Date Published: January 20, 2004
Publisher: Public Library of Science
Author(s): Ajit Janardhan, Tomek Swigut, Brian Hill, Michael P Myers, Jacek Skowronski
Abstract: The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.
Partial Text: Primate lentiviruses persist in the host by active replication and can reemerge from latent reservoirs that are established in cells of the immune system (Finzi and Siliciano 1998; Douek et al. 2003). The infectious cycle is intimately linked to interactions between circulating T cells and antigen-presenting cells (Stevenson et al. 1990; Bukrinsky et al. 1991; Embretson et al. 1993; Swingler et al. 1999; Geijtenbeek et al. 2000). These interactions involve T cell migration, adhesion, and antigen-initiated signaling, processes that are dependent on cytoskeletal dynamics regulated by the Rho subfamily of small GTPases (Hall 1998; Schmitz et al. 2000).
Nef is a multifunctional adaptor protein that modulates signal transduction and protein-sorting machineries. We purified to near homogeneity an abundant Nef-associated protein complex from T cells and identified by mass spectroscopy its major subunits as DOCK2 and ELMO1, a bipartite Rac activator (Sanui et al. 2003b). Notably, the extensive large-scale biochemical purification and sensitive proteomic analyses described in this report did not detect several cellular proteins previously reported to associate with HIV-1 Nef and mediate the effects of Nef in the cell (data not shown). This includes cellular proteins such as Vav (Fackler et al. 1999), PAKs (Sawai et al. 1995), inositol triphosphate receptor (Manninen and Saksela 2002), Lck protein-tyrosine kinase (Baur et al. 1997), clathrin adaptors (Greenberg et al. 1997; Le Gall et al. 1998; Piguet et al. 1998), and others.