In general, the interaction between Fas and FasL plays a important role in the homeostatic regulation of normal immune responses (37
). Stimulation of the TCR–CD3 complex in T cells causes upregulation of FasL and eventually leads to AICD or apoptosis (23
). A key molecule in this process is the TCR ζ chain and the three ITAMs contained therein. Cross-linking of the ζ chain or constructs containing individual ζ ITAMs alone were found to be sufficient to induce T cell activation and Fas-mediated apoptosis (28
). In agreement with these findings, we have shown here that TCR-ζ as well as the functional integrity of the ITAM signaling motifs of ζ were required for HIV-mediated upregulation of FasL. However, these findings further implied that HIV targets the TCR ζ chain directly through a viral protein.
To date, several lines of evidence indicated that the Nef protein exerted such a role. First, Nef-mediated activation of T cells has been demonstrated in a number of reports (8– 10). Second, expression of Nef in the cytoplasm of T cells interferes with early T cell signaling events emanating from the TCR–CD3 complex, including hypophosphorylation of TCR-ζ, whereas expression of a plasma membrane– associated form of Nef causes AICD in Jurkat cells (8
). Third, a very aggressive form of Nef from SIV, SIV-YE-Nef, basically functions like an ITAM domain of TCR-ζ (38
). Finally, SIV-induced upregulation of FasL in T cells depends on the expression of an intact Nef protein (22
), and Nef from a lethal SIV strain (smmPBj14) alone can directly cause FasL upregulation (39
). Thus, it appeared very likely that Nef acted at the level of the TCR. Indeed, our study confirms this assumption by showing that Nef can directly interact with the TCR ζ chain.
Strong evidence for the interaction of Nef with ζ came from a second, surprising finding. In Jurkat cells lacking the TCR, binding of the Nef-associated serine kinase p62/NAK was abolished. Conversely, reconstitution of these cells with the ζ ITAM 2 and 3 restored binding of p62/NAK with Nef. Furthermore, the integrity of the ITAM motif appeared to be important, since mutation of the ζ ITAMs greatly reduced the effect. As shown previously, the p62/NAK kinase has to be activated in order to bind to Nef (32
). These results suggest a dynamic interaction of Nef with the ζ ITAMs, ultimately resulting in the activation of p62/NAK, which in turn binds to Nef. In view of our and other studies, it is likely that activation of p62/NAK is part of Nef-mediated stimulation of T cell signaling pathways; however, at this point it is not clear whether p62/NAK has a role in the Nef-mediated upregulation of FasL. Notably, Nef binds to p62/NAK in cells lacking a TCR (31; e.g., COS cells). In these cells, the TCR ζ chain may be functionally replaced by other receptors, possibly containing ITAMs. This would explain why Nef has effects in cells usually not infected by HIV (40; e.g., NIH 3T3 cells).
More recently, Howe et al. showed that Nef from SIV or HIV-2 associated with the TCR ζ chain but failed to show an interaction with HIV-1 Nef (41
). Our study differs from that of Howe et al. in at least two respects. First we made constructs to target Nef to the plasma membrane where the TCR is located. Second, we have established functional consequence of the Nef–ζ interation which may have relevence to the pathogenesis of HIV interaction.
Induction of cell death by HIV could be mediated by different viral proteins. Cross-linking of CD4 by HIVgp120 in the presence of Tat protein can induce FasL expression and apoptosis of uninfected T cells (42
). Additionally, interaction of HIVgp120 with chemokine receptor CXCR4 on macrophages leads to death of CD8+
T cells mediated by TNF–TNFRII interaction (15
). In this study, we report an additional important mechanism of HIV-mediated apoptosis by demonstrating that Nef directly interacts with TCR-ζ and that both molecules are required for HIV-mediated upregulation of FasL. The interaction between Nef and TCR-ζ forms a signaling complex, bypassing the requirement for TCR ligation by antigen, and allowing HIV/SIV to activate T cells and upregulate FasL expression on the infected cells.
Thus, upregulation of FasL by Nef on HIV- or SIV- infected cells may, like FasL expression at sites of immune privilege and on some tumors, allow infected cells to evade the immune response. In addition, the effect of immune evasion is enhanced by Nef-mediated downregulation of surface MHC class I and CD4 expression (5, 6, 22, 43–46; see Fig. ). Taken together, our results provide additional insights into the molecular mechanism whereby the HIV accessory protein Nef regulates T cell activity and contributes to the pathogenesis of HIV.
Figure 7 Model describing mechanisms of immune evasion mediated by the HIV nef gene. Nef is expressed in the early viral life cycle and, after myristoylation, associates with the plasma membrane where several protein interactions take place. Nef interacts (more ...)