Reconstitution and Molecular Analysis of an Active Human Immunodeficiency Virus Type 1 Nef/p21-Activated Kinase 2 Complex

The Nef proteins encoded by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are major determinants of viral pathogenicity. The importance of Nef in viral pathogenesis was first shown in rhesus macaques, where a large deletion of the nef gene severely reduced SIV pathogenicity (28). Furthermore, in macaques infected with SIV containing a nef gene with a premature stop codon, the virus rapidly restored the nef open reading frame, showing that there is substantial selective pressure on the virus to express Nef (28). This finding was supported by the fact that a cohort consisting of one blood donor and eight transfusion recipients infected with Nef-defective HIV type 1 (HIV-1) demonstrated dramatically decreased rates of disease progression (13, 30, 32). Nef is a 27- to 34-kDa accessory protein expressed at high levels early in the viral life cycle (22, 41, 57). Nef is posttranslationally modified by N-terminal myristoylation, and this modification is essential for its different functions (9, 11, 35, 38). Nef is reported to alter signaling through the T-cell receptor (53), block apoptosis (60), activate Rac1 and Rac2 through DOCK2/ELMO1 (24), activate the serine/threonine kinase p21-activated kinase 2 (PAK-2) (4, 36, 44, 50), down-modulate cell surface receptors CD4 (1, 8, 20, 21) and major histocompatibility complex class I (52), and enhance viral infectivity (3, 11, 57). These last four activities of Nef are genetically separable on the basis of singly defective primary isolates and mutational analysis (19). The ability of Nef to interact with PAK-2 is conserved in a variety of SIV and HIV-1 Nefs, including SIVmac and SIVcpz, as well as in HIV-1 M, N, and O groups (31). The activation is also known to occur in different cell types and species, including human T cells and monocytic cells (4, 18, 36). Given this widespread conservation, Nef binding and activation of PAK-2 is likely to play an important role in viral pathogenesis, though at this time cellular substrates have yet to be identified. The cellular consequences of PAK-2 activation depend on the mechanism of activation and the cellular context in which activation occurs (47). For instance, cells respond to hyperosmolarity by inducing PAK-2 translocation to membranes prior to its activation (46). Activation of PAK-2 on membranes is transient and reversible and requires the activity of the p21-GTPase, Cdc42 (46). Cdc42 binds the p21 binding domain of PAK-2, but it is not known whether Cdc42 remains bound following activation (47). Some of the downstream consequences of PAK-2 activation by hyperosmolarity include activation of the stress-activated protein kinases SAPK/JNK and p38 (47). Stimulation of these pathways leads to phosphorylation of transcription factors and other proteins, which can eventually result in growth arrest or immune cell activation (23, 47). The activators of PAK-2, Cdc42 and Rac1, cycle between an active and an inactive state (47). In the inactive state they are bound to GDP, cytosolic, and complexed to RhoGDI, a GDP dissociation inhibitor (12, 58). After dissociation of RhoGDI, Rac1-GDP or Cdc42-GDP attaches to membrane via a C-terminal prenyl group, allowing binding of a guanine nucleotide exchange factor such as Vav or β-PIX (58). The guanine nucleotide exchange factor opens the nucleotide binding site of the p21-GTPase, allowing dissociation of GDP and binding of GTP (58). Inactivation occurs by hydrolysis of bound GTP to GDP through intrinsic GTPase activity and can be accelerated by a GTPase-activating protein (58). GTP-bound p21-GTPases are known to be required for PAK-2 activation by Nef on the basis of the following observations: (i) mutation of the p21-binding domain (PBD) of PAK-2 eliminates activation by Nef (45), (ii) cotransfection of Nef with constitutively active p21-GTPases enhances the Nef-associated PAK-2 activity (34, 37), and (iii) reduction of the steady-state level of activated p21-GTPases in the cell through the use of dominant negative (N17) mutants inhibits PAK-2 activation by Nef (34). The role of Nef could be to stabilize an interaction between the activated GTPase and PAK-2, to recruit PAK-2 to a membrane fraction where it can interact with activated GTPases, or to inhibit PAK-2 dephosphorylation. To examine the mechanism of PAK-2 activation by Nef, a eukaryotic cell-free system consisting of rabbit reticulocyte lysate, microsomal membranes, and in vitro-transcribed Nef RNA was characterized. This system faithfully recapitulated fundamental aspects of PAK-2 activation by Nef, including the need for activated p21 GTPases and for membrane association of Nef. This system was then employed to gain new insights into the mechanism of PAK-2 activation by Nef.