CELLULAR-DISTRIBUTION OF HIV TYPE-1 NEF PROTEIN - IDENTIFICATION OF DOMAINS IN NEF REQUIRED FOR ASSOCIATION WITH MEMBRANE AND DETERGENT-INSOLUBLE CELLULAR MATRIX

Cellular distribution of HIV-1 Nef protein was studied by expressing t he protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-rel ated proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef- 27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to de tergent solubilization. These two cellular fractions represent membran e- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-fra me AUG codon, was not modified with myristic acid at the amino terminu s. Consequently, this protein was present in a soluble form in the cyt osol. Furthermore, a mutant of Nef-27kD, in which the myristoylation s ignal is deleted, appears as a cytoplasmic soluble protein. To determi ne domains in Nef that are responsible for its subcellular distributio n, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), fro m which amino acids 73-88 were deleted, did not copurify with the dete rgent-insoluble fraction. The protein was, however, present in the par ticulate cytoplasmic fraction, presumably in association with membrane s. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton. Deletion of amino acids 73-88, on the other hand, specifically abolishes the a ssociation of Nef with the cytoskeleton matrix.