VIRION INCORPORATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF IS MEDIATED BY A BIPARTITE MEMBRANE-TARGETING SIGNAL - ANALYSIS OF ITS ROLEIN ENHANCEMENT OF VIRAL INFECTIVITY

The nef gene of primate immunodeficiency viruses is essential for high -titer virus replication and AIDS pathogenesis in vivo. In tissue cult ure, Nef is not required for human immunodeficiency virus (HN) infecti on but enhances viral infectivity, We and others have shown that Nef i s incorporated into HIV-1 particles and cleaved by the viral proteinas e, To determine the signal for Nef incorporation and to analyze whethe r virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion inco rporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV par ticles. Incorporation was reconstituted by targeting the respective pr oteins to the plasma membrane by using a heterologous signal. Mutation al analysis revealed that both myristoylation and an N-terminal cluste r of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domai n of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These result s indicate that Nef incorporation into HIV-I particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef co rrelated with enhanced infectivity of the respective viruses in a sing le-round replication assay. However, the phenotypes of HIV mutants wit h reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or dif ferent mechanisms may be involved in this system.