L Poulin1, J A Levy. 1. Cancer Research Institute, University of California, School of Medicine, San Francisco 94143.
Abstract
OBJECTIVE: To study the biochemical properties of the HIV-1 nef gene product. DESIGN: Earlier reports suggested that Nef protein is phosphorylated and has kinase activity. These properties were examined using an in vitro translated product. METHODS: A DNA fragment encoding Nef of HIV-1SF2 was transcribed in vitro under the control of T7 promoter. The generated nef messenger RNA (mRNA) was translated using a rabbit reticulocyte lysate system. Immunoprecipitation was performed with a specific monoclonal antibody to Nef. Kinase activity of the translated product was evaluated using [gamma-32P]GTP and ATP. RESULTS: Translated nef mRNA was found to encode a major protein of 27 kD with two other products, of 25 and 29 kD. Using Nef immune complexes for the kinase reaction, a protein of 46 kD that reacted with the anti-Nef monoclonal antibody was found to be strongly phosphorylated in the presence of Nef. This Nef-associated kinase activity on a p46 cellular substrate was dependent on ATP. CONCLUSIONS: The in vitro-translated nef gene product was found to be associated with phosphorylation of a p46 cellular protein present in rabbit reticulocyte lysate. The Nef protein has been linked to suppression of the replication of certain HIV strains. Identification of cellular targets of Nef activity could help to elucidate the mechanisms for this antiviral effect.
OBJECTIVE: To study the biochemical properties of the HIV-1nef gene product. DESIGN: Earlier reports suggested that Nef protein is phosphorylated and has kinase activity. These properties were examined using an in vitro translated product. METHODS: A DNA fragment encoding Nef of HIV-1SF2 was transcribed in vitro under the control of T7 promoter. The generated nef messenger RNA (mRNA) was translated using a rabbit reticulocyte lysate system. Immunoprecipitation was performed with a specific monoclonal antibody to Nef. Kinase activity of the translated product was evaluated using [gamma-32P]GTP and ATP. RESULTS: Translated nef mRNA was found to encode a major protein of 27 kD with two other products, of 25 and 29 kD. Using Nef immune complexes for the kinase reaction, a protein of 46 kD that reacted with the anti-Nef monoclonal antibody was found to be strongly phosphorylated in the presence of Nef. This Nef-associated kinase activity on a p46 cellular substrate was dependent on ATP. CONCLUSIONS: The in vitro-translated nef gene product was found to be associated with phosphorylation of a p46 cellular protein present in rabbit reticulocyte lysate. The Nef protein has been linked to suppression of the replication of certain HIV strains. Identification of cellular targets of Nef activity could help to elucidate the mechanisms for this antiviral effect.