DISSOCIATION OF THE CD4 DOWN-REGULATION AND VIRAL INFECTIVITY ENHANCEMENT FUNCTIONS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF

Recent evidence indicates that the nef gene of human immunodeficiency virus type 1 augments rather than inhibits viral replication in both c ell culture and in vivo models. In addition, nef alters various normal cellular processes, including the display of CD4 on the cell surface. However, it remains unknown whether the enhancement of infectivity an d the downregulation of CD4 represent linked or independent biologic p roperties of this single protein. In the present studies, mutational a nalyses were performed to define structure-function relationships with in the Nef protein that mediate these effects. To assess the functiona l consequences of these mutations, sensitive and reliable assays were developed to quantitate the viral infectivity enhancement and CD4 down regulation functions of Nef. The results indicate that membrane-target ing sequences at the N terminus of Nef are important for both function s of Nef, while certain other conserved regions are dispensable for bo th functions. A conserved proline-X-X repeat segment in the central co re of the protein, which is reminiscent of an SH3-binding domain, is c ritical for the enhancement of infectivity function but is dispensable for CD4 downregulation. However, the downregulation of CD4 by Nef app ears to involve a two-step process requiring the initial dissociation of p56(lck) from CD4 to permit engagement of the endocytic apparatus b y CD4. Together, these findings demonstrate that the infectivity enhan cement and CD4 dowregulation activities of human immunodeficiency viru s type 1 Nef can be dissociated. Thus, these processes may be independ ent of one another in the viral replication cycle.