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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

Proteomic Analysis of Vpu and Nef Targets and Identification of SNAT1 as a Vpu Substrate(A and B) SILAC-based quantitation of plasma membrane proteins in cells infected with Vpu-deficient (y axis) versus Nef-deficient (x axis) HIV-1 viruses (A) and cells transduced with Vpu (x axis) versus Nef (y axis) as single genes (B). Log2(fold change compared with uninfected [A] or GFP-transduced [B] cells) is shown for proteins from Cluster #35. Figures S3A and S3B selectively enlarge the lower left quadrant of each scatterplot. Proteins identified by >1 unique peptide are shown.(C) SNAT1 depletion by HIV-1 infection. CEM-T4s infected with WT NL4-3-deltaE-EGFP HIV-1 virus in the presence or absence of RTi were immunoblotted at the indicated time points. An MOI of 10 was used, and infection controls are shown in Figure S4C.(D) Rescue of SNAT1 in the absence of Vpu. CEM-T4s infected with WT, Vpu-deficient, or Nef-deficient HIV-1 NL4-3-deltaE-EGFP viruses were immunoblotted at 48 hr. An MOI of 10 was used, and infection controls are shown in Figure S4C.(E) SNAT1 depletion by Vpu. CEM-T4s stably transduced with GFP, Vpu, or Nef were immunoblotted. Untransduced CEM-T4s and CEM-T4s stably transduced with control or SNAT1-specific shRNAs were included as controls.(F) SNAT1 induction in activated primary T cells. Primary human CD4+ T cells activated with CD3/CD28 Dynabeads were immunoblotted at the indicated time points.(G and H) SNAT1 depletion by Vpu in activated primary T cells. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNA or Vpu constructs. After purification by AFMACS (Figure S4F), cells were either rested or re-stimulated with CD3/CD28 Dynabeads and immunoblotted (G) or analyzed by confocal microscopy (H) at 48 hr.See also Figures S3 and S4.
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fig3: Proteomic Analysis of Vpu and Nef Targets and Identification of SNAT1 as a Vpu Substrate(A and B) SILAC-based quantitation of plasma membrane proteins in cells infected with Vpu-deficient (y axis) versus Nef-deficient (x axis) HIV-1 viruses (A) and cells transduced with Vpu (x axis) versus Nef (y axis) as single genes (B). Log2(fold change compared with uninfected [A] or GFP-transduced [B] cells) is shown for proteins from Cluster #35. Figures S3A and S3B selectively enlarge the lower left quadrant of each scatterplot. Proteins identified by >1 unique peptide are shown.(C) SNAT1 depletion by HIV-1 infection. CEM-T4s infected with WT NL4-3-deltaE-EGFP HIV-1 virus in the presence or absence of RTi were immunoblotted at the indicated time points. An MOI of 10 was used, and infection controls are shown in Figure S4C.(D) Rescue of SNAT1 in the absence of Vpu. CEM-T4s infected with WT, Vpu-deficient, or Nef-deficient HIV-1 NL4-3-deltaE-EGFP viruses were immunoblotted at 48 hr. An MOI of 10 was used, and infection controls are shown in Figure S4C.(E) SNAT1 depletion by Vpu. CEM-T4s stably transduced with GFP, Vpu, or Nef were immunoblotted. Untransduced CEM-T4s and CEM-T4s stably transduced with control or SNAT1-specific shRNAs were included as controls.(F) SNAT1 induction in activated primary T cells. Primary human CD4+ T cells activated with CD3/CD28 Dynabeads were immunoblotted at the indicated time points.(G and H) SNAT1 depletion by Vpu in activated primary T cells. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNA or Vpu constructs. After purification by AFMACS (Figure S4F), cells were either rested or re-stimulated with CD3/CD28 Dynabeads and immunoblotted (G) or analyzed by confocal microscopy (H) at 48 hr.See also Figures S3 and S4.

Mentions: Depletion of most known HIV-1 cell surface targets has been attributed to Vpu and/or Nef (Haller et al., 2014; Tokarev and Guatelli, 2011). To assign downregulation of proteins in Cluster #35 to particular viral genes, we applied an unbiased, systematic approach to define Vpu and Nef substrates. SILAC-based PMP was used to compare expression levels of cell surface proteins in CEM-T4s infected with either Vpu- or Nef-deficient viruses (Figures 3A and S3A), or transduced with Vpu or Nef as single genes (Figures 3B and S3B). As positive controls, we found that Vpu depleted cell surface CD4 and tetherin, while Nef depleted cell surface CD4 and HLA-A (Figures 3A and 3B). Surprisingly, many HIV-1 cell surface targets were downregulated efficiently by both Vpu- and Nef-deficient viruses and not by overexpression of Vpu and Nef as single genes, suggesting Vpu- and Nef-independent mechanisms.


