Although unnecessary for the growth of HIV and SIV in cultured cells, the expression of Nef enhances viral replication and in adult macaques is essential for pathogenesis. The molecular host cell target for this viral protein is unknown, although Nef mediated phenotypic changes have been characterized, most notably, CD4 down modulation. When expressed in the human T cell line VB, Nef does not alter the CD4 glycoprotein maturation process or the early association of CD4 with p56[lck]. It is only after the CVD4 reaches the plasma membrane that a Nef-mediated effect can be measured; p56[lck] is dissociated from CD4, and the CD4 is rapidly endocytosed and degraded. Expression of SF2 Nef in T cell lines either has no effect or can enhance the T cell receptor - mediated activation pathway leading to IL2 generation. To identify the molecular associations with the Nef protein, we have generated a novel fusion protein, where the C-terminus of Nef possesses an octapeptide extension that is easily recognized by monoclonal antibodies against the octapeptide. This construction permits N-terminal myristylation and expression of this protein results in the normal Nef mediated CD4 modulation. Work by others has demonstrated that Nef associated with a serine kinase. Our present work confirms this finding, and characterizes this 65 kDa enzyme as belonging to the p21 GTPase-activated kinase (PAK) family. Altered activity of a PAK kinase could lead to either negative (e.g., growth inhibition and apoptosis) or positive (e.g., enhanced transcription) effects on the affected cell.

Antibodies, Monoclonal/IMMUNOLOGY Antigens, CD4/METABOLISM/PHYSIOLOGY Cell Line Down-Regulation (Physiology) Gene Products, nef/GENETICS/*PHYSIOLOGY Human Receptors, Antigen, T-Cell/PHYSIOLOGY src-Family Kinases/METABOLISM ABSTRACT

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