Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action

• Published in: Molecular biology of the cell 2010-10, Vol.21 (19), p.3279-3292
• Main Authors: Dikeakos, Jimmy D, Atkins, Katelyn M, Thomas, Laurel, Emert-Sedlak, Lori, Byeon, In-Ja L, Jung, Jinwon, Ahn, Jinwoo, Wortman, Matthew D, Kukull, Ben, Saito, Masumichi, Koizumi, Hirokazu, Williamson, Danielle M, Hiyoshi, Masateru, Barklis, Eric, Takiguchi, Masafumi, Suzu, Shinya, Gronenborn, Angela M, Smithgall, Thomas E, Thomas, Gary
• Format: Article
• Language: English
• Subjects: CD4-Positive T-Lymphocytes - drug effects; CD4-Positive T-Lymphocytes - enzymology; CD4-Positive T-Lymphocytes - virology; Cell Line; Down-Regulation - drug effects; Endocytosis - drug effects; Histocompatibility Antigens Class I - metabolism; HIV-1 - drug effects; Humans; Multienzyme Complexes - metabolism; nef Gene Products, Human Immunodeficiency Virus - metabolism; Phosphatidylinositol 3-Kinases - metabolism; Protein Binding - drug effects; Protein Transport - drug effects; PTEN Phosphohydrolase - metabolism; Signal Transduction - drug effects; Small Molecule Libraries - pharmacology; src-Family Kinases - metabolism; Time Factors; Transcription Factor AP-1 - metabolism; Vesicular Transport Proteins - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.e10-05-0470

HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1-mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2-dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1-dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.