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Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation
L X Liu1, F Margottin, S Le Gall
1Institut Cochin de Génétique Moléculaire, INSERM U332, Université Paris V, 24 Rue du Faubourg Saint-Jacques, 75014 Paris, France.
Abstract:
Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.
Insights
The human immunodeficiency virus type 1 (HIV-1) Nef protein interacts with a novel human thioesterase, mediating CD4 down-regulation crucial for viral pathogenesis. This interaction is essential for Nef's ability to reduce CD4 cell-surface expression.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Nef is a conserved protein in primate lentiviruses, essential for high viral loads and pathogenesis in vivo.
- Nef exhibits multiple functions in vitro, including CD4 down-regulation, altered T cell activation, and enhanced viral infectivity.
Purpose of the Study:
- To identify cellular proteins interacting with HIV-1 Nef that may mediate its functions.
- To investigate the role of a novel human thioesterase in Nef-induced CD4 down-regulation.
Main Methods:
- Yeast two-hybrid system to screen for interacting proteins with HIV-1 Nef.
- Isolation and characterization of a novel human thioesterase.
- In vitro interaction assays and co-immunoprecipitation in CEM cells.
- Analysis of Nef-thioesterase interaction in relation to CD4 down-regulation.
Main Results:
- A novel human thioesterase, homologous to E. coli thioesterase II, was identified.
- HIV-1 Nef and the novel human thioesterase interact in vitro and in vivo.
- Nef alleles unable to down-regulate CD4 showed poor or no interaction with the thioesterase.
- A Nef mutant selected for lack of thioesterase interaction also lost CD4 down-regulation capability.
Conclusions:
- The identified human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.
- The interaction between Nef and this thioesterase is critical for Nef's function in reducing cell-surface CD4 expression.