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.
Why were proteins previously reported to associate with Nef not detected in our studies? One possible explanation is that previously reported associations are unstable under the biochemical purification conditions used in our studies. This likely explains the apparent absence of clathrin adaptors in Nef preparations purified in our studies, since we know that Nef binds AP-2/AP-1 clathrin adaptors only weakly in the salt and pH conditions used here (data not shown). It is also possible that in some cases, epitope tags may be buried and therefore inaccessible to the monoclonal antibodies (mAbs) used for immunoaffinity purification. Moreover, some of the previously reported associations, especially those with protein kinases, such as PAKs, were best detected by an ultrasensitive in vitro kinase assay (Sawai et al. 1995
) and, as our data show, are likely of exceedingly low stoichiometry. (Of note, we did detect the presence of p62 phosphoprotein [PAK] by in vitro kinase assays of anti-Nef purifications [data not shown]). Finally, some of the described associations, such as that with thioesterase, are known to occur only with selected Nef variants (Cohen et al. 2000
), while those we report here occur with all Nef variants tested. Nonetheless, the specific isolation of the Nef–DOCK2–ELMO1–Rac complex reported here provides strong biochemical evidence to reinforce predictions from previous genetic studies that Nef functions through multiple independent interactions with different sets of downstream effector proteins.
Our observations strongly argue that DOCK2–ELMO1 is the major upstream regulator used by Nef to activate Rac in T cells and that through this interaction Nef can activate Rac in CD4+ T lymphocytes even in the absence of stimulation with antigen or chemokines. These data indicate that Nef targets a critical switch, DOCK2–ELMO1, that regulates Rac GTPases downstream of chemokine receptors and the TCR and uses it to modulate the downstream processes they control. Thus, the interaction of Nef with DOCK2 and ELMO1 provides an important mechanism by which Nef may impact pathogenesis by primate lentiviruses.
Our data strongly suggest that DOCK2–ELMO1 is the major activator of Rac targeted by Nef in T lymphocytes. This model is supported by the observations that Nef physically associates with a complex that contains DOCK2–ELMO1 and Rac, that specific mutations in Nef simultaneously disrupt its ability to bind this complex and to activate Rac, and that Nef fails to activate Rac in the absence of ELMO1. Although previous reports implicated Vav, a Rac1 guanine nucleotide exchange factor (GEF), as the critical downstream effector that Nef binds directly to activate PAK (Fackler et al. 1999
), our data do not support this possibility. Notably, we have been unable to detect the presence of Vav in anti-Nef immune complexes by both proteomic analyses and immunoblotting, indicating that these interactions are of low abundance relative to those with DOCK2 and ELMO1 and are therefore unlikely to mediate the bulk of Nef's impact on the Rac pathway. Significantly, ELMO1 is ubiquitously expressed (Gumienny et al. 2001
) and can associate with Nef in nonlymphoid cells in the absence of DOCK2 (data not shown). Thus, we postulate a general mechanism in which ELMO1, possibly in complex with another CDM family protein, mediates Rac activation and PAK recruitment by Nef that is observed in nonlymphoid cells (Sawai et al. 1995
; Fackler et al. 1999
; Arora et al. 2000
Our observation that the expression of Nef from the integrated HIV-1 provirus in primary CD4+
T cells did not alter IL-2 production in standard activation protocols was unexpected, because previous genetic evidence linked Nef binding to DOCK2–ELMO1 and the ensuing activation of Rac GTPases to the the ability of Nef to facilitate T cell activation. Specifically, the same mutations that we observed to disrupt Rac activation through the DOCK2–ELMO1 complex were previously reported to disrupt the stimulatory effect of Nef on aspects of T cell activation (Sawai et al. 1995
; Simmons et al. 2001
), and the observed effects were in some cases dramatic (Wang et al. 2000
). Moreover, Rac activation by DOCK2 facilitates T cell responsiveness to antigen, as disrupted Rac activation in DOCK2
(−/−) and Rac2
(−/−) mice is associated with defective immunological synapse formation and depressed antigen-specific responses (Yu et al. 2001
; Sanui et al. 2003a
). Since Nef deregulates DOCK2 function and DOCK2 is essential for proper signaling through the immunological synapse, further studies under a variety of conditions that modulate the formation and function of the immunological synapse may be required to reveal the effect of Nef in the context of HIV-1 infection.
Interestingly, the chemokine receptor system plays crucial roles in infection by primate lentiviruses. Previous studies revealed its essential role for virus entry into target cells (Deng et al. 1996
; Feng et al. 1996
). Primate lentiviruses also exploit this system to recruit uninfected target cells to sites of viral replication (Weissman et al. 1997
; Swingler et al. 1999
). Our observations reveal an additional level of complexity in primate lentivirus–chemokine receptor system interactions. Inhibiting chemotaxis of the infected T cells likely disrupts the generation of the immune response. During generation of the immune response, T cells are initially activated in the paracortex of lymph nodes and then migrate to the edges of follicles, where they interact with antigen receptor-activated B cells (Garside et al. 1998
). This physical interaction is required to drive B cells towards antibody production, isotype switching, and the affinity maturation of the antibody response. Hence, the development and maturation of the immune response require the ordered migration of activated T cells to specific sites within lymphoid tissue (Delon et al. 2002
). Notably, recent in vivo evidence documents Nef-dependent alterations in the distribution of SIV mac239-infected CD4+
T lymphocytes in the lymph nodes of experimentally infected rhesus macaques (Sugimoto et al. 2003
). In lymph nodes of monkeys infected with nef-
deleted SIV, most infected T cells were located in the B cell-rich follicles and in the border region between the paracortex and the follicles. In contrast, in monkeys infected with SIV harboring a functional nef
gene, most productively infected T cells remained in the T cell-rich paracortex and were only infrequently present in proximity to B cell follicles. This evidence shows that Nef disrupts the ordered migration patterns of infected CD4+
T lymphocytes in vivo and reinforces the possibility that this disruption impairs the generation and maturation of the immune response to antigens. Since a large fraction of HIV-1-infected CD4+
T cells is specific for HIV-1 antigens (Douek et al. 2002
), this effect of Nef provides another mechanism to suppress the antiviral immune response. Taken together, the functional interaction of Nef with DOCK2, ELMO1, and Rac enables Nef to modulate multiple aspects of T cell function.