HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice.
Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE(65) and PXXP(75). The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met(20). How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE(65)-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP(75) to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2(-/-) mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.
Atkins
Katelyn M
KM
Vollum Institute, Portland, Oregon 97239, USA.
Thomas
Laurel
L
Youker
Robert T
RT
Harriff
Melanie J
MJ
Pissani
Franco
F
You
Huihong
H
Thomas
Gary
G
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005805:Genes, MHC Class I; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D010766:Phosphorylation; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C499638:PACS2 protein, human; C529570:Pacs2 protein, mouse; D033921:Vesicular Transport Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus