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Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells.

Trinité B, Chan CN, Lee CS, Mahajan S, Luo Y, Muesing MA, Folkvord JM, Pham M, Connick E, Levy DN - PLoS ONE (2014)

Bottom Line: CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation.Partial T cell activation was further evident as an increase in CD69 expression.Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Science, New York University College of Dentistry, New York, New York, United States of America.

ABSTRACT
HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.

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Foxo1 inhibitor AS1842856 accelerates HIV-1 expression.AS1842856 (AS) was applied to cells 1 day before and 1 day after infection with single round GFP reporter virus. CD45RO- naïve T cells are shown. A. GFP and CD62L expression in infected naïve CD4+ T cells on the indicated day after infection. Donor A is shown. B. The number of GFP+ cells on the indicated day after infection. Data are average and SD of the two donors. C. Mean fluorescence intensity (MFI) of GFP in the GFP+ cells. Data are average and SD of the two donors. D. Fold increase in the number and the MFI of GFP+ cells, and total GFP fluorescence in the cells calculated as the product of the number of GFP+ cells multiplied by their MFI. Data are average and SD of the two donors. E. Near-full length reverse transcripts on day 2 after infection for one representative cell donor. Data are averages and SD for replicate PCR reactions for each donor. F. HIV-1 fully spliced RNA presented relative to day 1 DMSO condition. Data are averages and SD for replicate PCR reactions for each donor. G. Virus production measured by genomic RNA in culture medium. Data are averages and SD for replicate PCR reactions for each donor. H. CD62L MFI on the indicated day. Data are MFI of GFP+ cells divided by background staining. Data are average and SD of the two donors. I. CD69 MFI on day 7 post infection on naïve and memory resting CD4+ T cells for donor A.
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pone-0110719-g006: Foxo1 inhibitor AS1842856 accelerates HIV-1 expression.AS1842856 (AS) was applied to cells 1 day before and 1 day after infection with single round GFP reporter virus. CD45RO- naïve T cells are shown. A. GFP and CD62L expression in infected naïve CD4+ T cells on the indicated day after infection. Donor A is shown. B. The number of GFP+ cells on the indicated day after infection. Data are average and SD of the two donors. C. Mean fluorescence intensity (MFI) of GFP in the GFP+ cells. Data are average and SD of the two donors. D. Fold increase in the number and the MFI of GFP+ cells, and total GFP fluorescence in the cells calculated as the product of the number of GFP+ cells multiplied by their MFI. Data are average and SD of the two donors. E. Near-full length reverse transcripts on day 2 after infection for one representative cell donor. Data are averages and SD for replicate PCR reactions for each donor. F. HIV-1 fully spliced RNA presented relative to day 1 DMSO condition. Data are averages and SD for replicate PCR reactions for each donor. G. Virus production measured by genomic RNA in culture medium. Data are averages and SD for replicate PCR reactions for each donor. H. CD62L MFI on the indicated day. Data are MFI of GFP+ cells divided by background staining. Data are average and SD of the two donors. I. CD69 MFI on day 7 post infection on naïve and memory resting CD4+ T cells for donor A.

Mentions: We applied AS1842856 to cells from two donors prior to and one day after infection with a single round pseudotyped virus. We then measured GFP, CD62L and CD69 expression, viral cDNA and RNA within the cells, and de novo virus output (Figure 6). GFP+ cells emerged earlier in the AS1842856 treated cultures, and at two days after infection, the total GFP fluorescence in the culture was enhanced by an order of magnitude, consistent with the interpretation that Foxo1 suppression assists the establishment of infection in cells (Figure 6A–D). Reverse transcription was unaffected by AS1842856 (Figure 6E), but the production of HIV-1 RNA was accelerated (Figure 6F). Interestingly, AS1842856 did not increase the maximum RNA production from the cells but only accelerated the achievement of this peak. Application of AS1842856 to sorted GFP+ cells that had reached their maximum expression had little effect, increasing GFP fluorescence 16% after 3 days (Figure S3 in File S1). CD62L was modestly down-modulated in the GFP-negative (Figure 6A, 6H) and control uninfected cells by a single AS1842856 dosing on day 0 (not shown), which when left untreated increased their CD62L expression over time. GFP+ AS1842856 treated cells down-modulated CD62L more rapidly than untreated cells in parallel with accelerated HIV-1 expression. Repeated dosing with AS1842856 over 9 days resulted in down modulation similar to HIV-1 infection (not shown). AS1842856 synergized with HIV-1 in productively infected cells to increase CD69 expression (Figure 6I), especially in memory cells, indicating early T cell activation. There are no Foxo1 or KLF2 consensus binding sequences in this HIV-1 strain, so these transcription factors are unlikely to be enhancing virus expression through direct binding to the viral promoter.


Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells.

