Novel (n)PKC kinases phosphorylate Nef for increased HIV transcription, replication and perinuclear targeting

Abstract

The N-terminus of the human immunodeficiency virus (HIV) pathogenicity factor Nef associates with a protein complex (NAKC for Nef-associated kinase complex) that contains at least two kinases: the tyrosine kinase Lck and a serine kinase activity which was found to phosphorylate Lck and the Nef N-terminus. Here we show that this serine kinase activity is mediated by members of the novel Protein Kinase C (nPKC) subfamily, PKCdelta and theta. Association with the Nef N-terminus was sufficient to activate PKC leading to phosphorylation of Nef in vitro on a conserved serine residue at position 6. Mutation of serine 6 or coexpression of a transdominant negative PKC mutant significantly reduced Nef-stimulated HIV transcription and replication in resting PBMC. When analyzing the molecular mechanisms, we found that mutating serine 6 moderately affected myristoylation of Nef and its association with Pak2 activity, whereas CD4 downmodulation was not inhibited. More interestingly, this mutation abolished the typical perinuclear localization of Nef in T cells. We conclude that the activation of nPKCs by Nef is required to increase viral replication/infectivity and direct the subcellular localization of Nef.

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

“…1). As described previously (37,39,40,49), Nef localized to the plasma membrane, diffusely in the cytoplasm and at a perinuclear compartment, but was not detected in MCs. Quantification of the frequency of cells that displayed TCR MCs revealed that Nef did not significantly affect their overall occurrence (Fig.…”

HIV-1 Nef and Vpu Are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors, Including Tetraspanins

“…This is similar to the retargeting of Lck from the PM to RE/TGN compartments by Nef, for which association and colocalization are dispensable (38,65,(94)(95)(96). Nef may thus act in an indirect manner on TSPAN trafficking.…”

Viral Modulation of T-Cell Receptor Signaling

“…At least in the case of Lck, the physiological interaction involves Nef binding to the Lck SH2 and SH3 domains in a synergistic manner (24). The Nef/Lck interaction may not be direct; instead, it involves the formation of a complex between the PKC family members ␦ and and a conserved ␣-helix in the N terminus of Nef (13,159). The interaction with PAK2, a positive regulator of TcR signaling (23), involves a hydrophobic binding surface on the Nef core (1).…”