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Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef.

Matheson NJ, Sumner J, Wals K, Rapiteanu R, Weekes MP, Vigan R, Weinelt J, Schindler M, Antrobus R, Costa AS, Frezza C, Clish CB, Neil SJ, Lehner PJ - Cell Host Microbe (2015)

Bottom Line: SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS.We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis.Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK. Electronic address: njm25@cam.ac.uk.

No MeSH data available.


Related in: MedlinePlus

CoRe Metabolomics of Proliferating T Cells and Identification of Alanine Transport by SNAT1(A) Workflow of CoRe metabolomics experiment.(B) SNAT1 knockdown for CoRe metabolomics experiment. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNAs. After purification by AFMACS (Figure S6A), cells were either rested or re-stimulated with CD3/CD28 Dynabeads, then immunoblotted at 48 hr.(C) Defective proliferation of SNAT1-depleted primary T cells. Re-stimulated cells from (B) were seeded at equal densities and viable cells enumerated at the indicated time points using CytoCount beads. Data were obtained in triplicate. ∗∗p < 0.01. No difference in cell size between the two populations was seen by flow cytometry (Figure S6B).(D) CoRe metabolomic analysis of control and SNAT1-depleted primary T cells. Metabolite compositions of culture supernatants from (C) were determined by LC-MS at baseline, 24, and 48 hr. Data were obtained in triplicate, and Principal component analysis was used to compare net consumption or release of metabolites by control and SNAT1-depleted cells (upper panels). 95% confidence regions are shown. p values for differences in consumption or release of individual metabolites at 48 hr are shown on a negative log scale (middle panel). Net consumption or release of alanine and glutamine is shown scaled to a maximum change of 1 (lower panels). ∗∗p < 0.01.(E and F) Impaired alanine uptake by primary T cells depleted of SNAT1 by shRNA (E) or Vpu (F). Cells from Figures 3G–3H were re-stimulated for 48 hr with CD3/CD28 Dynabeads and uptake (counts per minutes; CPM) of 3H-alanine measured at time points from 30 s to 5 min. 3H-alanine transport in the presence of MeAIB is included as a control, and MeAIB-inhibitable uptake is highlighted (black arrows). 95% confidence bands on linear regression lines (indicating rates of uptake) are shown in gray. ∗∗p < 0.01.See also Figure S6.
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fig5: CoRe Metabolomics of Proliferating T Cells and Identification of Alanine Transport by SNAT1(A) Workflow of CoRe metabolomics experiment.(B) SNAT1 knockdown for CoRe metabolomics experiment. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNAs. After purification by AFMACS (Figure S6A), cells were either rested or re-stimulated with CD3/CD28 Dynabeads, then immunoblotted at 48 hr.(C) Defective proliferation of SNAT1-depleted primary T cells. Re-stimulated cells from (B) were seeded at equal densities and viable cells enumerated at the indicated time points using CytoCount beads. Data were obtained in triplicate. ∗∗p < 0.01. No difference in cell size between the two populations was seen by flow cytometry (Figure S6B).(D) CoRe metabolomic analysis of control and SNAT1-depleted primary T cells. Metabolite compositions of culture supernatants from (C) were determined by LC-MS at baseline, 24, and 48 hr. Data were obtained in triplicate, and Principal component analysis was used to compare net consumption or release of metabolites by control and SNAT1-depleted cells (upper panels). 95% confidence regions are shown. p values for differences in consumption or release of individual metabolites at 48 hr are shown on a negative log scale (middle panel). Net consumption or release of alanine and glutamine is shown scaled to a maximum change of 1 (lower panels). ∗∗p < 0.01.(E and F) Impaired alanine uptake by primary T cells depleted of SNAT1 by shRNA (E) or Vpu (F). Cells from Figures 3G–3H were re-stimulated for 48 hr with CD3/CD28 Dynabeads and uptake (counts per minutes; CPM) of 3H-alanine measured at time points from 30 s to 5 min. 3H-alanine transport in the presence of MeAIB is included as a control, and MeAIB-inhibitable uptake is highlighted (black arrows). 95% confidence bands on linear regression lines (indicating rates of uptake) are shown in gray. ∗∗p < 0.01.See also Figure S6.

Mentions: To identify endogenous SNAT1 substrates in primary human CD4+ T cells in an unbiased fashion, we combined an “activation-rest” strategy for shRNA knockdown (Monroe et al., 2014) with Consumption and Release (CoRe) metabolomics (Jain et al., 2012) (Figure 5A). Pure populations of transduced cells expressing control or SNAT1-specific shRNAs were generated by AFMACS (Figures 5B and S6A) (Matheson et al., 2014). After resting for 7–10 days, control and SNAT1-depleted cells were re-stimulated using CD3/CD28 Dynabeads. A marked reduction in proliferation of SNAT1-depleted cells was observed (Figures 5C and S6A). Culture supernatants were sampled at baseline, 24, and 48 hr, and extracellular metabolite fluxes were calculated on a per-cell basis. In total, data for consumption and release of 126 metabolites, including 19 natural amino acids, were used to derive CoRe metabolomic profiles of control and SNAT1-depleted cells. Principal component analysis readily distinguished these profiles, particularly at 48 hr (Figure 5D, upper panels). Surprisingly, across all measured metabolites, the most significant difference was in net alanine release, with no difference in net glutamine consumption (Figure 5D, middle and lower panels).


Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef.

Matheson NJ, Sumner J, Wals K, Rapiteanu R, Weekes MP, Vigan R, Weinelt J, Schindler M, Antrobus R, Costa AS, Frezza C, Clish CB, Neil SJ, Lehner PJ - Cell Host Microbe (2015)

CoRe Metabolomics of Proliferating T Cells and Identification of Alanine Transport by SNAT1(A) Workflow of CoRe metabolomics experiment.(B) SNAT1 knockdown for CoRe metabolomics experiment. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNAs. After purification by AFMACS (Figure S6A), cells were either rested or re-stimulated with CD3/CD28 Dynabeads, then immunoblotted at 48 hr.(C) Defective proliferation of SNAT1-depleted primary T cells. Re-stimulated cells from (B) were seeded at equal densities and viable cells enumerated at the indicated time points using CytoCount beads. Data were obtained in triplicate. ∗∗p < 0.01. No difference in cell size between the two populations was seen by flow cytometry (Figure S6B).(D) CoRe metabolomic analysis of control and SNAT1-depleted primary T cells. Metabolite compositions of culture supernatants from (C) were determined by LC-MS at baseline, 24, and 48 hr. Data were obtained in triplicate, and Principal component analysis was used to compare net consumption or release of metabolites by control and SNAT1-depleted cells (upper panels). 95% confidence regions are shown. p values for differences in consumption or release of individual metabolites at 48 hr are shown on a negative log scale (middle panel). Net consumption or release of alanine and glutamine is shown scaled to a maximum change of 1 (lower panels). ∗∗p < 0.01.(E and F) Impaired alanine uptake by primary T cells depleted of SNAT1 by shRNA (E) or Vpu (F). Cells from Figures 3G–3H were re-stimulated for 48 hr with CD3/CD28 Dynabeads and uptake (counts per minutes; CPM) of 3H-alanine measured at time points from 30 s to 5 min. 3H-alanine transport in the presence of MeAIB is included as a control, and MeAIB-inhibitable uptake is highlighted (black arrows). 95% confidence bands on linear regression lines (indicating rates of uptake) are shown in gray. ∗∗p < 0.01.See also Figure S6.
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fig5: CoRe Metabolomics of Proliferating T Cells and Identification of Alanine Transport by SNAT1(A) Workflow of CoRe metabolomics experiment.(B) SNAT1 knockdown for CoRe metabolomics experiment. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNAs. After purification by AFMACS (Figure S6A), cells were either rested or re-stimulated with CD3/CD28 Dynabeads, then immunoblotted at 48 hr.(C) Defective proliferation of SNAT1-depleted primary T cells. Re-stimulated cells from (B) were seeded at equal densities and viable cells enumerated at the indicated time points using CytoCount beads. Data were obtained in triplicate. ∗∗p < 0.01. No difference in cell size between the two populations was seen by flow cytometry (Figure S6B).(D) CoRe metabolomic analysis of control and SNAT1-depleted primary T cells. Metabolite compositions of culture supernatants from (C) were determined by LC-MS at baseline, 24, and 48 hr. Data were obtained in triplicate, and Principal component analysis was used to compare net consumption or release of metabolites by control and SNAT1-depleted cells (upper panels). 95% confidence regions are shown. p values for differences in consumption or release of individual metabolites at 48 hr are shown on a negative log scale (middle panel). Net consumption or release of alanine and glutamine is shown scaled to a maximum change of 1 (lower panels). ∗∗p < 0.01.(E and F) Impaired alanine uptake by primary T cells depleted of SNAT1 by shRNA (E) or Vpu (F). Cells from Figures 3G–3H were re-stimulated for 48 hr with CD3/CD28 Dynabeads and uptake (counts per minutes; CPM) of 3H-alanine measured at time points from 30 s to 5 min. 3H-alanine transport in the presence of MeAIB is included as a control, and MeAIB-inhibitable uptake is highlighted (black arrows). 95% confidence bands on linear regression lines (indicating rates of uptake) are shown in gray. ∗∗p < 0.01.See also Figure S6.
Mentions: To identify endogenous SNAT1 substrates in primary human CD4+ T cells in an unbiased fashion, we combined an “activation-rest” strategy for shRNA knockdown (Monroe et al., 2014) with Consumption and Release (CoRe) metabolomics (Jain et al., 2012) (Figure 5A). Pure populations of transduced cells expressing control or SNAT1-specific shRNAs were generated by AFMACS (Figures 5B and S6A) (Matheson et al., 2014). After resting for 7–10 days, control and SNAT1-depleted cells were re-stimulated using CD3/CD28 Dynabeads. A marked reduction in proliferation of SNAT1-depleted cells was observed (Figures 5C and S6A). Culture supernatants were sampled at baseline, 24, and 48 hr, and extracellular metabolite fluxes were calculated on a per-cell basis. In total, data for consumption and release of 126 metabolites, including 19 natural amino acids, were used to derive CoRe metabolomic profiles of control and SNAT1-depleted cells. Principal component analysis readily distinguished these profiles, particularly at 48 hr (Figure 5D, upper panels). Surprisingly, across all measured metabolites, the most significant difference was in net alanine release, with no difference in net glutamine consumption (Figure 5D, middle and lower panels).

Bottom Line: SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS.We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis.Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK. Electronic address: njm25@cam.ac.uk.

No MeSH data available.


Related in: MedlinePlus