HIV-1 Nef Binds the DOCK2–ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis

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.

Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef

“…In addition to its essential roles in both clathrin-mediated and clathrin-independent endocytic events, Dyn2 has been implicated in the regulation of actin dynamics (48,49), in signaling (50), and in centrosome cohesion (51). In particular, Dyn2 controls cytoskeletal remodeling by Rac (52), a small GTPase that has been implicated in the function of Nef (12,53). However, the modulation of Rac function by Dyn2 appears unrelated to clathrin-mediated endocytosis (52), whereas our results imply that Dyn2 and clathrin are both required for the enhancement of infectivity by Nef, suggesting that this activity of Nef depends on the function of Dyn2 in the budding of clathrin-coated vesicles.…”

“…Nef induces the clathrin-mediated endocytosis and degradation of cell surface CD4 (2)(3)(4)(5), and also triggers the down-regulation of other cell surface molecules, including MHC class I antigens (6), which may facilitate the evasion of an antiviral immune response (7). Nef also modulates T cell function and apoptosis by engaging components of cellular signaling pathways, including Vav, Pak2, ASK1, and the DOCK2-ELMO1 complex (8)(9)(10)(11)(12).…”

Lentiviral Vpr usurps Cul4–DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle

“…Protein complexes were purified by two sequential immunoprecipitations via the FLAG and HA epitope tags from 7 g to 12 g of U937 cells stably expressing hfa-tagged Vpr proteins (or control cells), or transiently transfected HEK293T cells as described previously (45). MudPIT analysis of protein complexes was performed as previously described (48,49) and is described in detail in SI Text.…”

HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo

“…Nef-PAK2 association depends on a critical phenylalanine at position 195 of Nef (or 191 depending on the HIV-1 nef allele analyzed) that is dispensable for other Nef activities (9). Disruption of host cell actin remodeling by Nef does not affect intrinsic replication properties of HIV-1 (10, 11) but Nef impairs T-lymphocyte chemotaxis in vitro (12)(13)(14). Whether Nef also affects T-lymphocyte motility in mammals, which is only in part driven by chemokines (15), has not yet been addressed.…”