The HIV-1 Nef Protein and Phagocyte NADPH Oxidase Activation*
- Frederik Vilhardt‡§,
- Olivier Plastre‡,
- Makoto Sawada¶,‖,
- Kazuo Suzuki**,
- Maciej Wiznerowicz‡,
- Etsuko Kiyokawa‡,
- Didier Trono‡ and
- Karl-Heinz Krause‡
- From the ‡Biology of Ageing Laboratory, Department of Geriatrics, Geneva University Hospitals, 1225 Geneva, Switzerland, the ¶Joint Research Division for Therapies against Intractable Disease, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, ‖Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Tokyo 332-0012, Japan, the **National Institute of Infectious Diseases (NIID-NIH) Toyama-1-chome, Shinjuku, Tokyo 8640, Japan, and the ‡Department of Genetics and Microbiology, Faculty of Medicine, Geneva University, 1211 Geneva, Switzerland
Abstract
Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca2+ ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.
- HIV-1
- human immunodeficiency virus, type 1
- CM-DCF
- 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate
- CNF1
- cytotoxic-necrotizing factor 1
- CRIB
- Cdc42/Rac-interactive binding
- DPI
- diphenyl iodonium
- GM-CSF
- granulocyte macrophage colony-stimulating factor
- l-NMMA
- NG-monomethyl-l-arginine monoacetate salt
- PAK
- p21-activated kinase
- PMA
- phorbol myristate acetate
- RhoGDI
- Rho GDP dissociation inhibitor
- GST
- glutathione S-transferase
- HBSS. Hanks'-buffered saline solution
- FITC, fluorescein isothiocyanate
- Received January 28, 2002.
- Revision received August 19, 2002.
- The American Society for Biochemistry and Molecular Biology, Inc.
