Abstract
The HIV Nef protein is an important pathogenic factor that modulates cell surface receptor trafficking and impairs cell motility, presumably by interfering at multiple steps with chemotactic receptor signaling. Here, we report that a dominant effect of Nef is to trigger AIP4 E3 ligase-mediated Gα(i2) ubiquitination, which leads to Gα(i2) endolysosomal sequestration and destruction. The loss of the Gα(i2) subunit was demonstrable in many cell types in the context of gene transfection, HIV infection, or Nef protein transduction. Nef directly interacts with Gα(i2) and ternary complexes containing AIP4, Nef, and Gα(i2) form. A substantial reversal of Gα(i2) loss and a partial recovery of impaired chemotaxis occurred following siRNA knockdown of AIP4 or NEDD4 or by inhibiting dynamin. The N-terminal myristoyl group, (62)EEEE(65) motif, and (72)PXXP(75) motif of Nef are critical for this effect to occur. Nef expression does not affect a Gq(i5) chimera where the five C-terminal residues of Gq are replaced with those of Gα(i2). Lysine at position 296 of Gα(i2) was identified as the critical determinant of Nef-induced degradation. By specifically degrading Gα(i2), Nef directly subverts leukocyte migration and homing. Impaired trafficking and homing of HIV Nef-expressing lymphocytes probably contributes to early immune dysfunction following HIV infection.
Highlights
HIV Nef disrupts chemotaxis of immune cells by targeting multiple steps
Nef-induced Loss of G␣i2 Was Partially Reversed by Expression of Enzymatically Defective AIP4 or by siRNA Knockdown of AIP4 or NEDD4—To firmly establish the role of AIP4 in the Nef-induced G␣i2 ubiquitination and degradation, Jurkat cells were co-transfected with Nef or an empty vector and WT or an enzymatically defective HECT domain mutant, AIP4-C830A [43, 48]
We have shown here that HIV Nef impairs G-protein signaling from chemokine receptors by selectively targeting G␣i2 for ubiquitination and rapid endolysosomal destruction
Summary
HIV Nef disrupts chemotaxis of immune cells by targeting multiple steps. Results: Nef recruits AIP4 E3 ligase, to ubiquitinate G␣i2 of heterotrimeric G-proteins for subsequent lysosomal degradation. Ligand-occupied chemoattractant receptors, trigger G␣ subunits of the heterotrimeric G-protein Gi to exchange GTP for GDP This leads to the release of G␣i-associated G␥ subunits, activation of downstream effectors, and directed cell migration [1]. During an adaptive immune response, activated paracortical T cells use CCR7 and CXCR5 to migrate to the follicle edge and eventually into the lymph node follicle, where they help coordinate B lymphocyte responses [11, 12] Impaired signaling through these different receptors can cause immune dysfunction and lymphoid tissue disorganization. A hallmark of HIV infection is severe disruption of the cellular microenvironment of lymph nodes and the gastrointestinal associated lymphoid tissue This occurs in part as a consequence of CD4ϩ T cell depletion, a major contributing factor is compromised chemoattractant receptor signaling [13, 14]. The consequence of the loss of G␣i2 is a marked impairment in chemoattractant receptor signaling and directed leukocyte migration
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