Limits...
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

Mechanism of SNAT1 Depletion by Vpu(A) Interaction of SNAT1 with Vpu. HeLa cells stably transduced with Vpu-HA were immunoprecipitated with anti-SNAT1 (G63; first panel) or anti-HA (second panel) antibodies and immunoblotted with anti-SNAT1 (H60) or anti-Vpu antibodies. Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls.(B) Ubiquitination of SNAT1 by Vpu. HeLa cells stably transduced with Vpu-HA were either immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (first panel) or immunoprecipitated with anti-SNAT1 (G63) antibody, re-immunoprecipitated with anti-SNAT1 (H60) antibody, and immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (second panel). Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls. Ubiquitinated SNAT1 in control (blue arrow) and Vpu-expressing (red arrow) HeLas is highlighted.(C) β-TrCP-dependent depletion of SNAT1. HeLa cells stably transduced with Vpu-HA were transfected with control or β-TrCP-specific siRNA then immunoblotted.(D and E) SNAT1 depletion via an endolysosomal pathway. HeLa cells stably transduced with Vpu-HA were either treated with MG132, lactacystin, concanamycin, or bafilomycin (D) or transfected with control or TSG101-specific siRNA (E) then immunoblotted.(F) Molecular determinants of SNAT1 downregulation. Jurkats stably expressing Vpu WT or indicated Vpu mutants were immunoblotted. Cells transduced with empty vector (blue), Vpu WT (red), and Vpu A14L (pink) are highlighted. The same cells stained with anti-CD4 or anti-tetherin antibodies and analyzed by flow cytometry are shown in Figure S5A.See also Figure S5.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608997&req=5

fig4: Mechanism of SNAT1 Depletion by Vpu(A) Interaction of SNAT1 with Vpu. HeLa cells stably transduced with Vpu-HA were immunoprecipitated with anti-SNAT1 (G63; first panel) or anti-HA (second panel) antibodies and immunoblotted with anti-SNAT1 (H60) or anti-Vpu antibodies. Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls.(B) Ubiquitination of SNAT1 by Vpu. HeLa cells stably transduced with Vpu-HA were either immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (first panel) or immunoprecipitated with anti-SNAT1 (G63) antibody, re-immunoprecipitated with anti-SNAT1 (H60) antibody, and immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (second panel). Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls. Ubiquitinated SNAT1 in control (blue arrow) and Vpu-expressing (red arrow) HeLas is highlighted.(C) β-TrCP-dependent depletion of SNAT1. HeLa cells stably transduced with Vpu-HA were transfected with control or β-TrCP-specific siRNA then immunoblotted.(D and E) SNAT1 depletion via an endolysosomal pathway. HeLa cells stably transduced with Vpu-HA were either treated with MG132, lactacystin, concanamycin, or bafilomycin (D) or transfected with control or TSG101-specific siRNA (E) then immunoblotted.(F) Molecular determinants of SNAT1 downregulation. Jurkats stably expressing Vpu WT or indicated Vpu mutants were immunoblotted. Cells transduced with empty vector (blue), Vpu WT (red), and Vpu A14L (pink) are highlighted. The same cells stained with anti-CD4 or anti-tetherin antibodies and analyzed by flow cytometry are shown in Figure S5A.See also Figure S5.

Mentions: To probe the mechanism of Vpu-mediated SNAT1 depletion, we confirmed that Vpu binds endogenous SNAT1 (Figure 4A, lanes 2 and 4) and leads to SNAT1 ubiquitination (Figure 4B, lane 5). As with CD4 and tetherin, downregulation of SNAT1 is rescued by mutation of the Vpu phosphodegron responsible for β-TrCP recruitment (S52, 56A) (Figures 3G, panel 6, 3H, lane 6, 4F, and S5A) and by RNAi-mediated depletion of β-TrCP (Figure 4C, lane 3).


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)

Mechanism of SNAT1 Depletion by Vpu(A) Interaction of SNAT1 with Vpu. HeLa cells stably transduced with Vpu-HA were immunoprecipitated with anti-SNAT1 (G63; first panel) or anti-HA (second panel) antibodies and immunoblotted with anti-SNAT1 (H60) or anti-Vpu antibodies. Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls.(B) Ubiquitination of SNAT1 by Vpu. HeLa cells stably transduced with Vpu-HA were either immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (first panel) or immunoprecipitated with anti-SNAT1 (G63) antibody, re-immunoprecipitated with anti-SNAT1 (H60) antibody, and immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (second panel). Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls. Ubiquitinated SNAT1 in control (blue arrow) and Vpu-expressing (red arrow) HeLas is highlighted.(C) β-TrCP-dependent depletion of SNAT1. HeLa cells stably transduced with Vpu-HA were transfected with control or β-TrCP-specific siRNA then immunoblotted.(D and E) SNAT1 depletion via an endolysosomal pathway. HeLa cells stably transduced with Vpu-HA were either treated with MG132, lactacystin, concanamycin, or bafilomycin (D) or transfected with control or TSG101-specific siRNA (E) then immunoblotted.(F) Molecular determinants of SNAT1 downregulation. Jurkats stably expressing Vpu WT or indicated Vpu mutants were immunoblotted. Cells transduced with empty vector (blue), Vpu WT (red), and Vpu A14L (pink) are highlighted. The same cells stained with anti-CD4 or anti-tetherin antibodies and analyzed by flow cytometry are shown in Figure S5A.See also Figure S5.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608997&req=5

fig4: Mechanism of SNAT1 Depletion by Vpu(A) Interaction of SNAT1 with Vpu. HeLa cells stably transduced with Vpu-HA were immunoprecipitated with anti-SNAT1 (G63; first panel) or anti-HA (second panel) antibodies and immunoblotted with anti-SNAT1 (H60) or anti-Vpu antibodies. Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls.(B) Ubiquitination of SNAT1 by Vpu. HeLa cells stably transduced with Vpu-HA were either immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (first panel) or immunoprecipitated with anti-SNAT1 (G63) antibody, re-immunoprecipitated with anti-SNAT1 (H60) antibody, and immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (second panel). Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls. Ubiquitinated SNAT1 in control (blue arrow) and Vpu-expressing (red arrow) HeLas is highlighted.(C) β-TrCP-dependent depletion of SNAT1. HeLa cells stably transduced with Vpu-HA were transfected with control or β-TrCP-specific siRNA then immunoblotted.(D and E) SNAT1 depletion via an endolysosomal pathway. HeLa cells stably transduced with Vpu-HA were either treated with MG132, lactacystin, concanamycin, or bafilomycin (D) or transfected with control or TSG101-specific siRNA (E) then immunoblotted.(F) Molecular determinants of SNAT1 downregulation. Jurkats stably expressing Vpu WT or indicated Vpu mutants were immunoblotted. Cells transduced with empty vector (blue), Vpu WT (red), and Vpu A14L (pink) are highlighted. The same cells stained with anti-CD4 or anti-tetherin antibodies and analyzed by flow cytometry are shown in Figure S5A.See also Figure S5.
Mentions: To probe the mechanism of Vpu-mediated SNAT1 depletion, we confirmed that Vpu binds endogenous SNAT1 (Figure 4A, lanes 2 and 4) and leads to SNAT1 ubiquitination (Figure 4B, lane 5). As with CD4 and tetherin, downregulation of SNAT1 is rescued by mutation of the Vpu phosphodegron responsible for β-TrCP recruitment (S52, 56A) (Figures 3G, panel 6, 3H, lane 6, 4F, and S5A) and by RNAi-mediated depletion of β-TrCP (Figure 4C, lane 3).

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