title: Analysis of HIV-1 Gag protein interactions via biotin ligase tagging.
abstract: We have examined the interactions of wild-type (WT) and matrix protein-deleted (ΔMA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ΔMA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ΔMA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference.
  
  
  OBJECTIVE
  We have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild-type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled, and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1 pol gene and neighbor Gag proteins, but, surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over 50 cellular proteins that were biotinylated, including membrane and cytoskeletal proteins and proteins involved in lipid metabolism, nuclear import, translation, and RNA processing. Our results offer new insights into HIV-1 Gag protein trafficking and activities and provide new potential targets for antiviral interference.
authors: Department of Molecular Microbiology and Immunology, Oregon Health Science University, Portland, Oregon, USA.
authors: Christopher
authors: C
authors: Ritchie
authors: Department of Molecular Microbiology and Immunology, Oregon Health Science University, Portland, Oregon, USA.
authors: Isabel
authors: I
authors: Cylinder
authors: Department of Molecular Microbiology and Immunology, Oregon Health Science University, Portland, Oregon, USA.
authors: Emily J
authors: EJ
authors: Platt
authors: Department of Molecular Microbiology and Immunology, Oregon Health Science University, Portland, Oregon, USA barklis@ohsu.edu.
authors: Eric
authors: E
authors: Barklis
mesh_terms: D001710:Biotin / Q000032:analysis; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D015497:HIV-1 / Q000502:physiology*; D054884:Host-Pathogen Interactions; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
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