Trinité B, Chan CN, Lee CS, Mahajan S, Luo Y, Muesing MA, Folkvord JM, Pham M, Connick E, Levy DN - PLoS ONE (2014)

Foxo1 inhibitor AS1842856 accelerates HIV-1 expression.AS1842856 (AS) was applied to cells 1 day before and 1 day after infection with single round GFP reporter virus. CD45RO- naïve T cells are shown. A. GFP and CD62L expression in infected naïve CD4+ T cells on the indicated day after infection. Donor A is shown. B. The number of GFP+ cells on the indicated day after infection. Data are average and SD of the two donors. C. Mean fluorescence intensity (MFI) of GFP in the GFP+ cells. Data are average and SD of the two donors. D. Fold increase in the number and the MFI of GFP+ cells, and total GFP fluorescence in the cells calculated as the product of the number of GFP+ cells multiplied by their MFI. Data are average and SD of the two donors. E. Near-full length reverse transcripts on day 2 after infection for one representative cell donor. Data are averages and SD for replicate PCR reactions for each donor. F. HIV-1 fully spliced RNA presented relative to day 1 DMSO condition. Data are averages and SD for replicate PCR reactions for each donor. G. Virus production measured by genomic RNA in culture medium. Data are averages and SD for replicate PCR reactions for each donor. H. CD62L MFI on the indicated day. Data are MFI of GFP+ cells divided by background staining. Data are average and SD of the two donors. I. CD69 MFI on day 7 post infection on naïve and memory resting CD4+ T cells for donor A.
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Related In: Results  -  Collection

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pone-0110719-g006: Foxo1 inhibitor AS1842856 accelerates HIV-1 expression.AS1842856 (AS) was applied to cells 1 day before and 1 day after infection with single round GFP reporter virus. CD45RO- naïve T cells are shown. A. GFP and CD62L expression in infected naïve CD4+ T cells on the indicated day after infection. Donor A is shown. B. The number of GFP+ cells on the indicated day after infection. Data are average and SD of the two donors. C. Mean fluorescence intensity (MFI) of GFP in the GFP+ cells. Data are average and SD of the two donors. D. Fold increase in the number and the MFI of GFP+ cells, and total GFP fluorescence in the cells calculated as the product of the number of GFP+ cells multiplied by their MFI. Data are average and SD of the two donors. E. Near-full length reverse transcripts on day 2 after infection for one representative cell donor. Data are averages and SD for replicate PCR reactions for each donor. F. HIV-1 fully spliced RNA presented relative to day 1 DMSO condition. Data are averages and SD for replicate PCR reactions for each donor. G. Virus production measured by genomic RNA in culture medium. Data are averages and SD for replicate PCR reactions for each donor. H. CD62L MFI on the indicated day. Data are MFI of GFP+ cells divided by background staining. Data are average and SD of the two donors. I. CD69 MFI on day 7 post infection on naïve and memory resting CD4+ T cells for donor A.
Mentions: We applied AS1842856 to cells from two donors prior to and one day after infection with a single round pseudotyped virus. We then measured GFP, CD62L and CD69 expression, viral cDNA and RNA within the cells, and de novo virus output (Figure 6). GFP+ cells emerged earlier in the AS1842856 treated cultures, and at two days after infection, the total GFP fluorescence in the culture was enhanced by an order of magnitude, consistent with the interpretation that Foxo1 suppression assists the establishment of infection in cells (Figure 6A–D). Reverse transcription was unaffected by AS1842856 (Figure 6E), but the production of HIV-1 RNA was accelerated (Figure 6F). Interestingly, AS1842856 did not increase the maximum RNA production from the cells but only accelerated the achievement of this peak. Application of AS1842856 to sorted GFP+ cells that had reached their maximum expression had little effect, increasing GFP fluorescence 16% after 3 days (Figure S3 in File S1). CD62L was modestly down-modulated in the GFP-negative (Figure 6A, 6H) and control uninfected cells by a single AS1842856 dosing on day 0 (not shown), which when left untreated increased their CD62L expression over time. GFP+ AS1842856 treated cells down-modulated CD62L more rapidly than untreated cells in parallel with accelerated HIV-1 expression. Repeated dosing with AS1842856 over 9 days resulted in down modulation similar to HIV-1 infection (not shown). AS1842856 synergized with HIV-1 in productively infected cells to increase CD69 expression (Figure 6I), especially in memory cells, indicating early T cell activation. There are no Foxo1 or KLF2 consensus binding sequences in this HIV-1 strain, so these transcription factors are unlikely to be enhancing virus expression through direct binding to the viral promoter.

Bottom Line: CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation.Partial T cell activation was further evident as an increase in CD69 expression.Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Science, New York University College of Dentistry, New York, New York, United States of America.

ABSTRACT
HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.

Show MeSH
Related in: MedlinePlus