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)

Proteomic Analysis of Vpu and Nef Targets and Identification of SNAT1 as a Vpu Substrate(A and B) SILAC-based quantitation of plasma membrane proteins in cells infected with Vpu-deficient (y axis) versus Nef-deficient (x axis) HIV-1 viruses (A) and cells transduced with Vpu (x axis) versus Nef (y axis) as single genes (B). Log2(fold change compared with uninfected [A] or GFP-transduced [B] cells) is shown for proteins from Cluster #35. Figures S3A and S3B selectively enlarge the lower left quadrant of each scatterplot. Proteins identified by >1 unique peptide are shown.(C) SNAT1 depletion by HIV-1 infection. CEM-T4s infected with WT NL4-3-deltaE-EGFP HIV-1 virus in the presence or absence of RTi were immunoblotted at the indicated time points. An MOI of 10 was used, and infection controls are shown in Figure S4C.(D) Rescue of SNAT1 in the absence of Vpu. CEM-T4s infected with WT, Vpu-deficient, or Nef-deficient HIV-1 NL4-3-deltaE-EGFP viruses were immunoblotted at 48 hr. An MOI of 10 was used, and infection controls are shown in Figure S4C.(E) SNAT1 depletion by Vpu. CEM-T4s stably transduced with GFP, Vpu, or Nef were immunoblotted. Untransduced CEM-T4s and CEM-T4s stably transduced with control or SNAT1-specific shRNAs were included as controls.(F) SNAT1 induction in activated primary T cells. Primary human CD4+ T cells activated with CD3/CD28 Dynabeads were immunoblotted at the indicated time points.(G and H) SNAT1 depletion by Vpu in activated primary T cells. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNA or Vpu constructs. After purification by AFMACS (Figure S4F), cells were either rested or re-stimulated with CD3/CD28 Dynabeads and immunoblotted (G) or analyzed by confocal microscopy (H) at 48 hr.See also Figures S3 and S4.
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fig3: Proteomic Analysis of Vpu and Nef Targets and Identification of SNAT1 as a Vpu Substrate(A and B) SILAC-based quantitation of plasma membrane proteins in cells infected with Vpu-deficient (y axis) versus Nef-deficient (x axis) HIV-1 viruses (A) and cells transduced with Vpu (x axis) versus Nef (y axis) as single genes (B). Log2(fold change compared with uninfected [A] or GFP-transduced [B] cells) is shown for proteins from Cluster #35. Figures S3A and S3B selectively enlarge the lower left quadrant of each scatterplot. Proteins identified by >1 unique peptide are shown.(C) SNAT1 depletion by HIV-1 infection. CEM-T4s infected with WT NL4-3-deltaE-EGFP HIV-1 virus in the presence or absence of RTi were immunoblotted at the indicated time points. An MOI of 10 was used, and infection controls are shown in Figure S4C.(D) Rescue of SNAT1 in the absence of Vpu. CEM-T4s infected with WT, Vpu-deficient, or Nef-deficient HIV-1 NL4-3-deltaE-EGFP viruses were immunoblotted at 48 hr. An MOI of 10 was used, and infection controls are shown in Figure S4C.(E) SNAT1 depletion by Vpu. CEM-T4s stably transduced with GFP, Vpu, or Nef were immunoblotted. Untransduced CEM-T4s and CEM-T4s stably transduced with control or SNAT1-specific shRNAs were included as controls.(F) SNAT1 induction in activated primary T cells. Primary human CD4+ T cells activated with CD3/CD28 Dynabeads were immunoblotted at the indicated time points.(G and H) SNAT1 depletion by Vpu in activated primary T cells. Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and mock transduced or transduced with the indicated shRNA or Vpu constructs. After purification by AFMACS (Figure S4F), cells were either rested or re-stimulated with CD3/CD28 Dynabeads and immunoblotted (G) or analyzed by confocal microscopy (H) at 48 hr.See also Figures S3 and S4.
Mentions: Depletion of most known HIV-1 cell surface targets has been attributed to Vpu and/or Nef (Haller et al., 2014; Tokarev and Guatelli, 2011). To assign downregulation of proteins in Cluster #35 to particular viral genes, we applied an unbiased, systematic approach to define Vpu and Nef substrates. SILAC-based PMP was used to compare expression levels of cell surface proteins in CEM-T4s infected with either Vpu- or Nef-deficient viruses (Figures 3A and S3A), or transduced with Vpu or Nef as single genes (Figures 3B and S3B). As positive controls, we found that Vpu depleted cell surface CD4 and tetherin, while Nef depleted cell surface CD4 and HLA-A (Figures 3A and 3B). Surprisingly, many HIV-1 cell surface targets were downregulated efficiently by both Vpu- and Nef-deficient viruses and not by overexpression of Vpu and Nef as single genes, suggesting Vpu- and Nef-independent mechanisms.

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