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Pulsed stable isotope labeling of amino acids in cell culture uncovers the dynamic interactions between HIV-1 and the monocyte-derived macrophage.

Kraft-Terry SD, Engebretsen IL, Bastola DK, Fox HS, Ciborowski P, Gendelman HE - J. Proteome Res. (2011)

Bottom Line: Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased.Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication.We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198-5880, USA.

ABSTRACT
Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host-cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus-cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in de novo protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection.

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Related in: MedlinePlus

pSILAC experimental design for HIV-1 infection of primary human macrophages. Control cells were treated for 48 h with [13C6]-l-Arginine and [D4]-l-Lysine, while at the same time, HIV-1-infected cells were treated with [13C6, 15N4]-l-Arginine and [13C6, 15N2]-l-Lysine. MDM were lysed and protein was quantified. Equal amounts of protein were combined, separated by OFFGEL electrophoresis, sequenced by LC–MS/MS and quantified using MaxQuant.
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fig1: pSILAC experimental design for HIV-1 infection of primary human macrophages. Control cells were treated for 48 h with [13C6]-l-Arginine and [D4]-l-Lysine, while at the same time, HIV-1-infected cells were treated with [13C6, 15N4]-l-Arginine and [13C6, 15N2]-l-Lysine. MDM were lysed and protein was quantified. Equal amounts of protein were combined, separated by OFFGEL electrophoresis, sequenced by LC–MS/MS and quantified using MaxQuant.

Mentions: SILAC medium was prepared using two different combinations of isotopically labeled l-Arginine and l-Lysine: “Medium” complete macrophage media, where l-Arginine and l-Lysine were replaced with [13C6]-l-Arginine and [D4]-l-Lysine (SIGMA-Isotec, St Louis, MO), and “Heavy” complete macrophage media, where l-Arginine and l-Lysine were replaced with [13C6, 15N4]-l-Arginine and [13C6, 15N2]-l-Lysine (SIGMA-Isotec, St Louis, MO).(12) SILAC media without MCSF was added on days 1, 3, or 5 following HIV-1 infection and cells lysed 48 h following SILAC medium addition. “Medium” macrophage media was applied to uninfected control cells and “Heavy” was applied to infected cells (Figure 1). This was repeated with three independent donors.


Pulsed stable isotope labeling of amino acids in cell culture uncovers the dynamic interactions between HIV-1 and the monocyte-derived macrophage.

Kraft-Terry SD, Engebretsen IL, Bastola DK, Fox HS, Ciborowski P, Gendelman HE - J. Proteome Res. (2011)

pSILAC experimental design for HIV-1 infection of primary human macrophages. Control cells were treated for 48 h with [13C6]-l-Arginine and [D4]-l-Lysine, while at the same time, HIV-1-infected cells were treated with [13C6, 15N4]-l-Arginine and [13C6, 15N2]-l-Lysine. MDM were lysed and protein was quantified. Equal amounts of protein were combined, separated by OFFGEL electrophoresis, sequenced by LC–MS/MS and quantified using MaxQuant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108467&req=5

fig1: pSILAC experimental design for HIV-1 infection of primary human macrophages. Control cells were treated for 48 h with [13C6]-l-Arginine and [D4]-l-Lysine, while at the same time, HIV-1-infected cells were treated with [13C6, 15N4]-l-Arginine and [13C6, 15N2]-l-Lysine. MDM were lysed and protein was quantified. Equal amounts of protein were combined, separated by OFFGEL electrophoresis, sequenced by LC–MS/MS and quantified using MaxQuant.
Mentions: SILAC medium was prepared using two different combinations of isotopically labeled l-Arginine and l-Lysine: “Medium” complete macrophage media, where l-Arginine and l-Lysine were replaced with [13C6]-l-Arginine and [D4]-l-Lysine (SIGMA-Isotec, St Louis, MO), and “Heavy” complete macrophage media, where l-Arginine and l-Lysine were replaced with [13C6, 15N4]-l-Arginine and [13C6, 15N2]-l-Lysine (SIGMA-Isotec, St Louis, MO).(12) SILAC media without MCSF was added on days 1, 3, or 5 following HIV-1 infection and cells lysed 48 h following SILAC medium addition. “Medium” macrophage media was applied to uninfected control cells and “Heavy” was applied to infected cells (Figure 1). This was repeated with three independent donors.

Bottom Line: Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased.Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication.We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198-5880, USA.

ABSTRACT
Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host-cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus-cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in de novo protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection.

Show MeSH
Related in: MedlinePlus