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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

TMT-Based Proteomic Time Course of Plasma Membrane Protein Expression in HIV-1-Infected Cells(A) Workflow of TMT-based 6-plex PMP time course experiment. In subsequent figures, time points 1–5 show plasma membrane protein expression 0, 6, 24, 48, and 72 hr after HIV-1 infection (where 0 hr = uninfected cells), and time point 6 shows plasma membrane protein expression 72 hr after HIV-1 infection in the presence of reverse transcriptase inhibitors (RTi). NL4-3-deltaE-EGFP HIV-1 viruses at an MOI of 10 were used for all proteomic experiments.(B) Comparison of temporal profiles of CD4 and tetherin obtained by proteomic (TMT) versus flow cytometric quantitation. Cells from (A) were stained with anti-CD4 and anti-tetherin antibodies at the indicated time points and analyzed by flow cytometry. Relative abundance is expressed as a fraction of maximum TMT reporter ion or fluorescence intensity. For linear regression, log2(fold change compared with uninfected cells) is shown.(C) Temporal profiles of previously reported targets for HIV-mediated downregulation.(D) Identification of enriched temporal profiles by STEM. Model temporal profiles (black) and matched experimental protein expression profiles (red) are shown. Each box includes a profile identification number (top left) and an unadjusted p value (bottom left). Colored boxes indicate model profiles assigned more proteins than expected by chance alone (Bonferroni-adjusted p values < 0.05).See also Figure S1 and Tables S1 and S2.
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fig1: TMT-Based Proteomic Time Course of Plasma Membrane Protein Expression in HIV-1-Infected Cells(A) Workflow of TMT-based 6-plex PMP time course experiment. In subsequent figures, time points 1–5 show plasma membrane protein expression 0, 6, 24, 48, and 72 hr after HIV-1 infection (where 0 hr = uninfected cells), and time point 6 shows plasma membrane protein expression 72 hr after HIV-1 infection in the presence of reverse transcriptase inhibitors (RTi). NL4-3-deltaE-EGFP HIV-1 viruses at an MOI of 10 were used for all proteomic experiments.(B) Comparison of temporal profiles of CD4 and tetherin obtained by proteomic (TMT) versus flow cytometric quantitation. Cells from (A) were stained with anti-CD4 and anti-tetherin antibodies at the indicated time points and analyzed by flow cytometry. Relative abundance is expressed as a fraction of maximum TMT reporter ion or fluorescence intensity. For linear regression, log2(fold change compared with uninfected cells) is shown.(C) Temporal profiles of previously reported targets for HIV-mediated downregulation.(D) Identification of enriched temporal profiles by STEM. Model temporal profiles (black) and matched experimental protein expression profiles (red) are shown. Each box includes a profile identification number (top left) and an unadjusted p value (bottom left). Colored boxes indicate model profiles assigned more proteins than expected by chance alone (Bonferroni-adjusted p values < 0.05).See also Figure S1 and Tables S1 and S2.

Mentions: To gain a comprehensive, unbiased overview of plasma membrane protein regulation by HIV-1, we used PMP to measure expression levels of cell surface proteins in CEM-T4 T cells infected with HIV-1 (Figure 1A) (Weekes et al., 2013, 2014). By spinoculating cells with Env-deficient, VSVg-pseudotyped virus, we ensured a synchronous, single-round infection, and by using a multiplicity of infection (MOI) sufficient to infect >90% of cells, we minimized confounding effects from bystander (uninfected) cells (Figure S1A). We exploited 6-plex TMT quantitation to compare plasma membrane protein abundance in uninfected cells (0 hr), at 4 time points following HIV-1 infection (6, 24, 48, and 72 hr) and, to control for cellular changes occurring in the absence of de novo viral gene expression, in cells infected for 72 hr in the presence of reverse transcriptase inhibitors (RTi). In total, 2,320 proteins were quantitated, including 804 proteins previously reported to localize to the plasma membrane (Figure S1B). The complete dataset is shown in interactive Table S1, which allows generation of temporal profiles for any quantitated genes of interest.


