different approaches: the first approach (validation 1) was a confirmation of the hits in HeLa CD4++ using Nef as an inducer of CD4 down-modulation. Screening hits confirmed in HeLa cells were subsequently investigated in a more relevant system (validation 2): SupT1 T cells infected with HIV-1 engineered to express an eGFP reporter gene (Table 1). Our final interest is primarily in host proteins that are important for CD4 trafficking in HIV-1-infected cells, but in epithelial cells such as HeLa cells, CD4 trafficking might differ in some aspects from that in T cells, the main targets of HIV-1 [18]. In validation 1, we confirmed that knock-down of 20 genes out of 75 selected hits affected CD4 down-regulation (see Figure 2). These genes were further evaluated in validation 2. These 20 selected genes are briefly described in Additional file 2. In validation 2, cell surface expression of CD4 was analyzed two, four and seven days after HIV infection of SupT1 cells expressing shRNA targeting one of these 20 genes. For this, we used an HIV NL4-3 construct expressing all the HIV genes and a GFP marker gene expressed in frame with Nef, separated through an IRES sequence. The residual CD4 expression in the HIV-infected fraction (eGFP+) compared to uninfected cells in the same culture (eGFP−) was calculated as described in Methods. Seven genes were found to significantly contribute to CD4 down-regulation in HIV-infected SupT1 (Figure 3A). These genes are: DNM3 (dynamin 3; 1.7 fold increase of CD4 residual expression compared to non-targeting shRNA-expressing cells), SNX22 (sorting nexin 22; 1.5 fold), HRBL (HIV-Rev binding protein Like; 1.6 fold), IDH3G (Isocitrate dehydrogenase; 1.9 fold), HSP90B1 (Heat shock protein 90 kDa beta member 1; 1.3 fold), EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15; 4 fold) and ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1; 1.25 fold). The knock-down efficiencies at mRNA level were determined via qPCR for the selected shRNA clones (see Additional file 3). To investigate whether the effects observed were Nef-dependent we performed two additional sets of experiments, using different viruses and vectors. In the first, we compared CD4 down-modulation, either induced by wild type reporter virus (HIV-1 NL4-3 IRES-EGFP) or by a vpu -deleted derivative virus (HIV-1 NL4-3 IRES-EGFP, dVpu) in SupT1 cells knocked-down for the target genes. The residual CD4 expression in the HIV-infected cells was calculated as described above. The fold increase of CD4-expression in cells knocked down for the target genes relative to the control non-targeting shRNA-expressing cells was comparable between WT and dVpu viruses (Figure 3C), suggesting little contribution of these factors to Vpu-mediated CD4 down-regulation.Table 1


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

Landi A, Vermeire J, Iannucci V, Vanderstraeten H, Naessens E, Bentahir M, Verhasselt B - Retrovirology (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269872&req=5

Fig3: 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.
Mentions: To confirm the biological relevance of the selected gene candidates, we used an independent validation strategy using different shRNA sequences (Mission RNAi consortium) to target the genes identified during the screening. For each of the selected genes, we individually evaluated 2 to 5 lentiviral clones, each expressing different shRNA sequences to target the gene transcript (depending on the validation status and availability by the supplier). The validation set-up involved two different approaches: the first approach (validation 1) was a confirmation of the hits in HeLa CD4++ using Nef as an inducer of CD4 down-modulation. Screening hits confirmed in HeLa cells were subsequently investigated in a more relevant system (validation 2): SupT1 T cells infected with HIV-1 engineered to express an eGFP reporter gene (Table 1). Our final interest is primarily in host proteins that are important for CD4 trafficking in HIV-1-infected cells, but in epithelial cells such as HeLa cells, CD4 trafficking might differ in some aspects from that in T cells, the main targets of HIV-1 [18]. In validation 1, we confirmed that knock-down of 20 genes out of 75 selected hits affected CD4 down-regulation (see Figure 2). These genes were further evaluated in validation 2. These 20 selected genes are briefly described in Additional file 2. In validation 2, cell surface expression of CD4 was analyzed two, four and seven days after HIV infection of SupT1 cells expressing shRNA targeting one of these 20 genes. For this, we used an HIV NL4-3 construct expressing all the HIV genes and a GFP marker gene expressed in frame with Nef, separated through an IRES sequence. The residual CD4 expression in the HIV-infected fraction (eGFP+) compared to uninfected cells in the same culture (eGFP−) was calculated as described in Methods. Seven genes were found to significantly contribute to CD4 down-regulation in HIV-infected SupT1 (Figure 3A). These genes are: DNM3 (dynamin 3; 1.7 fold increase of CD4 residual expression compared to non-targeting shRNA-expressing cells), SNX22 (sorting nexin 22; 1.5 fold), HRBL (HIV-Rev binding protein Like; 1.6 fold), IDH3G (Isocitrate dehydrogenase; 1.9 fold), HSP90B1 (Heat shock protein 90 kDa beta member 1; 1.3 fold), EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15; 4 fold) and ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1; 1.25 fold). The knock-down efficiencies at mRNA level were determined via qPCR for the selected shRNA clones (see Additional file 3). To investigate whether the effects observed were Nef-dependent we performed two additional sets of experiments, using different viruses and vectors. In the first, we compared CD4 down-modulation, either induced by wild type reporter virus (HIV-1 NL4-3 IRES-EGFP) or by a vpu -deleted derivative virus (HIV-1 NL4-3 IRES-EGFP, dVpu) in SupT1 cells knocked-down for the target genes. The residual CD4 expression in the HIV-infected cells was calculated as described above. The fold increase of CD4-expression in cells knocked down for the target genes relative to the control non-targeting shRNA-expressing cells was comparable between WT and dVpu viruses (Figure 3C), suggesting little contribution of these factors to Vpu-mediated CD4 down-regulation.Table 1

Bottom Line: 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.The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University and Ghent University Hospital, De Pintelaan 185, B-9000, Gent, Belgium. alessia.landi@ugent.be.

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.

Show MeSH
Related in: MedlinePlus