Cellular Distribution of HIV Type 1 Nef Protein: Identification of Domains in Nef Required for Association with Membrane and Detergent-Insoluble Cellular Matrix

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Abstract
Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related 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 detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, 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), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. 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 association of Nef with the cytoskeleton matrix.