HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane

Abstract

The HIV-1 virulence factor Nef promotes high-titer viral replication, immune escape, and pathogenicity. Nef interacts with interleukin-2–inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk), two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages, respectively). Using a cell-based bimolecular fluorescence complementation assay, here we demonstrate that Nef recruits both Itk and Btk to the cell membrane and induces constitutive kinase activation in transfected 293T cells. Nef homodimerization-defective mutants retained their interaction with both kinases but failed to induce activation, supporting a role for Nef homodimer formation in the activation mechanism. HIV-1 infection up-regulates endogenous Itk activity in SupT1 T cells and donor-derived peripheral blood mononuclear cells. However, HIV-1 strains expressing Nef variants with mutations in the dimerization interface replicated poorly and were significantly attenuated in Itk activation. We conclude that direct activation of Itk and Btk by Nef at the membrane in HIV-infected cells may override normal immune receptor control of Tec-family kinase activity to enhance the viral life cycle.

Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes

“…Using the same approach, Li et al also established that SIV Nef (mac239 allele) interacted strongly with Itk and Btk at the plasma membrane and induced constitutive kinase autophosphorylation (86). This observation is significant because SIV Nef does not bind to the SH3 domain of Hck 87, providing further evidence that the mechanism of Tec-family kinase interaction with Nef may involve contacts in addition to the SH3 domain (82).…”

“…More recent studies combined BiFC with anti-phosphotyrosine immunofluorescence microscopy to demonstrate that Nef recruits both Itk and Btk to the cell membrane, resulting in sustained kinase autophosphorylation (86). A myristoylation-defective Nef mutant failed to recruit the kinases to the membrane, providing evidence that Nef also induces kinase relocalization.…”

“…Mutants of HIV-1 Nef that are defective for homodimer formation have been re-analyzed in recent work (86). These mutants involve three hydrophobic residues that contribute to the Nef dimer interface in the crystal complexes with both the SH3 domain alone and with the dual SH3-SH2 domain of Hck (Nef L112, Y115, and F121; Figures 5 and 6).…”

“…These mutants involve three hydrophobic residues that contribute to the Nef dimer interface in the crystal complexes with both the SH3 domain alone and with the dual SH3-SH2 domain of Hck (Nef L112, Y115, and F121; Figures 5 and 6). Recombinant full-length Nef proteins with these mutations are predominantly (Y115D) or exclusively (L112D and F121A) monomers by analytical size exclusion chromatography and show reduced propensity to form homodimers in the cell-based BiFC assay (86). The observation that these mutations do not completely abolish Nef homodimer formation in the BiFC assay likely relates to enhanced local concentrations of Nef at the cell membrane plus the fact that the YFP fluorophore is irreversibly reconstituted once formed (83).…”

“…The observation that these mutations do not completely abolish Nef homodimer formation in the BiFC assay likely relates to enhanced local concentrations of Nef at the cell membrane plus the fact that the YFP fluorophore is irreversibly reconstituted once formed (83). Nevertheless, each of these mutants showed greatly diminished capacity to activate the Tec-family kinases Itk and Btk at the cell membrane (86). Similar results were shown with Hck using the same cell-based assay (96), suggesting that activation of both kinase families requires a Nef dimer.…”