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)

TMT-Based Proteomic Time Course of Plasma Membrane Protein Expression in HIV-1-Infected Cells(A) Workflow of TMT-based 6-plex PMP time course experiment. In subsequent figures, time points 1–5 show plasma membrane protein expression 0, 6, 24, 48, and 72 hr after HIV-1 infection (where 0 hr = uninfected cells), and time point 6 shows plasma membrane protein expression 72 hr after HIV-1 infection in the presence of reverse transcriptase inhibitors (RTi). NL4-3-deltaE-EGFP HIV-1 viruses at an MOI of 10 were used for all proteomic experiments.(B) Comparison of temporal profiles of CD4 and tetherin obtained by proteomic (TMT) versus flow cytometric quantitation. Cells from (A) were stained with anti-CD4 and anti-tetherin antibodies at the indicated time points and analyzed by flow cytometry. Relative abundance is expressed as a fraction of maximum TMT reporter ion or fluorescence intensity. For linear regression, log2(fold change compared with uninfected cells) is shown.(C) Temporal profiles of previously reported targets for HIV-mediated downregulation.(D) Identification of enriched temporal profiles by STEM. Model temporal profiles (black) and matched experimental protein expression profiles (red) are shown. Each box includes a profile identification number (top left) and an unadjusted p value (bottom left). Colored boxes indicate model profiles assigned more proteins than expected by chance alone (Bonferroni-adjusted p values < 0.05).See also Figure S1 and Tables S1 and S2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig1: TMT-Based Proteomic Time Course of Plasma Membrane Protein Expression in HIV-1-Infected Cells(A) Workflow of TMT-based 6-plex PMP time course experiment. In subsequent figures, time points 1–5 show plasma membrane protein expression 0, 6, 24, 48, and 72 hr after HIV-1 infection (where 0 hr = uninfected cells), and time point 6 shows plasma membrane protein expression 72 hr after HIV-1 infection in the presence of reverse transcriptase inhibitors (RTi). NL4-3-deltaE-EGFP HIV-1 viruses at an MOI of 10 were used for all proteomic experiments.(B) Comparison of temporal profiles of CD4 and tetherin obtained by proteomic (TMT) versus flow cytometric quantitation. Cells from (A) were stained with anti-CD4 and anti-tetherin antibodies at the indicated time points and analyzed by flow cytometry. Relative abundance is expressed as a fraction of maximum TMT reporter ion or fluorescence intensity. For linear regression, log2(fold change compared with uninfected cells) is shown.(C) Temporal profiles of previously reported targets for HIV-mediated downregulation.(D) Identification of enriched temporal profiles by STEM. Model temporal profiles (black) and matched experimental protein expression profiles (red) are shown. Each box includes a profile identification number (top left) and an unadjusted p value (bottom left). Colored boxes indicate model profiles assigned more proteins than expected by chance alone (Bonferroni-adjusted p values < 0.05).See also Figure S1 and Tables S1 and S2.
Mentions: To gain a comprehensive, unbiased overview of plasma membrane protein regulation by HIV-1, we used PMP to measure expression levels of cell surface proteins in CEM-T4 T cells infected with HIV-1 (Figure 1A) (Weekes et al., 2013, 2014). By spinoculating cells with Env-deficient, VSVg-pseudotyped virus, we ensured a synchronous, single-round infection, and by using a multiplicity of infection (MOI) sufficient to infect >90% of cells, we minimized confounding effects from bystander (uninfected) cells (Figure S1A). We exploited 6-plex TMT quantitation to compare plasma membrane protein abundance in uninfected cells (0 hr), at 4 time points following HIV-1 infection (6, 24, 48, and 72 hr) and, to control for cellular changes occurring in the absence of de novo viral gene expression, in cells infected for 72 hr in the presence of reverse transcriptase inhibitors (RTi). In total, 2,320 proteins were quantitated, including 804 proteins previously reported to localize to the plasma membrane (Figure S1B). The complete dataset is shown in interactive Table S1, which allows generation of temporal profiles for any quantitated genes of interest.

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