THE HIV-1 NEF PROTEIN DOES NOT HAVE GUANINE-NUCLEOTIDE BINDING, GTPASE, OR AUTOPHOSPHORYLATING ACTIVITIES

Recombinant HIV-1 Nef proteins with either thr-15 or ala-15 have been constructed and expressed in the T7 bacterial system. From the soluble portion of bacterial lysates both Nef(thr-15) and Nef(ala-15) have been purified to near homogeneity through 6 nondenaturing chromatographic steps in the presence of MgCl2. Neither purified proteins display the previously reported GTP binding activity. Additionally Nef(thr-15) does not have autophosphorylating activity with either [gamma-P-32]GTP or [gamma-P-32]ATP. Although GTPase activity is present in the preparations of Nef proteins, it does not increase during purification and is attributed to bacterial contaminations.