Nef-mediated Clathrin-coated Pit Formation

Abstract

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

The Tyrosine Kinase ACK1 Associates with Clathrin-coated Vesicles through a Binding Motif Shared by Arrestin and Other Adaptors

“…We show here that elevated ACK1 can both stimulate and inhibit the rate of transferrin uptake (depending on the level of the protein). Such stimulated transferrin uptake might result from a general stimulation of clathrin assembly on the plasma membrane, similar to overexpression of transferrin receptor itself (36) or HIV Nef protein (37). Although tyrosine phosphorylation of integral membrane proteins can mediate endocytosis (38) we do not observe any requirement for ACK1 catalytic activity.…”

Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef

“…Dyn2 is a ubiquitously expressed GTPase that is essential for clathrin-mediated endocytosis, and is thought to function as a scission factor that mediates the separation of coated vesicles from the plasma membrane (26). Interestingly, Nef has been shown to trigger the de novo formation of clathrincoated pits when tethered to the plasma membrane (44), and may thus act as a connector between cargo such as CD4 and the endocytic machinery.…”

“…The K 44 A mutants of Dyn1 and Dyn2 inhibit clathrin-mediated endocytosis (36). Dyn1(K 44 A) also inhibits the Nef-induced down-regulation of CD4 but not of MHC class I molecules (37,38).…”

The Effects of HIV-1 Nef on CD4 Surface Expression and Viral Infectivity in Lymphoid Cells Are Independent of Rafts

“…Nef misroutes CD4 and other proteins through an interaction with a number of sorting proteins, including ␤-COP, AP complexes, and PACS-1 (18 -21). Overexpression of Nef also provokes pleiotropic effects on sorting organelles, inducing the accumulation of clathrin-coated pits, endosomes, lysosomes, and multivesicular bodies (22)(23)(24).…”